AN ABSTRACT OF THE DISSERTATION OF
Paul J. Carini for the degree of Doctor of Philosophy in Microbiology presented on May
23, 2013.
Title: Genome-Enabled Investigation of the Minimal Growth Requirements and
Phosphate Metabolism for Pelagibacter Marine Bacteria
Abstract approved:
Stephen J. Giovannoni
Members of the SAR11 clade of heterotrophic α-proteobacteria are ubiquitous
and abundant in the world’s oceans where they are thought to play a pivotal role in the
global carbon cycle. The first SAR11 bacterium cultivated in vitro, ‘Candidatus
Pelagibacter ubique’ HTCC1062 (Ca. P. ubique), was isolated by dilution into sterile
natural seawater, from which undefined dissolved organic carbon supplied nutrients for
heterotrophic growth. However, variation in the composition of such native dissolved
organic matter hindered efforts to identify the specific nutrient requirements of Ca. P.
ubique and elucidate its metabolism. For this dissertation work, genome-enabled
metabolic reconstruction was used to develop a defined artificial seawater medium for
Ca. P. ubique that consisted of inorganic salts, defined types and amounts of organic
carbon, reduced sulfur and vitamins. Subsequent in vitro experimentation was used to
show that Ca. P. ubique requires simultaneous additions of glycine and methionine to
meet cellular requirements for glycine and sulfur, respectively. A new requirement for
pyruvate was identified and linked to the production of alanine. We found that pyruvate
could be replaced by additions of glucose or oxaloacetate and that glycine could be
replaced with serine or glycine betaine. Interestingly, glycolate partially fulfilled the
glycine requirement of Ca. P. ubique, likely because of dual use as a carbon and glycine
source. Once these major organic nutrient requirements were established, the defined
medium was used to identify specific trace requirements of Ca. P. ubique for vitamins or
vitamin precursors. Previously, analysis of the Ca. P. ubique genome did not identify
complete biosynthetic pathways for thiamine (vitamin B1), pantothenate (vitamin B5),
pyridoxine (vitamin B6), biotin (vitamin B7) or cobalamin (vitamin B12), suggesting that
these vitamins cannot be synthesized de novo. We describe requirements for the
thiamine-pyrimidine precursor 4-amino-5-hydroxymethyl-2-methylpyrimidine (HMP)
and vitamin B7 as determined from laboratory cultures. We determined in situ (the
Sargasso Sea) concentrations of HMP by liquid chromatography coupled tandem mass
spectrometry. Our measurements show that in situ HMP concentrations are ample for the
native SAR11 population. Genomic and experimental evidence that vitamins B5 and B6
are synthesized de novo is presented and genomic evidence that vitamin B12 is not
required by Ca. P. ubique is discussed. A second Pelagibacter isolate, Pelagibacter sp.
str. HTCC7211 was also grown on a defined medium. Pelagibacter sp. str. HTCC7211
encodes a suite of genes, absent in Ca. P. ubique, dedicated to the acquisition and storage
of inorganic phosphate and the utilization of organic phosphorus. On a defined medium,
the growth of both Ca. P. ubique and Pelagibacter sp. str. HTCC7211 was limited by
excluding inorganic phosphate (Pi) and we show that Ca. P. ubique has an apparent
requirement for 10.2 attomoles Pi cell-1 and Pelagibacter sp. str. HTCC7211 has an
apparent requirement for 45.7 attomoles Pi cell-1. Discrete phosphorus utilization
physiotypes were evident, whereby Pelagibacter sp. str. HTCC7211, but not Ca. P.
ubique, utilized assorted Pi-esters and phosphonates in place of Pi to meet its cellular Prequirement. When grown on methylphosphonic acid (Mpn), the apparent P-requirement
decreased in Pelagibacter sp. str. HTCC7211 to 11.4 attomoles cell-1. We investigated
the underlying transcriptional dynamics that give rise to the observed physiotypes using
DNA microarrays. Ca. P. ubique responded rapidly to P-limitation by upregulating a
high-affinity transport system operon (pstSCAB-phoUB) and stress response genes
suggestive of a stringent response. Conversely, Pelagibacter sp. str. HTCC7211
responded slowly to the onset of P-limitation; no transcripts were differentially regulated
for 68 hours, after which, phosphonate transport and utilization genes showed the greatest
increases in expression. Collectively, these experiments exemplify the utility of growing
Ca. P. ubique (and other Pelagibacter isolates) on a defined medium and have opened the
door for additional experiments that were previously impossible to conduct on natural
seawater. This work also represents an important step towards developing Ca. P. ubique
into a model system that can be used to understand the cellular adaptations that make
oligotrophy a successful life strategy in the sea.
©Copyright by Paul J. Carini
May 23, 2013
All Rights Reserved
Genome-Enabled Investigation of the Minimal Growth Requirements and Phosphate
Metabolism for Pelagibacter Marine Bacteria
by
Paul J. Carini
A DISSERTATION
submitted to
Oregon State University
in partial fulfillment of
the requirements for the
degree of
Doctor of Philosophy
Presented May 23, 2013
Commencement June 2014
Doctor of Philosophy dissertation of Paul J. Carini presented on May 23, 2013.
APPROVED:
Major Professor, representing Microbiology
Chair of the Department of Microbiology
Dean of the Graduate School
I understand that my dissertation will become part of the permanent collection of Oregon
State University libraries. My signature below authorizes release of my dissertation to
any reader upon request.
Paul J. Carini, Author
ACKNOWLEDGEMENTS
I express my sincerest appreciation and love to my wife Kiri for her unwavering
love and support (and tolerance) that has been the foundation for my successful
completion of this degree. To my daughter Lucia (arguably my greatest experiment to
date), whose smiles and hugs reverse even the most discouraging of days, thank you for
your unconditional love. I thank Stephen Giovannoni for the opportunity to excel under
his tutelage; as an advisor, he didn’t push me to become a better scientist, instead he
challenged me, which is exactly what I needed to grow into the scientist I leave as today
(although I didn’t always realize it). Thank you to Emily O. Campbell for her belief in me
as a mentor and hard work necessary to complete the experiments described in Chapters
3-5. H. James Tripp has been instrumental to my knowledge of the methodologies of
metabolic reconstruction and cultivation of SAR11 cells. Through the years, our
professional relationship has grown into a close friendship that I value greatly. Thank you
to my family in Wisconsin who has endured (or reveled in?) my six years away from
home and to my in-laws in Oregon who have given me a new sense of home. A special
thank you to Carmen for introducing me to Kiri and Mariah for being there to keep Kiri
and Lucia company when I was busy doing experiments.
CONTRIBUTION OF AUTHORS
Dr. Laura Steindler and Dr. Sara Beszteri performed experiments related to the
cell division patterns linked to alanine use in chapter 2. Emily O. Campbell performed
some of the growth experiments in chapter 3, chapter 4 and chapter 5. Jeff Morré and Dr.
Samuel Bennett assisted with the extraction and quantification of vitamins from seawater
described in chapter 3. Dr. Sergio Sañudo-Wilhelmy performed vitamin measurements
important to the results obtained in chapters 3 and chapter 5. Dr. J. Cameron Thrash
constructed the phylogenetic trees in chapter 3. Dr. Taghd Begley synthesized the 4amino-5-hydroxymethyl-2-methylpyrimidine and 4-amino-5-aminomethyl-2methylpyrimidine for experiments in chapter 3. Dr. Angelicque E. White assisted with
methane measurements described in chapter 4. Dr. Stephen J. Giovannoni and the author
conceptualized all experiments.
TABLE OF CONTENTS
Page
Chapter 1 General Introduction .......................................................................................... 1
The foundations of microbial ecology ............................................................................ 2
Marine microbiology ...................................................................................................... 2
The ‘great plate count anomaly’ ..................................................................................... 4
Discovery and cultivation of SAR11 .............................................................................. 5
The SAR11 genome sequence and metabolic reconstruction ......................................... 8
Unraveling the metabolism of Ca. P. ubique .................................................................. 8
Genome streamlining .................................................................................................... 13
Growth on a defined medium ....................................................................................... 15
Chapter 2 Nutrient Requirements for the Growth of the Extreme Oligotroph ‘Candidatus
Pelagibacter ubique’ HTCC1062 on a Defined Medium .................................................. 19
Abstract ......................................................................................................................... 20
Introduction ................................................................................................................... 21
Methods......................................................................................................................... 24
Results ........................................................................................................................... 27
Discussion of results ..................................................................................................... 29
Chapter 3 ‘Candidatus Pelagibacter ubique’ HTCC1062 is Dependent on 4-Amino-5Hydroxymethyl-2-Methylpyrimidine, An Abundant Thiamine Precursor In The Sea ..... 46
Abstract ......................................................................................................................... 47
Introduction ................................................................................................................... 48
Results & Discussion .................................................................................................... 49
Methods......................................................................................................................... 58
Chapter 4 Distinct Phosphate-Acquisition Physiotypes and Adaptations for Extreme
Marine Oligotrophy in SAR11 Bacteria ........................................................................... 66
Abstract ......................................................................................................................... 67
Introduction ................................................................................................................... 68
Methods......................................................................................................................... 71
Results ........................................................................................................................... 75
Discussion ..................................................................................................................... 78
Chapter 5 Analysis of the Vitamin B5, B6, B7 And B12 Requirements of ‘Candidatus
Pelagibacter ubique’ HTCC1062: Evidence for B7 Auxotrophy. ..................................... 97
Abstract ......................................................................................................................... 98
Introduction ................................................................................................................... 99
Methods....................................................................................................................... 104
Results ......................................................................................................................... 106
Conclusions ................................................................................................................. 109
Chapter 6 Conclusions .................................................................................................... 121
Bibliography ................................................................................................................... 123
TABLE OF CONTENTS (Continued)
Page
Appendices...................................................................................................................... 139
Appendix 1 .................................................................................................................. 140
Appendix 2 .................................................................................................................. 145
Appendix 3 .................................................................................................................. 164
LIST OF FIGURES
Figure
Page
Figure 2-1: Simplified illustration of central metabolism in Ca. P. ubique. ..................... 41
Figure 2-2: Growth of Ca. P. ubique in AMS1 with organic carbon additions. ............... 42
Figure 2-3: Maximum cell yields of Ca. P. ubique in response to pyruvate and glycine
additions…………………………………………………………………………….43
Figure 2-4: DNA content and morphology of SYBR Green I-stained stationary phase
cells from pyruvate-deplete and replete batch cultures............................................. 44
Figure 2-5: Glycolate assimilation gene organizations in E. coli and Ca. P. ubique........ 45
Figure 3-1: Comparative genomics of thiamine biosynthesis........................................... 63
Figure 3-2: Growth of Ca. P. ubique under thiamine-limiting conditions and responses to
thiamine pyrimidines. ............................................................................................... 64
Figure 3-3: Depth distribution of dissolved 4-amino-5-hydroxymethyl-2methylpyrimidine (HMP) and thiamine in the Sargasso Sea. ................................... 65
Figure 4-1: Linear dose responses of Ca. P. ubique and Pelagibacter sp. str. HTCC7211
to P-sources. .............................................................................................................. 93
Figure 4-2: P-source utilization by Ca. P. ubique and Pelagibacter sp. str. HTCC7211. 94
Figure 4-3: Comparative genomics of gene expression during Pi-starvation in Ca. P.
ubique and Pelagibacter sp. str. HTCC7211. ........................................................... 95
Figure 4-4: Methane (CH4) production by Pelagibacter sp. str. HTCC7211 utilizing Mpn
as a sole P-source. ..................................................................................................... 96
Figure 5-1: Simplified illustration of B-vitamin-requiring reactions in Ca. P. ubique... 113
Figure 5-2: Comparative genomics of vitamin B5 biosynthesis in Ca. P. ubique. ......... 114
Figure 5-3: Comparative genomics of vitamin B6 biosynthesis in Ca. P. ubique. ......... 115
Figure 5-4: Comparative genomics of vitamin B7 biosynthesis in Ca. P. ubique. ......... 116
Figure 5-5: Routes of canonical vitamin B12 biosynthesis…………………………...…117
Figure 5-6: Ca. P. ubique is bradytrophic for vitamin B5 (pantothenate) in the presence of
vitamin B7 (biotin). ................................................................................................. 118
Figure 5-7: Vitamin B7-scrubbed medium allows for demonstration of vitamin B7requirement in Ca. P. ubique. ................................................................................. 119
LIST OF TABLES
Table
Page
Table 2-1: Constituents of the Artificial Medium for SAR11 (AMS1) ............................ 38
Table 2-2: Potential sources of pyruvate for Ca. P. ubique when grown in AMS1 with
25 µM glycine and 10 µM methionine...................................................................... 39
Table 2-3: Potential sources of glycine for Ca. P. ubique when grown in AMS1 with
50 µM pyruvate and 10 µM methionine ................................................................... 40
Table 5-1: Common vitamin B12-requiring enzymes are absent in Ca. P. ubique.......... 120
LIST OF APPENDIX FIGURES
Figure
Page
Figure A1- 1: Transfer of Ca. P. ubique batch cultures from natural seawater to AMS1
................................................................................................................................. 140
Figure A1- 2: Flow cytometry profiles and corresponding microscopic images at different
pyruvate:glycine molar ratios. ................................................................................ 141
Figure A1- 3: DNA fluorescence profiles and maximum cell densities in response to
alanine additions. .................................................................................................... 142
Figure A1- 4: Effect of vitamin additions on Ca. P. ubique growth............................... 143
Figure A2- 1: Simplified illustration of Ca. P. ubique’s central metabolism, highlighting
thiamine-requiring biosynthetic reactions…………………………………………145
Figure A2- 2: Reproduction of Figure 3-2a in main text with microarray sample points
labeled. .................................................................................................................... 146
Figure A2- 3: Growth responses of Ca. P. ubique to potential thiamine precursors. ..... 146
Figure A2- 4: Maximum-likelihood phylogenetic reconstruction of the SAR11_0811
amino acid sequence. .............................................................................................. 147
Figure A2- 5: Illustration of predicted thiamine regulatory elements or thiamine
pyrimidine salvage genes genetically associated with the putative HMP transport
protein coding sequence. ......................................................................................... 149
Figure A2- 6: Profiles of in situ temperature and chlorophyll a fluorescence at
Hydrostation S in the Sargasso Sea during water collection for vitamin analysis...156
Figure A3- 1: Growth curves of Pelagibacter sp. str. HTCC7211 on alternate phosphorus
sources showing diauxic growth pattern…………………………………………..164
Figure A3- 2: Growth curves for linear dose responses of Pelagibacter sp. str.
HTCC7211 grown on Pi. ......................................................................................... 165
Figure A3- 3: Growth curves for linear dose responses of Ca. P. ubique grown on Pi...166
Figure A3- 4: Growth curves for linear dose responses of Pelagibacter sp. str.
HTCC7211 grown on Mpn. .................................................................................... 167
Figure A3- 5: Simplified illustration of phospholipid biosynthetic routes and predicted
reactions involved in lipid remodeling in Pelagibacter sp. str. HTCC7211. ......... 169
Figure A3- 6: Scanning transmission electron microscopy images of Pelagibacter sp. str.
HTCC7211 cells grown under different P-regimes. ............................................... 170
LIST OF APPENDIX TABLES
Table
Page
Table A2- 1: Gene transcripts more abundant in thiamine-limited conditions ............... 157
Table A2- 2: Gene transcripts more abundant in thiamine-replete conditions ............... 160
Table A3- 1: Specific growth rates of Ca. P. ubique & Pelagibacter sp. str. HTCC7211 in
AMS1 with different amounts of Pi or Mpn……………………………………….171
Table A3- 2: Genes differentially regulated in P-limited Ca. P. ubique 4 hours after resuspension. .............................................................................................................. 172
Table A3- 3: Genes differentially regulated in P-limited Ca. P. ubique 20 hours after resuspension. .............................................................................................................. 173
Table A3- 4: Genes differentially regulated in P-limited Ca. P. ubique 38 hours after resuspension. .............................................................................................................. 175
Table A3- 5: Genes differentially regulated in P-limited Pelagibacter sp. str. HTCC7211
68 hours after re-suspension. .................................................................................. 177
Table A3- 6: Genes differentially regulated in P-limited Pelagibacter sp. str. HTCC7211
96 hours after re-suspension. .................................................................................. 178
DEDICATION
During my time in Oregon I have come to realize that my love for nature and my
interest in how the world works is the result of years of listening to my grandfather
Joseph Horbas. Although not formally educated in science, he is a Naturalist, in the truest
sense of the word. His love for nature and penchant for observation has resonated with
me as long as I can remember. And, on days where I’m working outside or walking with
Lucia, I think of the times I spent working in his yard fondly, or find myself passing on
this love of nature to the next generation of Naturalists. This dissertation work is
dedicated to him.
Genome-Enabled Investigation of the Minimal Growth Requirements and Phosphate
Metabolism for Pelagibacter Marine Bacteria
Chapter 1
General Introduction
For over a century, the field of microbial ecology has been predicated on the
ability of scientists to cultivate and study environmentally relevant microbes under
controlled laboratory conditions. The goal of the research contained herein is to develop
isolates of the abundant and ubiquitous SAR11 clade of α-proteobacteria into model
systems that can be used to understand the metabolism of oligotrophs and the
biogeochemical processes they mediate in situ. Studies such as these have traditionally
been at the forefront of microbial ecology. Though, with the 20th century advances in
molecular biology and biochemistry - primarily nucleic acid sequencing and the
expression of foreign genes in heterologous hosts - cultivation of environmentally
important organisms has been minimized, effectively having been replaced by metabolic
models of uncultivated microbes deduced from DNA sequence information. However,
sequence data is merely descriptive, requiring experimental evidence to contextualize it
in terms of organismal and ecosystem function. Therefore, as is presented here, the ideas
and hypotheses constructed from genomes and metagenomic analyses should be
addressed by experimentation for a truer understanding of microbial metabolism and the
roles of microbes in ecosystem dynamics.
2
The foundations of microbial ecology
The understanding that bacteria act as the functional agents of geochemical
change was largely derived from the experiments of Sergio Winogradsky and Martinus
Beijerinck in the late 19th century. Winogradsky discovered that key steps in the sulfur
and nitrogen cycles were mediated by specific groups of bacteria. Through isolation and
study of pure bacterial cultures, Winogradsky proposed the idea of chemolithotrophy – a
form of metabolism that derives energy from inorganic compounds to fuel CO2 fixation.
Winogradsky’s contemporary, Beijerinck, also understood that specific assemblages of
bacteria in the natural environment exploited distinct geochemical niches. Through his
own work designing and testing different microbial growth media, Beijerinck developed
the concept of the enrichment culture. He reasoned that by altering the nutritional
composition of growth media, one could select for (that is, enrich for) specific nutrient
transformations or consortia of bacteria that share similar growth requirements. By
studying both pure and enriched cultures under laboratory conditions, Winogradsky and
Beijerinck pioneered what are now foundational ideas and methodology in microbial
ecology. By linking laboratory results to environmental observations, they proposed that
microbes had an active and important role in environmental nutrient cycling.
Marine microbiology
The prevailing view in the late 19th century was that although microbes were
abundant in soils, they could not survive in the harsh conditions of the sea. High salt
concentrations, extremely low amounts of organic carbon and other nutrients, together
with large doses of ultraviolet light from the sun, were thought to render the open ocean
3
devoid of microbial life. Reports from Bernard Fischer in the late 1800’s changed this
perspective (reviewed in (1)). Fischer experimentally developed growth media for marine
microbes and used it to cultivate, characterize and study environmental distributions of
bacteria in the sea. Fischer determined that an ideal medium for the growth of marine
bacteria should contain 1.0% peptone, 0.5% fish extract, and 10% gelatin (or 2% agar) in
a seawater base. Using this medium, he isolated and characterized a number of marine
organisms, including the first species of luminescent bacteria (Photobacterium indicum).
Fischer also identified ecological relationships related to bacterial distributions. For
example, he observed that nearly all samples of seawater contained cultivatable bacteria
and that seawater contained an average of 1,083 bacteria per milliliter. In 1894 he noted
that there were more bacteria in coastal waters than in the open ocean and that bacteria
were more abundant at depth (200-400 meters) than at the surface.
In 1941 Claude ZoBell identified a reliable growth medium that produced higher
cell counts per unit of seawater than the media described by Fischer (2). Known as
ZoBell medium 2216 (and currently known commercially as ‘Marine Agar 2216’), it
consisted of Bacto™ peptone (5.0 g L-1), ferric phosphate (0.1 g L-1) and Bacto™ agar (15
g L-1), dissolved in aged or “rotted” seawater. Variations on ZoBell medium 2216
included the addition specific carbon sources or inorganic nutrients such as glucose (for
example (3)) to further enrich for specific bacterial groups or activities. However, ZoBell
himself understood the limitations of his medium – specifically, that no single growth
medium could meet the growth requirements of all cells in a seawater sample (1, 2).
4
The ‘great plate count anomaly’
The culture-based methods used to enumerate marine bacteria as exemplified by
Fischer, ZoBell and others, routinely produced lower cell counts per unit volume than
direct counts of planktonic cells by microscopy. For example, Waksman (4) counted
1000-fold more bacteria per milliliter of seawater under the microscope than were
identified when using agar plate-based quantification methods. Jannasch and Jones (5)
found that direct counts of seawater were 13 to 9,700-fold higher than those obtained by
cultural methods. Similar observations were made in other aquatic ecosystems (6). The
incongruence between direct counts and plate counts was presented by Staley and
Konopka as “the great plate count anomaly” (7), and was believed to result from one of
two paradigms: 1) the cells observed by microscopy were dead or otherwise nonviable; or
2) the growth medium used to cultivate bacteria did not support their growth
requirements. ZoBell succinctly summarized the topic in his 1946 monograph (1): “At
best, direct counts give data which only supplement and aid in the interpretation of
results obtained by cultural procedures.”
Departing from approaches that used high-nutrient growth media, Button
developed a new methodology called “dilution culturing” for use in marine ecosystems
(8, 9). Button hypothesized that abundant marine bacteria are probably in a constant state
of starvation due to the paucity of nutrients in their oligotrophic environments (8). This
paradoxical phenomenon, known as ‘oligotrophy’ (reviewed in (10)), describes the
metabolism of cells that grow optimally in nutrient-deplete conditions. Button reasoned
that sterilized seawater (usually autoclaved) could supply sufficient native dissolved
5
organic matter to support the growth of oligotrophs. He calculated that his method of
cultivation should favor numerically dominant organisms (which he hypothesized were
oligotrophic) over cells that persist in natural waters at lower densities but tend to grow
well on high-nutrient media. This method of dilution culturing was successfully applied
by Button and colleagues to isolate the marine oligotrophs Sphingopyxis alaskensis
RB2256 and Cycloclasticus oligotrophus (9, 11).
Discovery and cultivation of SAR11
In the late 1970’s and 1980’s a trio of discoveries were made that would forever
revolutionize microbial ecology – i) the development of more reliable and convenient
DNA sequencing (‘Sanger sequencing’ (12)); ii) the polymerase chain reaction (PCR)
(13); iii) and a microbial classification system based on rRNA gene sequences (14). With
these new molecular tools and insights into microbial evolution, researchers began
investigating microbial diversity in the environment not with a microscope or agar plates,
but with PCR primers and sequence analysis tools (15). Stephen Giovannoni used this
type of cultivation-independent approach to assay the microbial diversity of the Sargasso
Sea, an extremely oligotrophic ocean gyre in the North Atlantic Ocean (16). Giovannoni
used PCR to amplify 16S ribosomal RNA genes from DNA extracted from Sargasso Sea
bacterioplankton. The resulting PCR products were cloned, sequenced and used as a basis
for molecular phylogeny. Molecular phylogeny revealed that many of the sequences
belonged to a deeply-rooted group of uncultivated α-proteobacteria (16). A DNA
sequence, representative of the abundant group of sequences was named ‘SAR11’ (short
for clone number 11 identified from the SARgasso sea). Hybridization of SAR11
6
sequence probes to total rRNA collected from bacterioplankton obtained at different
locations showed that the SAR11 rRNA was widely distributed and comprised a
relatively large amount of total rRNA in the locations assayed (16). Continued study of
SAR11 distributions, using fluorescent in situ hybridization (FISH) of SAR11 probes to
filtered bacterioplankton cells, showed that cells that hybridized to the SAR11 probes
were extremely small, planktonic cells that can account for up to 50% of all cells near the
ocean’s surface and up to 25% of all planktonic cells at depth (17). Today it is understood
that the SAR11 cells are widely distributed in marine waters (17-19) and some freshwater
ecosystems (20).
For nearly a decade after the discovery of its DNA in the environment,
researchers struggled to cultivate SAR11 in the laboratory on traditional marine growth
media (for example, ZoBell media 2216). Prior to the use of sterile seawater by Button,
most growth media contained large amounts of enzymatically digested protein, fish
extract, or other suitable constituents, in hopes to boost the number and diversity of
cultivatable isolates. In contrast to this, Button hypothesized that unamended (or
minimally amended) seawater could supply carbon (in the form of complex and
undefined, but importantly, native, dissolved organic matter) and other nutrients in
sufficient quantities to favor the growth of numerically abundant oligotrophs. Button’s
dilution culture technique was improved by Giovannoni and colleagues by miniaturizing
cultivation chambers and developing high-throughput arrays for filtering, enumerating
and identifying cells at lower densities (21). The speed and efficiency of enumeration was
later enhanced by implementation of a 96-well microtiter plate-based flow cytometer,
7
which allowed for the rapid and accurate determination of cell counts with a limit of
detection of about 1000 cells ml-1 (22). This approach, one of high-throughput cultivation
by dilution, would be used repeatedly to successfully cultivate cells that were routinely
observed by the microscopists, but hypothesized to be dead or dormant by ZoBell and
others.
In 2002, Giovannoni and colleagues isolated the first SAR11-clade bacterium
from the environment (the northeast Pacific Ocean off of the coast of Oregon, USA)
using dilution culturing into autoclaved natural seawater (23). The name proposed for the
first SAR11 isolate was ‘Candidatus Pelagibacter ubique’ strain HTCC1062 (Ca. P.
ubique, herein). Although cultivated, Ca. P. ubique was given (and still retains)
‘Candidatus’ status because growth experiments required for classification by the
Bacteriological Code (24) could not be performed for technical reasons. Analyses of Ca.
P. ubique’s growth kinetics and metabolism in natural seawater showed the compounding
factors that had contributed to previous cultivation difficulties. First, growth was
completely inhibited in the presence of 0.001% (w/v) protease peptone – a complex
mixture of nutrients in most conventional marine media preparations (23). Additionally,
the maximum cell yield was under 106 cells ml-1, well below densities for optical density
measurements (a technique typically used for assessment of broth cultures) and colony
formation (for visualization on agar plates). Low specific growth rates of 0.4 - 0.58 d-1
may have also contributed to cultivation difficulties. Thereafter, Ca. P. ubique was
routinely propagated on a base medium of autoclaved natural seawater, usually obtained
from the northeast Pacific Ocean.
8
The SAR11 genome sequence and metabolic reconstruction
Since its initial discovery, SAR11 cells have been identified worldwide and the
clade is generally inferred to have a large role in the trophic interactions related to the
global carbon cycle because of their abundance and ubiquity. However, identifying the
specific metabolic nuances of SAR11 cells proved to be a challenge, owing to their
inherent growth characteristics. The genome of Ca. P. ubique was sequenced in 2005
with the goal of identifying clues from metabolic reconstruction that could be used to
unravel the growth requirements of these cells (25). The closed genome was just over 1.3
Mbp in length and is one of the smallest genomes of any free-living organism. Metabolic
reconstruction showed that Ca. P. ubique likely encodes complete metabolic pathways
for all 20 amino acids and all other cellular constituents (except for five B-vitamins,
discussed in subsequent chapters) (25). Cells appeared to be non-glycolytic (although
some, but not all genes encoding proteins used in the Entner-Doudoroff glycolytic
pathway were identified), aerobic heterotrophs that made efficient use of dissolved
organic matter in the ocean. Although metabolic reconstruction did not identify obvious
reasons for the inability to grow Ca. P. ubique on defined medium, clever analysis and
design of laboratory experiments were successful in uncovering nutrient requirements for
increased cell yields on natural seawater.
Unraveling the metabolism of Ca. P. ubique
Using the reconstructed metabolism of Ca. P. ubique as a guide, Tripp and
Schwalbach conducted a series of growth experiments to test hypotheses generated from
genomic information, and ultimately identified three classes of nutrients that were
9
required for growth: reduced organosulfur compounds, serine (or glycine), and low
molecular weight organic acids. The first anomaly identified in the Ca. P. ubique genome
was that the genes for assimilatory sulfate reduction (cysDNCHIJ) were absent (26). This
was unusual because sulfate is not only extremely abundant in the ocean, but at the time,
only one other aerobic heterotrophic bacterium (Idiomarina iloihiensis) lacked
assimilatory sulfate reduction genes (26). Tripp hypothesized that because Ca. P. ubique
was deficient in assimilatory sulfate reduction, an already reduced source of sulfur was
required for growth. In the sea, methionine and dimethylsulfoniopropionate (DMSP) are
relatively abundant reduced sulfur compounds (27), and may act as sources of reduced
sulfur for Pelagibacter cells. Previously, Malmstrom showed that SAR11 cells compete
effectively for DMSP and free amino acids, providing support for the hypothesis that
DMSP is metabolized by Ca. P. ubique (28). To test this hypothesis in vitro, Pelagibacter
was grown in a natural seawater-based medium with amendments of inorganic nutrients
and mixed carbon, but no reduced sulfur additions. Ca. P. ubique cell densities responded
linearly to DMSP additions and the sulfur from 35S-DMSP was incorporated into cellular
biomass, showing that it is required and used for growth (26). DMSP could be replaced
by methionine, implying that either of these compounds can serve as a reduced sulfur
source for Ca. P. ubique. Other SAR11-clade genomes lack the genes encoding proteins
that are necessary for sulfate reduction (29), suggesting that the reduced sulfur
requirement of Ca. P. ubique is common among wild SAR11-clade cells.
Additional metabolic reconstruction revealed an unusual gene complement and
chromosomal organization of genes encoding for enzymes involved in the synthesis and
10
regulation of serine, glycine and threonine. Typically, serine is derived from the
gluconeogenic precursor 3-phosphoglycerate (3PG) through the action of the SerA (D-3phosphoglycerate dehydrogenase), SerB (3-phosphoserine phosphatase), and SerC (3phosphoserine aminotransferase) proteins. Glycine is derived directly from serine by
action of the GlyA (serine hydroxymethyl-transferase) protein that removes a methyl
group from serine and transfers it to tetrahydrofolate (THF), forming 5,10methylenetetrahydrofolate (CH3-THF), glycine and water. Ca. P. ubique lacks the serB
and serC genes encoding proteins required for serine biosynthesis. However, a putative
secondary route to glycine and serine synthesis was identified in Ca. P. ubique via a
threonine aldolase. In Saccharomyces cerevisiae, the threonine aldolase cleaves threonine
to form glycine (30). Therefore, because threonine synthesis is complete, a threonine
aldolase is present and the GlyA reaction is reversible (that is, when there is sufficient
CH3-THF and glycine present, serine can be formed), the initial metabolic reconstruction
of Ca. P. ubique’s glycine and serine synthesis appeared to be complete.
However, because the threonine aldolase is a low-specificity enzyme in E. coli
(31), Tripp hypothesized that Ca. P. ubique was ‘effectively auxotrophic’ for glycine and
serine synthesis. To test this, Ca. P. ubique was grown in a natural seawater medium with
amendments of inorganic nutrients, mixed carbon and a source of reduced sulfur. Ca. P.
ubique cell yields responded linearly to additions of glycine, suggesting that glycine is a
required nutrient (32). Serine, but not threonine, replaced glycine in this natural seawater
medium, providing evidence that the threonine aldolase is not active in Ca. P. ubique, or
its activity was too low to detect under the experimental conditions (32).
11
In addition to effective serine/glycine auxotrophy, unusual features pertaining to
the regulation of glycine metabolism were identified. Similar to other organisms, Ca. P.
ubique contains a glycine-activated riboswitch preceding the coding sequence of the
glycine cleavage system T-protein (gcvT). The GcvT protein is part of the glycine
cleavage complex that catalyzes the cleavage of glycine to form ammonia and a methyl
group that is transferred to the methyl pool (via THF), where it can be oxidized for
energy (reviewed in (33)). Riboswitches are mRNA leader sequences that fold to form a
2° structure capable of binding specific effector molecules that affect transcription and/or
translation of the downstream mRNA (reviewed in (34)). When ample glycine is present,
the gcvT transcript containing the riboswitch binds glycine, allowing for translation of the
GcvT protein. Conversely, when the glycine concentration is low, the switch is
configured to prevent translation of gcvT. The system acts to regulate the degradation of
excess glycine and to use the products of glycine degradation as a source of ammonia and
energy.
A second glycine riboswitch was predicted in the Ca. P. ubique genome in an
uncommon position preceding the coding sequence of the glcB gene, encoding the malate
synthase-G (32). In E. coli, glcB is in an operon with genes encoding the glycolate
oxidase (glcDEFGB) which, when expressed, allows for the oxidation of glycolate to
glyoxylate (via GlcDEF) (35) and the subsequent condensation of glyoxylate with acetylCoA (via GlcB) to form the tricarboxylic acid (TCA) cycle intermediate malate (36).
Through this mechanism, carbon from glycolate can be shunted into the TCA cycle
where it is converted to biomass or burned for energy. However, in the Ca. P. ubique
12
genome, glcB is not co-localized with the glcDEF genes (32). Instead, it appeared that
GlcB was involved in completing the glyoxylate bypass in Ca. P. ubique (along with the
isocitrate lyase) and that the glyoxylate bypass of the TCA cycle was regulated by
glycine (32). Tripp theorized that the unusual arrangement of riboswitches – one
preceding glcB and one preceding gcvT - aided Ca. P. ubique cells in balancing its energy
and carbon requirements (32).
The primary reconstruction of Ca. P. ubique’s carbohydrate metabolism identified
unusual features for an aerobic heterotroph. The hallmark pathway of glycolysis – the
Embden–Meyerhof–Parnas (EMP) pathway of glycolysis - was missing, and instead, an
unusual variant of the Entner–Doudoroff (ED) glycolytic pathway was proposed to be
present. Schwalbach explored the unusual nature of carbohydrate utilization with two
SAR11 isolates – the Pacific Ocean isolate Ca. P. ubique and Pelagibacter sp. str.
HTCC7211, isolated from the oligotrophic Sargasso Sea in the Atlantic Ocean. Genomic
comparison of the two strains showed that Pelagibacter sp. str. HTCC7211 lacked the
genes predicted to be involved in the ED-variant glycolytic pathway. Schwalbach
hypothesized that because of these genomic differences; Ca. P. ubique but not
Pelagibacter sp. str. HTCC7211 would metabolize glucose. Radiotracer experiments
supported this by showing that only Ca. P. ubique assimilated and oxidized glucose to
CO2 (37). Through elegant use of DNA microarrays and growth experiments, Schwalbach
showed that when grown on glucose, the predicted ED-variant genes were significantly
upregulated in Ca. P. ubique, relative to cultures grown with pyruvate as a primary
carbon source. Perhaps more importantly, Schwalbach noted that neither isolate
13
metabolized a range of hexose or pentose sugars (except for glucose in Ca. P. ubique)
that are generally thought of as ‘high-value’ carbon sources in nature. Instead, both Ca. P.
ubique and Pelagibacter sp. str. HTCC7211 utilized a range of low molecular weight
organic acids, including pyruvate, lactate, taurine, acetate and oxaloacetate (37). Taken
together, these findings showed that low molecular weight organic acids are important
carbon sources for SAR11, and that not all SAR11 isolates are able to utilize glucose.
Further support for these findings comes from comparative genomics studies of SAR11
genomes, where poor conservation of genes used for carbohydrate metabolism was
observed across seven SAR11 genomes, suggesting that variation in carbohydrate
metabolism is not unusual within the SAR11 clade (29).
Genome streamlining
The studies by Tripp and Schwalbach identified three distinct classes of
compounds that Ca. P. ubique depends on for cellular metabolism: reduced sulfur
compounds, glycine (or serine), and low molecular weight organic acids (and sometimes
glucose). Only when constituents from all three classes were present did they observe
increases in maximal cell densities (10-fold above those observed in unamended natural
seawater) (26, 32, 37). The apparent co-requirement for such distinct compounds is
unusual compared to the physiology of marine bacteria cultivated on standard growth
media. The uncommon co-requirements have been described within the context of
adaptive selection for gene loss in the Pelagibacter lineage by a process termed ‘genome
streamlining.’ Reduction of genome size is not uncommon within small bacterial
populations that experience a high degree of genetic drift such as obligate symbionts
14
(reviewed in (38)). In cases of obligate symbiosis, the effect of relaxed selection to
maintain gene function leads to the accumulation of mutations in non-essential genes.
Because the effective population of symbionts is small, these mutations are more likely to
become fixed within the population by genetic drift. Over time, the combination of
mutation, deletion and genetic drift leads to genome degradation and a decrease in size.
In contrast to the small population sizes of obligate symbionts, the Pelagibacter
population is massive, and as such, genetic drift is not expected to have had such a
distinct effect. Instead, it is proposed that selection is responsible for the reduction of
Pelagibacter genomes. This leads to a logical question: what trait is selection acting upon
to keep Pelagibacter genomes small? One hypothesis is that by reducing the size of the
genome, Pelagibacter saves on the cost of maintaining DNA and expressing genes. That
is, the cost of maintaining certain genes (or the pathways their products are involved in)
has become too metabolically expensive from either an energetic point of view or from
the availability of the raw materials (for example, core nutrients such as N, P and C) (25,
39, 40).
Morris has put forward an explanation regarding a putative selective pressure that
would result in adaptive gene loss and termed it the ‘black queen hypothesis’ (41). The
hypothesis, named by analogy to the queen of spades in the card game of Hearts, states
that within a given mixed-member community, a metabolically expensive function (e.g. sulfate reduction) can be lost from a portion of the community (Pelagibacter cells, for
example) if the remaining members of the community can compensate for that lost
function (provide a reduced sulfur source for Pelagibacter). In the context of the
15
parenthetical example above, phytoplankton reduce sulfur and make methionine and
DMSP as compatible solutes. Presumably the amount of these compounds that leak from
cells or are released upon cell death is sufficient to support the SAR11 populations
observed in nature. Thus, if maintenance of the sulfate reduction genes (or running the
sulfate reduction pathway) was metabolically expensive to SAR11, loss of the genes
where environmental reduced sulfur is ample may have conferred a competitive
advantage.
Growth on a defined medium
Although headway was made by the identification of the three major ‘food
groups’ that Ca. P. ubique needs to grow, further advances were necessary in order to
develop Ca. P. ubique into a system for understanding marine oligotrophy and the trophic
role of SAR11 cells in the environment. Though an abundance of data has been collected
for numerous microbes on ‘complex’ (multiple constituents) and ‘undefined’ (unknown
constituents) growth media, the compositions of those media were relatively stable, and
the types of microbes grown in such media were robust enough to withstand the small
amount of nutritional variability that may have been encompassed between different
media preparations. This has not been the case for Ca. P. ubique and other SAR11
isolates. Immediately after cultivation on natural seawater, ‘batch effects’ were observed
with SAR11 cells grown on seawater collected at different times whereby different
batches of seawater supported varying final cell yields (23). The specific reasons for the
batch effects are unknown, but one presumes that it is the result of native nutrients (or
inhibitory factors) being present in one water sample but absent (or found in different
16
amounts) in another. The observation that different batches of seawater have varying
effects on growth of marine microbes had been made previously (42). The lack of
consistency among batches of seawater early on during Ca. P. ubique growth studies
made the reproduction of experiments difficult because each batch of seawater was finite
and had its own ‘character’ that tended to supersede the effects of nutrient addition
experiments. Moreover, it was unknown if natural seawater provided a required, but
unidentified, trace nutrient for Ca. P. ubique growth. Without a defined medium, the
requirement for an unknown nutrient could not be ruled out.
One of the archetypal methodologies for understanding microbial physiology is to
exclude a nutrient of interest and study the growth effect of removal on a particular
organism. Hence, another problem with utilizing natural seawater, beyond that its
composition is unknown, is that specific nutrients cannot be excluded from seawater to
study the effect on cellular metabolism. Currently, removal of a single compound from
seawater (for nutrient exclusion experiments), without altering the rest of the makeup, is
not possible. And finally, the issue of seawater collection is extremely problematic. It is
rather impractical to collect seawater in sufficient volumes to do routine lab work unless
the sea is directly accessible.
Minimal defined media have been instrumental tools at the disposal of
microbiologists studying a variety of systems, including enterobacteria and Gram positive
bacilli (43, 44). To understand Ca. P. ubique’s role in environmental nutrient cycling and
to determine a minimal set of requirements for Ca. P. ubique growth, experiments were
designed to identify constituents for growth on a defined minimal medium (see Chapter
17
2). The defined medium was used to explore the trace requirements for five B-vitamins
(B1 (Chapter 3), B5, B6, B7 and B12 (Chapter 5)) for which complete canonical
biosynthetic pathways were not identified in the Ca. P. ubique genome. By doing such
experiments, we have determined a minimal set of carbon and sulfur sources that Ca. P.
ubique requires for growth and show that at least one other Pelagibacter isolate
(Pelagibacter sp. str. HTCC7211) utilizes the same nutrients. We have uncovered a
vitamin B7 (biotin) requirement and an unusual requirement for the vitamin B1 (thiamine)
precursor 4-amino-5-hydroxymethyl-2-methylpyrimidine (HMP). We did not find
physiological support for a vitamin B5 (pantothenate), B6 (pyridoxine) or B12 (cobalamin)
requirement, but instead offer alternative evidence for why Ca. P. ubique appears to be
prototrophic (or in the case of pantothenate, bradytrophic) for these vitamins.
In addition to studying the nutrient requirements of Pelagibacter cells, specific
adaptations that Ca. P. ubique employs to effectively compete for trace levels of Pi are
described (Chapter 4). These adaptations include the use of alternate phosphorus
compounds and the probable rearrangement of lipid polar head groups to reduce
phospholipid (and hence cellular Pi) content. Moreover, we verify that the phosphonate
degradation pathway (C-P lyase) in Pelagibacter sp. str. HTCC7211 confers the ability to
utilize phosphonates and phosphite, and show that when methylphosphonate (Mpn) is
used for phosphorus, methane is stoichiometrically released. The release of methane from
the aerobic sea is well described and we propose that in certain regions of the ocean,
Pelagibacter sp. str. HTCC7211-like SAR11 cells may be responsible for the production
of much of the aerobically produced methane.
18
These studies represent important first steps in the development of Ca. P. ubique
(and other SAR11 isolates) into a model system for understanding the role of the SAR11
clade as an agent of biogeochemical change in the environment. Likewise, because Ca. P.
ubique has an unusual physiology, encoded by divergent metabolic pathways with
unfamiliar genetic arrangements and regulatory schemes, it provides researchers with an
opportunity to study the underpinnings of oligotrophy in this abundant clade. Oligotrophy
is characterized, somewhat loosely, as the paradoxical requirement for low concentrations
of nutrients for optimal growth. We show herein that Ca. P. ubique clearly conforms to
this conceptual definition, but that it can be manipulated in vitro to grow at high nutrient
concentrations. Model systems are essential to push our knowledge forward about new
and unusual types of metabolism and physiology, such as oligotrophy. Today, the same
cultures Winogradsky and Beijerinck isolated and domesticated for laboratory use have
been developed into model systems from which environmental, biochemical,
physiological and geochemical discoveries are rooted. Finally, the strategies described
here are meant to outline how to use DNA sequence information, from both genomes and
from the environment, to construct testable hypotheses about microbial metabolism and
understand how microbes affect nutrient cycles in the ocean.
19
Chapter 2
Nutrient Requirements for the Growth of the Extreme Oligotroph ‘Candidatus
Pelagibacter ubique’ HTCC1062 on a Defined Medium
Paul Carini1, Laura Steindler1,2, Sara Beszteri1,3 and Stephen J. Giovannoni1
1
2
3
Department of Microbiology Oregon State University Corvallis, OR 97331
Current address: Department of Marine Biology, The Leon H Charney School of Marine
Science, University of Haifa, Haifa, Israel
Current address: Alfred Wegener Institute for Polar and Marine Research, Bremerhaven,
Germany
Published: The ISME journal. doi:10.1038/ismej.2012.122
20
Abstract
Chemoheterotrophic marine bacteria of the SAR11 clade are Earth’s most
abundant organisms. Following the first cultivation of a SAR11 bacterium, ‘Candidatus
Pelagibacter ubique’ strain HTCC1062 (Ca. P. ubique) in 2002, unusual nutritional
requirements were identified for reduced sulfur compounds and glycine or serine. These
requirements were linked to genome streamlining resulting from selection for efficient
resource utilization in nutrient-limited ocean habitats. Here we report the first successful
cultivation of Ca. P. ubique on a defined artificial seawater medium (AMS1), and an
additional requirement for pyruvate or pyruvate precursors. Optimal growth was
observed with the collective addition of inorganic macro- and micronutrients, vitamins,
methionine, glycine and pyruvate. Methionine served as the sole sulfur source but
methionine and glycine were not sufficient to support growth. Optimal cell yields were
obtained when the stoichiometry between glycine and pyruvate was 1:4, and incomplete
cell division was observed in cultures starved for pyruvate. Glucose and oxaloacetate
could fully replace pyruvate, but not acetate, taurine or a variety of tricarboxylic acid
cycle intermediates. Moreover, both glycine betaine and serine could substitute for
glycine. Interestingly, glycolate partially restored growth in the absence of glycine. We
propose that this is the result of the use of glycolate, a product of phytoplankton
metabolism, as both a carbon source for respiration and as a precursor to glycine. These
findings are important because they provide support for the hypothesis that some microorganisms are challenging to cultivate because of unusual nutrient requirements caused
by streamlining selection and gene loss. Our findings also illustrate unusual metabolic
21
rearrangements that adapt these cells to extreme oligotrophy, and underscore the
challenge of reconstructing metabolism from genome sequences in organisms that have
non-canonical metabolic pathways.
Introduction
Laboratory studies of ecologically important organisms are important for
understanding the principles and mechanisms that govern ecosystem behavior (45). For
example, several studies have reported high connectedness in marine plankton
communities (46, 47). ‘Connectance’ is a general measure of the degree to which
populations within a community display correlated behavior, and, in terrestrial
ecosystems, has been linked to ecosystem stability (48). The SAR11 clade of αproteobacteria are the most abundant heterotrophs in marine euphotic zones worldwide
(17) and have been shown to contribute significantly to the overall connectedness of
marine microbial plankton communities (47). Specific mechanisms that might explain
SAR11 connectedness are slowly emerging from studies of genomes and the metabolism
of cells in culture (26, 32, 37, 49).
‘Candidatus Pelagibacter ubique’ HTCC1062 (Ca. P. ubique), a member of the
SAR11 clade, has a small genome and displays characteristic signatures of streamlining
selection (25). According to the genome streamlining theory, when effective population
sizes are large, extreme selection for the efficient use of resources in nutrient-poor
environments can result in genome reduction (25, 50, 51). The metabolic consequences
of genome reduction in Ca. P. ubique have been the subject of several reports (26, 32,
37).
22
Metabolic reconstruction from the Ca. P. ubique genome highlighted the
conspicuous absence of genes common to aerobic chemoorganoheterotrophs (Figure 2-1).
Subsequent batch culture experiments, using a natural seawater medium, showed that Ca.
P. ubique required an unusual combination of nutrients for growth (26, 32, 37).
Specifically, Ca. P. ubique lacks genes necessary for assimilatory sulfate reduction, and
as a result, requires reduced sulfur compounds such as methionine or 3dimethylsulphoniopropionate for growth (26). Similarly, the unusual absence of common
genes for serine and glycine biosynthesis resulted in a conditional requirement for either
of these amino acids (32). Concomitantly, an uncommon arrangement of two glycineactivated riboswitches was reported; one located upstream of the gene encoding for the
glycine cleavage system T-protein (gcvT) and the other upstream of the malate synthase
gene (glcB) (32). The first of these is a common riboswitch involved in glycine cleavage
to CO2 and NH4+, but the second is a rare riboswitch configuration that was implicated in
glyoxylate metabolism (32). Although Ca. P. ubique is missing the complete Embden–
Meyerhof–Parnas glycolytic pathway (genes encoding pyruvate kinase and
phosphofructokinase are absent, Figure 2-1), a putative operon encoding genes involved
in a predicted variant of the Entner–Doudoroff glycolytic pathway is present, as is a
complete gluconeogenic pathway (Figure 2-1; see (37)). Experiments showed that low
molecular weight organic acids were important carbon sources for multiple SAR11
isolates and that Ca. P. ubique, but not all SAR11 isolates, oxidized glucose (37).
Moreover, genes coding for proteins involved in carbohydrate metabolism are not
consistently conserved across the SAR11 clade, suggesting that the metabolism of sugars
23
and other carbohydrates may be involved in niche partitioning or ecotype variation (29).
Previously, Ca. P. ubique was grown exclusively in natural seawater-based media.
However, seawater contains an undefined mixture of naturally occurring organic
compounds that vary in composition and quantity between seasons, locations and depths.
The consequences of this nutrient variability on Ca. P. ubique’s physiology was initially
observed in the form of ‘batch effects’, in which seawater collected at different times
yielded different cell densities or growth rates independent of nutrient additions (23). The
three main limitations of conducting experiments with Ca. P. ubique cells on natural
seawater-based media are: (i) the precise composition and concentrations of organic
matter in seawater is unknown and variable; (ii) the finite supply of any single seawater
batch limits comparisons between experiments conducted with different batches; and (iii)
native concentrations of nutrients (for example, nitrogen, sulfur and carbon) cannot be
excluded.
We developed a defined medium to use as a tool for studying the nutrient
requirements and metabolism of Ca. P. ubique. Ultimately, without a defined medium, it
was impossible to rule out that unknown compounds in natural organic matter were
essential for growth of Ca. P. ubique. A simple approach to solve this problem is the
addition of a diverse mixture of compounds, under the assumption that the organism will
use only what it needs. This approach is commonly implemented by adding mixtures of
nutrients such as yeast extract or digests of protein to growth media. However, complex,
high-nutrient mixtures inhibit the growth of many oligotrophic chemoheterotrophs,
making this strategy impractical (23, 26, 32). Building on previous metabolic models for
24
Ca. P. ubique, we hypothesized that the minimum nutrient requirements for growth on a
defined medium would include pyruvate in addition to a source of reduced sulfur and
glycine. We report here the propagation of Ca. P. ubique to high cell density on a defined
artificial seawater medium containing inorganic micro- and macronutrients, vitamins,
methionine (for reduced sulfur), glycine and pyruvate. Pyruvate, or its precursors, was
identified as the last essential macronutrient needed by Ca. P. ubique for growth. We also
demonstrate that several environmentally relevant compounds can be used by Ca. P.
ubique in place of pyruvate and glycine, and describe unusual rearrangements of central
metabolic pathways that confer these properties.
Methods
Organism source
Ca. P. ubique HTCC1062 was originally isolated from the northeast Pacific
Ocean as described elsewhere (23). Frozen glycerol stocks from the original isolation of
Ca. P. ubique were used as the source inoculum for all experiments.
Media preparation
Artificial seawater medium AMS1 (Table 2-1) was derived from the artificial
seawater medium AMP1 (52). Organic buffers (for example, HEPES and EDTA) were
excluded to avoid potential toxic effects (53) and the possibility that they might be used
as substrates for growth (54). After autoclaving, AMS1 was sparged with 0.1 µm-filtered
CO2 for 5 h followed by sparging with air for 10 h to establish a bicarbonate-based buffer
system (21). All vitamins and organics were added after autoclaving and sparging. The
25
pH of the resulting AMS1 typically ranged from 7.5 to 7.7.
Cultivation details
All cultures were grown in acid-washed and autoclaved polycarbonate flasks at
20°C with shaking at 60 r.p.m. under a 12/12-h light (140-180 µmol photons m-2 s-1)/dark
cycle.
Measurement of growth
Cells were stained with SYBR Green I (Molecular Probes, Inc., Eugene, OR,
USA) and counted with a Guava Technologies flow cytometer (Millipore, Billerica, MA,
USA) at 48- to 72-h intervals as described elsewhere (22, 26).
Acclimation to growth on AMS1
Natural seawater collected from the Newport Hydroline station NH-05 (latitude:
44.651, longitude: -124.181) from a depth of 10 m in June 2008 was amended with
glycine (1 µM), methionine (1 µM), pyruvate (50 µM), FeCl3 (1 µM) and vitamins and
inoculated with Ca. P. ubique from glycerol stocks. When exponentially growing cells in
this amended natural seawater medium exceeded a density of 2.0 × 106 cells ml-1, they
were diluted (1:100) with fresh AMS1 supplemented with glycine (1 µM) (as a
glycine/serine source), methionine (as a sulfur source) (1µM), pyruvate (as a carbon
source) (50 µM), FeCl3 (1 µM) and vitamins. We observed no lag phase in cells that were
transferred into amended AMS1 (Supplementary Figure A1-1 in appendix 1). All cultures
described herein are derived from this lineage and have been maintained exclusively on
AMS1 for >15 consecutive batch culture transfers (approximately 150 generations).
26
Pyruvate substitution experiments
Potential pyruvate precursors (Table 2-2) were selected because they were either
(i) ‘common’ sole carbon sources for chemoorganoheterotrophs or (ii) present in
seawater as products of phytoplankton metabolism. Each substitute was tested at a
concentration of 50 µM in AMS1 amended with 25 µM glycine, 10 µM methionine, 1µM
FeCl3 and vitamins. The positive control was amended with 50 µM pyruvate and the
same concentrations of glycine, methionine, FeCl3 and vitamins. The negative control
contained no pyruvate, but was otherwise identical.
Glycine substitution experiments
Potential glycine precursors (Table 2-3) were selected because they were either (i)
metabolic precursors of glycine in other organisms or (ii) predicted to be a precursor
based on metabolic reconstruction in Ca. P. ubique. Each substitute was tested at a
concentration of 25 µM in AMS1 with pyruvate (50 µM), methionine (10 µM), FeCl3
(1µM) and vitamins. The positive control was amended with 25 µM glycine and the
negative control contained no glycine.
Cell division experiments
Growth media consisted of AMS1 amended with glycine (1 µM), methionine
(1 µM), FeCl3 (1 µM), vitamins and either 0.5 µM pyruvate (deplete conditions) or 50
µM pyruvate (replete conditions). SYBR Green I-stained cultures that exhibited relative
DNA fluorescence values of 300–325 and 475–500, were independently filtered on to 0.2
µm black polycarbonate filters, and imaged using a Leica DMRB epifluorescence
27
microscope (Wetzlar, Germany) equipped with filter sets appropriate for SYBR Green I
(excitation: 450–490 nm; emission: 580 nm). Images were captured with a Hamamatsu
ORCA-ER CCD digital camera (Hamamatsu City, Japan) and Scanalytics IPLab v3.5.5
scientific imaging software (Fairfax, VA, USA).
Chemicals
All inorganic salts were obtained from Sigma-Aldrich Co. (St Louis, MO, USA)
and were of the highest available quality (typically labeled ‘ultrapure’). All other
compounds were obtained from Sigma-Aldrich Co. or other commercial vendors and
were of reagent grade quality.
Results
When grown on AMS1 with methionine, glycine and pyruvate, Ca. P. ubique’s
maximum specific growth rate was 0.41 ± 0.01 day-1 (mean ± s.d., n=3), and batch
cultures reached maximum cell densities of 9.18 ± 0.02 × 107 cells ml-1 (mean ± s.d.,
n=3) (Figure 2-2). Additions of methionine or carbon (as pyruvate and glycine) alone did
not result in increased cell densities beyond those of the negative control (Figure 2-2).
Cell densities responded linearly to both pyruvate (R2=0.998) and glycine (R2=0.995)
additions in AMS1 when other nutrients were in excess (Figure 2-3). The maximum cell
density increased by 2.6 × 106 cells ml-1 µM-1 pyruvate (Figure 2-3a) and
1.0 ×107 cells ml-1 µM-1 glycine (Figure 2-3b).
Ca. P. ubique utilized oxaloacetate and glucose in place of pyruvate on AMS1
(Table 2-2). As observed previously (37), Ca. P. ubique’s specific growth rate was slower
28
with glucose as a sole pyruvate source. Addition of taurine or lactate resulted in cell
densities in excess of fivefold greater than the negative control, but did not achieve the
cell densities of the pyruvate treatment. Notably, in the absence of pyruvate, additions of
alanine or glycine did not improve growth yield.
Glycine betaine and serine were able to fully replace glycine in AMS1 and
glycolate partially substituted for glycine (Table 2-3). Ca. P. ubique grew slower but to
slightly higher cell densities when glycine betaine was the sole glycine source. Glycolate
led to cell density increases fourfold greater than those of the negative control. The
addition of pyruvate, without glycine, did not result in higher cell densities.
While developing the AMS1 medium, unusual cell division patterns were
observed under pyruvate-deplete conditions. When SYBR Green was used to stain DNA
in early stationary-phase cells, fluorescence from pyruvate-limited cells was about twofold higher than from cells raised in a pyruvate-replete medium (Figures 2-4a and b).
Microscopic images from these cultures showed that the increase in DNA fluorescence
was caused by elongated cells containing two nucleoids (‘doublets’) (Figures 2-4c and d).
We observed this unusual cell division pattern when cells entered pyruvate-limited
stationary-phase across a range of pyruvate:glycine ratios (Supplementary Figure A1-2 in
appendix 1). This phenomenon was previously observed when Ca. P. ubique cells were
grown in a natural seawater medium without added pyruvate (unpublished data).
Experiments conducted in natural seawater found alanine induced the division of cell
doublets (unpublished data). The effect of different alanine concentrations on Ca. P.
ubique cell morphology in stationary-phase cultures grown in AMS1 was tested using
29
relative DNA fluorescence as a proxy for the formation of cell doublets (Supplementary
Figure A1-3 in appendix 1). DNA fluorescence profiles and cell counts suggested that
alanine induced cell division in pyruvate-limited cultures.
Discussion of results
Similar to the goals of Neidhardt et al. (43), who developed a defined medium for
the growth of enterobacteria, one of our objectives was to prepare a ‘physiologically
optimal and experimentally useful’ medium in which Ca. P. ubique, and other SAR11
isolates, could be propagated reproducibly. This artificial medium for the growth of Ca.
P. ubique contains only methionine, glycine, pyruvate, vitamins and inorganic salts. The
specific growth rates observed on AMS1 (0.41 ± 0.01 day-1) were comparable to those
previously published for Ca. P. ubique grown in seawater batch cultures (23, 26, 37) and
to those from microcosm experiments with natural assemblages of plankton (55, 56).
Results observed with cells growing in AMS1 support the previous conclusions
that Ca. P. ubique requires a reduced sulfur source, glycine and an organic acid for
growth. Our results also confirm that methionine meets Ca. P. ubique’s requirement for
exogenous reduced sulfur compounds (26) but cannot substitute for glycine or pyruvate
as a sole carbon source. The linear responses to both pyruvate and glycine additions,
when other constituents were in excess, indicated that both pyruvate and glycine were
necessary for optimal growth and used in the molar ratio of 4.0:1 (pyruvate:glycine). In
addition, we showed that alanine is required for septation, the final step in cell division,
and that in the absence of pyruvate, Ca. P. ubique cells were not able to synthesize
alanine by other routes, thus implicating pyruvate as a primary precursor for alanine
30
biosynthesis. In Escherichia coli, the rate of cell wall biosynthesis increases during
septation in order to accommodate the synthesis of the new daughter cell poles (57).
Because alanine is a major component of cell walls in Gram-negative bacteria (58), we
propose that in pyruvate-limiting conditions, Ca. P. ubique is unable to synthesize
sufficient alanine to complete septation, resulting in the formation of cell doublets.
Our observations show that pyruvate or its precursors are required to synthesize
alanine, but that alanine cannot replace pyruvate (Table 2-2). In Ca. P. ubique, L-alanine
is putatively formed by an alanine dehydrogenase (encoded by ald, SAR11_0809—
Figure 2-1). Ald catalyzes the formation of alanine from pyruvate and ammonia via the
oxidation of reduced nicotinamide adenine dinucleotide, and has been shown to be
involved in both the catabolism and anabolism of alanine in Pseudomonas, Rhodobacter
and Sphingopyxis species (59-61). Although Ald catalyzes a reversible reaction in some
organisms, our data suggest that in Ca. P. ubique, Ald does not catalyze the formation of
pyruvate from alanine under the conditions tested.
The observation that both glucose and oxaloacetate could replace pyruvate
suggests that pyruvate is a metabolic intermediate formed during the catabolism of these
compounds. Both compounds were previously identified as carbon sources for Ca. P.
ubique in natural seawater (37). The use of oxaloacetate in place of pyruvate is consistent
with the presence of a predicted malic enzyme gene (maeB, SAR11_0375) that may be
involved in the decarboxylation of oxaloacetate to form pyruvate. Our results are also
consistent with Schwalbach’s proposed glycolytic pathway in Ca. P. ubique that
predicted pyruvate was an end product of glucose metabolism (37). Previously, in natural
31
seawater-based media, taurine, acetate and lactate additions resulted in cell density
increases similar to those observed with pyruvate additions (37). However, in our
experiments on artificial seawater media, maximum cell densities decreased in this order:
pyruvate >> taurine > lactate >> acetate (Table 2-2). Taurine is putatively metabolized to
acetyl-CoA in Ca. P. ubique, suggesting that taurine catabolism supplies two-carbon units
for biosynthesis. In our experiments, acetate, a direct precursor to acetyl-CoA, could not
replace pyruvate (Table 2-2). This contradicts the prediction of a metabolic pathway for
acetate assimilation as a sole carbon source via the glyoxylate bypass in Ca. P. ubique
(Figure 2-1). We postulate that the glyoxylate bypass has instead been recruited to
function in glycine metabolism, as described below.
We show for the first time that glycine betaine, in addition to serine, can meet Ca.
P. ubique’s glycine requirement. Previously, Tripp et al. (32) identified serine as the only
compound able to replace glycine in natural seawater-based batch cultures of Ca. P.
ubique. Consistent with this finding, we found that serine was an effective replacement
for glycine on AMS1 (Table 2-3). Glycine betaine is an important osmolyte that can be
degraded by marine bacteria (49, 62, 63). Ca. P. ubique’s genome encodes a full suite of
genes predicted to be involved in the acquisition and stepwise oxidation of glycine
betaine to form glycine (32, 49). Experiments also showed that Ca. P. ubique can oxidize
methyl groups derived from glycine betaine to CO2 (49). Although glycine betaine fully
replaced glycine, we observed a reduction in growth rate when glycine betaine was
supplied as a sole glycine source. Tripp et al. (32, 49) previously reported that large
amounts of glycine betaine had unpredictable or deleterious effects on the growth of Ca.
32
P. ubique in natural seawater. We propose that growth rate reduction may be in part due
to toxicity resulting from the production of formaldehyde (by the predicted
dimethylglycine dehydrogenase, EC:1.5.99.2, SAR11_1253) and hydrogen peroxide (by
the predicted sarcosine oxidase, EC:1.5.3.1; for genes, see (49)) during glycine betaine
catabolism.
We also report the new finding that Ca. P. ubique partially utilized glycolate to
meet its glycine requirement. Glycolate is commonly formed by phytoplankton as a result
of photorespiration (64-68). In E. coli, carbon from glycolate is assimilated into biomass
after it is oxidized to glyoxylate by the glycolate oxidase (glcDEF) (35, 36). Glyoxylate
is then condensed with acetyl-CoA by the malate synthase (GlcB) to form the
tricarboxylic acid cycle intermediate malate (35). In Ca. P. ubique, genes encoding the
glycolate oxidase are found in a putative operon with a pyridoxal-phosphate-dependent
aminotransferase, annotated as an aspartate aminotransferase (aspC), but separate from
the malate synthase gene (Figure 2-5). Previously, Tripp et al. (32) had proposed that this
aminotransferase produces glycine from glyoxylate, but, working with natural seawater
media, were unable to demonstrate replacement of glycine by glycolate to validate this
hypothesis.
Results presented here help resolve an enigmatic arrangement of glycine-activated
riboswitches associated with genes of the glyoxylate cycle and glycine catabolism. Ca. P.
ubique has genes for the proteins required to channel glycolate into the tricarboxylic acid
cycle where it can be assimilated into biomass or oxidized (glcDEF and glcB; (32)). In
Ca. P. ubique, a glycine-activated riboswitch is located in a very unusual position
33
upstream of the glcB coding sequence, and a second glycine-activated riboswitch is
located in a common arrangement, upstream of gcvT, where it functions to regulate
glycine cleavage to CO2 and NH4+. It is now apparent that this unusual configuration of
two riboswitches results in glycine concentrations regulating the fate of glyoxylate, as
illustrated in Figure 2-5. We postulate that when intracellular glycine concentrations fall
too low, the switch on glcB closes, shunting glycolate through glyoxylate to form glycine.
When intracellular glycine concentrations are ample, the glyoxylate bypass opens to
channel glycolate-derived carbon into the tricarboxylic acid cycle. We attribute the
observation that glycolate did not support as high a cell yield as glycine (Table 2-3) to its
dual role in these pathways. This model also helps explain why Ca. P. ubique does not
respond vigorously to acetate addition; the glyoxylate bypass, which in most cells is used
for acetate assimilation, has been recruited to functions that are adaptive to the ocean
environment.
One of the mysteries yet to be fully explained is the growth of Ca. P. ubique cells
to 106 cells ml-1 in the absence any additional carbon compounds except vitamins. We
determined the maximum amount of methionine, glycine and pyruvate carried over with
the source inoculum to be 500, 500 and 156 pM, respectively. Based on the calculated
per-cell glycine and pyruvate requirement (regression lines in Figure 2-3), and the
previously published sulfur requirement (26), we conclude that the carryover of these
nutrients does not sufficiently explain the growth of the negative controls observed in
Figures 2-2 and 2-3. One alternative explanation for this growth is that Ca. P. ubique is
able to utilize the carbon and sulfur originating from one or more of the vitamins, vitamin
34
degradation products or traces of contaminating carbon in the vitamin stocks. We tested
this and found that Ca. P. ubique responded in a dose-dependent manner to increasing
amounts of freshly prepared vitamins (Supplementary Figure A1-4 and discussion in
appendix 1). It is not clear whether Ca. P. ubique can utilize the vitamins themselves for
growth or if the vitamins enable more efficient use of other traces of contaminating
carbon in AMS1. Regardless of the source of the contaminating nutrients, we show that
maximum cell densities are two orders of magnitude greater when methionine, glycine
and pyruvate are added to the medium.
Metabolic reconstruction has shown Ca. P. ubique’s unusual requirement for a
balanced supply of organic matter is a consequence of streamlining selection for minimal
genome size and metabolic simplicity (Figures 2-1 and 2-5; (26, 32, 37)). We speculate
that the unusual arrangement of central metabolism in Ca. P. ubique is adaptive not only
because it is small and simple, and therefore requires fewer nutrients to replicate, but also
because it is suited to planktonic environments where the flux of labile dissolved organic
matter is low but continuous most of the time. In addition to the potential supply of
pyruvate from glycolysis in some strains, pyruvate and its precursors oxaloacetate and
glyoxylate are common metabolic intermediates in other organisms and also are formed
by the photooxidation of dissolved organic matter (69-73). Taurine, a compound
produced by both phytoplankton and animals (74), also substituted for pyruvate, but at
greatly reduced efficiency. Previously it was shown that the Ca. P. ubique glycine
requirement could be met by glycine or serine, which are found in seawater at nanomolar
concentrations (32). We also found that the common osmolyte glycine betaine and the
35
photorespiration product glycolate could serve as precursors for glycine biosynthesis.
Glycolate is produced by oxygenic phototrophs when they become carbon limited including both eukaryotic phytoplankton (64, 66, 67) and marine cyanobacteria (68) - the
dominant marine phototrophs in temperate and tropical oceans. Tripp showed previously
that either methionine or 3-dimethylsulphoniopropionate, a phytoplankton osmolyte,
could meet the Ca. P. ubique requirement for organosulfur (26, 75). Therefore, the results
of this study suggest that Ca. P. ubique has evolved to efficiently use a combination of
ubiquitous, low molecular weight metabolites produced by phytoplankton, or resulting
from the photooxidation of dissolved organic carbon, which may partially explain its high
abundance in the euphotic zone.
A number of definitions have been proposed for the term ‘oligotroph’ based on
optimal and inhibitory nutrient concentrations (reviewed in (10)). These definitions do
not easily fit the metabolic behaviors being observed in the experiments presented here.
The observation that alanine, derived from pyruvate or its precursors, is required for cell
division (Figure 2-4; Supplementary Figures A1-2 and A1-3 in appendix 1), but that large
amounts of alanine adversely affect growth (Table 2-2), is an example of a deleterious
imbalance caused by the presentation of a metabolic substrate to cells at either unnatural
concentrations or in unnatural combinations. Unusual nutrient requirements, together
with cryptic patterns of nutrient substitution and inhibition, present the experimentalist
with a complex problem. This problem can be solved by the stepwise process of
metabolic reconstruction coupled with experimentation if a defined medium is available.
Although this approach is time consuming (especially with slow-growing cells, such as
36
Ca. P. ubique), it has been successfully applied in other systems (76). The capacity of Ca.
P. ubique for growth with the ambient concentrations of organic matter found in
autoclaved seawater (23), and in mineral salts without added carbon (Figure 2-2 and
Supplementary Figure A1-4 in appendix 1) are ample evidence of the adaptation of these
cells to growth at low nutrient concentrations. More importantly, members of the SAR11
clade are the most successful chemoorganoheterotrophs in oligotrophic ocean systems.
Therefore, although Pelagibacter defies conventional definitions of the term ‘oligotroph’
because it can tolerate some compounds at relatively high concentrations, it clearly
conforms to the concept of oligotrophy.
Metabolic reconstruction in silico results in metabolic models that are subject to
uncertainties that can only be resolved by studying the physiology and metabolism of
cells in vitro. The defined medium presented here for Ca. P. ubique, provides a platform
from which such controlled experiments can occur. Ca. P. ubique and other cells with
streamlined metabolism are particularly challenging to model because of uncertainties
arising from the loss of genes that function in canonical pathways (Figure 2-1). For
instance, the requirements for glycine and alanine were not evident from the initial
genome analysis of Ca. P. ubique (25). In addition, the hypothesis that pyruvate could
serve as the sole carbon precursor for both biosynthesis and energy production was not
predicted by previous metabolic models. Uncommon gene arrangements also complicate
the interpretation of metabolic models in Ca. P. ubique (Figure 2-5). In E. coli, the genes
coding for proteins required for the assimilation of carbon from glycolate are found at a
single locus and are transcribed to a polycistronic message. In Ca. P. ubique, the same
37
genes are physically separated from one another—but their functions are linked through
the metabolic intermediate glycine (Figure 2-5). Experiments with cultures growing on a
defined medium are an important step in the refinement of metabolic models such as the
one presented in Figures 2-1 and 2-5.
The original success of dilution to extinction approaches was founded on the
principle that if cells could be detected and cultured at low concentrations, on low
nutrient media, their physiological responses to specific nutrient additions could be
studied in subsequent experiments. The results presented in Tables 2-2 and 2-3, in light of
the previously reported metabolic abilities of Ca. P. ubique (32, 37), highlight one of the
challenges associated with cultivating specialist oligotrophic organisms. Our results
suggest that perhaps the largest obstacle to overcome in the cultivation of marine
oligotrophs is the specific, unusual and often combinatorial or conditional nutrient
requirements. There is growing evidence that a number of abundant microbial plankton
species that are not yet cultivated, or are cultivated but difficult to propagate, are similar
to Ca. P. ubique in that they are abundant, small cells that contain relatively small
genomes (<2.0 Mbp) (39, 77, 78). The success of cultivating Ca. P. ubique on artificial
media relied on a targeted, minimal combination of nutrients deduced from metabolic
reconstruction from a sequenced genome. If, as is likely, such requirements are common
among organisms with streamlined genomes, it is hoped that this approach may serve as a
blueprint for the cultivation of other important taxa that are difficult to grow in the
laboratory.
38
Table 2-1: Constituents of the Artificial Medium for SAR11 (AMS1)1
Compound
Final Concentration
Base Salts:
NaCl
481 mM
MgCl2•6H2O
27 mM
CaCl2•2H2O
10 mM
KCl
9 mM
NaHCO3
6 mM
MgSO4•7H2O
2.8 mM
Macronutrients:
(NH4)2SO4
400 µM
NaH2PO4 (pH 7.5)
50 µM
Trace Metals:
FeCl3•6H2O
117 nM
MnCl2•4H2O
9 nM
ZnSO4•7H2O
800 pM
CoCl2•6H2O
500 pM
Na2MoO4•2H2O
300 pM
Na2SeO3
1 nM
NiCl2•6H2O
1 nM
Vitamins:
B1
6 µM
B3
800 nM
B5
425 nM
B6
500 nM
B7
4 nM
B9
4 nM
B12
700 pM
myo-inositol
6 µM
4-aminobenzoic acid
60 nM
1
The AMS1 medium does not include
nutrients to meet the requirements for reduced
sulfur (see (26)), glycine (see text), or
pyruvate (see text).
39
Table 2-2: Potential sources of pyruvate for Ca. P. ubique when grown in AMS1 with
25 µM glycine and 10 µM methionine
potential pyruvate source
glucose
pyruvate (positive control)
oxaloacetate
taurine
lactate
ribose
malate
citrate
acetate
no pyruvate (negative control)
succinate
alanine
glycine
1
Presented as mean ± s.d. × 107 cells ml-1, n=3
2
Presented as mean ± s.d. cells d-1, n=3
maximum
density1
12.1±3.83
8.91±0.74
6.92±0.04
0.46±0.14
0.11±0.09
0.05±0.01
0.03±0.01
0.03±0.00
0.02±0.01
0.02±0.01
0.02±0.00
0.01±0.00
0.01±0.00
growth
rate2
0.27±0.02
0.33±0.01
0.33±0.04
0.21±0.02
0.18±0.04
0.23±0.01
0.14±0.04
0.12±0.01
0.10±0.05
0.13±0.02
0.11±0.01
0.01±0.01
0.06±0.01
40
Table 2-3: Potential sources of glycine for Ca. P. ubique when grown in AMS1 with
50 µM pyruvate and 10 µM methionine
maximum
density1
glycine betaine
13.1±0.34
glycine (positive control)
10.2±0.28
serine
8.34±0.15
glycolate
1.88±0.13
acetate
0.66±0.10
malate
0.51±0.03
glyoxylate
0.50±0.01
succinate
0.46±0.06
pyruvate
0.44±0.05
citrate
0.44±0.01
no glycine (negative control)
0.43±0.02
2-oxoglutarate
0.39±0.05
1
7
-1
Presented as mean ± s.d. × 10 cells ml , n=3
2
Presented as mean ± s.d. cells d-1, n=3
potential glycine source
growth rate2
0.18±0.01
0.38±0.01
0.40±0.01
0.35±0.01
0.30±0.01
0.32±0.03
0.38±0.01
0.31±0.01
0.35±0.01
0.29±0.01
0.34±0.00
0.33±0.01
41
α-D-glucose
gluconate
β-D-glucose
β-D-glucose-6P 6-phosphogluconate
β-D-fructose-6P
α-D-glucose-6P
β-D-fructose-1,6P2
serine
ribulose-5P
2,4-keto-3-deoxy6-phosphogluconate
3-HP G3-P
glycine
SO42-
PEP
ald
pyruvate
CO2
alanine
NH3
CO2
homocysteine
CO2
malate
succinate
O-acetyl- PAPS
homoserine
H2S SO32-
acetyl-CoA
oxaloacetate
APS
citrate
methionine
glcB
glyoxylate
isocitrate
CO2
2-oxoglutarate
CO2
Figure 2-1: Simplified illustration of central metabolism in Ca. P. ubique. Black lines:
Reactions predicted to occur in Ca. P. ubique based on genome content. Red lines:
Reactions predicted to occur in E. coli, but missing from Ca. P. ubique. Blue lines:
Putative glucose oxidation pathway (see ref. 37). 3-HP: 3-hydroxypyruvate, G3-P:
glyceraldehyde-3-phosphate, APS: adenosine 5'-phosphosulfate, PAPS: 3'phosphoadenylyl sulfate, PEP: phosphoenolpyruvate. Gene names in green are discussed
further in this manuscript. glcB: malate synthase; ald: alanine dehydrogenase. Bolded
compounds were previously identified as growth substrates for Ca. P. ubique.
42
methionine + glycine + pyruvate
glycine and pyruvate
no added nutrients
methionine
cell density (cells ml-1)
108
107
106
105
104
0
5
10
15
time (days)
20
25
30
Figure 2-2: Growth of Ca. P. ubique in AMS1 with organic carbon additions. Black:
Cells grown without additions of glycine, methionine or pyruvate. Red: Cells amended
with methionine (10 µM), glycine (50 µM), and pyruvate (50 µM). Blue: Cells amended
with methionine (10 µM) only. Green: Cells amended with glycine (50 µM) and pyruvate
(50 µM) only. Points are the average density of triplicate cultures. Error bars indicate
± 1.0 s.d. (n=3). When error bars are not visible, they are smaller than the size of the
symbols.
maximum cell density (× 107 cells ml-1)
43
1.6
A
y=2.6×106x + 1.9×106
R2=0.998
1.2
0.8
0.4
0
0
1
2
3
4
5
maximum cell density (× 107 cells ml-1)
pyruvate added (μM)
B
5.0
y=1.0×107x + 2.9×106
R2=0.995
4.0
3.0
2.0
1.0
0
0
1
2
3
4
glycine added (μM)
5
Figure 2-3: Maximum cell yields of Ca. P.
ubique in response to pyruvate and glycine
additions. A) Pyruvate titration in AMS1
supplemented with glycine (50 µM) and
methionine (10 µM). Using the formula of the
regression line, we calculated the maximum
cell density achievable from pyruvate
carryover with source inoculum (156 pM) to
be 400 cells ml-1 (filled star). B) Glycine
titration into AMS1 supplemented with
pyruvate (50 µM) and methionine (10 µM).
Using the formula of the regression line, we
calculated the maximum cell density
achievable from glycine carryover with source
inoculum (500 pM) to be 5,000 cells ml-1
(filled star). Points are the average maximum
cell densities of triplicate batch cultures. Error
bars indicate ± 1.0 s.d. (n=3). When error bars
are not visible, they are smaller than the size
of the symbols.
44
counts
300-325
event-1
A
C
475-500
event-1
B
D
relative DNA
fluorescence intensity
Figure 2-4: DNA content and morphology of SYBR Green I-stained stationary phase
cells from pyruvate-deplete and replete batch cultures. Red dashed line in A and B
represents the minimum threshold of fluorescence detection. Black dashed lines in A and
B represent relative DNA fluorescence values of 300-325 per event and 475-500 per
event, as indicated with black arrows. A) Relative DNA fluorescence of cells from
pyruvate-replete (50 µM) stationary phase cultures, and B) pyruvate-deplete (0.5 µM)
stationary phase cultures. C) Fluorescent microscopy image of cells from (A).
Arrowheads point to single cells. D) Fluorescent microscopy image of cells from (B).
Arrowheads point to cell doublets.
45
Escherichia coli K-12 MG1655
glcC
glcD
glcE
glcF
G
‘Candidatus Pelagibacter ubique’ HTCC1062
aspC
glcD
glcE
glcF
glcB
ldh
0274
yghK
~220 kbp
accA
glcB
recA
acetyl-CoA
O2
glycolateout
H2O2
glycolatein
glyoxylate
GlcB
amino donor
(amino acid)
malate
TCA cycle
glycine-activated
translation of glcB
2-oxo-acid
glycinein
glycineout
NH3+ CO2
protein
serine
glcB mRNA
Figure 2-5: Glycolate assimilation gene organizations in E. coli and Ca. P. ubique.
Reaction arrows are are colored by the genes predicted to catalyze the reaction. Green
stem-loop images represent the glycine-activated riboswitch (32). Gene annotations are
as described in NCBI, however we predict the reaction catalyzed by AspC to be as
described in the figure. glcC: DNA-binding transcriptional dual regulator, glycolatebinding; glcD: glycolate oxidase subunit, FAD-linked; glcE: glycolate oxidase, FADbinding subunit; glcF: glycolate oxidase, iron-sulfur subunit; G: glcG; Putative glc
operon gene, function unknown; glcB: malate synthase G; yghK: glycolate transporter;
aspC: probable aspartate transaminase; 0275: SAR11_0275; probable 2-hydroxyacid
dehydrogenase; 0274: SAR11_0274; major facilitator superfamily transporter, possible
sugar-phosphate transporter; accA: acetyl-CoA carboxylase; recA: recombinase A.
46
Chapter 3
‘Candidatus Pelagibacter ubique’ HTCC1062 is Dependent on 4-Amino-5Hydroxymethyl-2-Methylpyrimidine, An Abundant Thiamine Precursor In The Sea
Paul Carini1, Emily O. Campbell1, Jeff Morré5, Sergio Sañudo-Wilhelmy3, Samuel
Bennett2, J. Cameron Thrash1, Taghd Begley4 and Stephen Giovannoni1
1
2
3
Department of Microbiology, Oregon State University Corvallis, OR 97331
Department of Environmental & Molecular Toxicology, Oregon State University,
Corvallis, OR 97331
Department of Biological Sciences, Marine Environmental Biology, and Earth Science,
University of Southern California, Los Angeles, CA 90089
4
Department of Chemistry, Texas A&M University, College Station, Texas 77843
5
Department of Chemistry, Oregon State University, Corvallis, OR 97331
47
Abstract
The essential B-vitamin thiamine (B1) has complex distribution patterns in the sea
and is often low or undetectable in marine surface waters. Despite this, analysis of
sequenced genomes belonging to surface-dwelling organisms in the SAR11 clade of
marine heterotrophic bacteria revealed incomplete thiamine synthesis pathways. A
representative SAR11 isolate, ‘Candidatus Pelagibacter ubique’ HTCC1062, lacks thiC,
encoding the 4-amino-5-hydroxymethyl-2-methylpyrimidine (HMP) synthase, required
for de novo synthesis of thiamine’s pyrimidine moiety, but retains the remainder of the
thiamine biosynthetic pathway. Here we show that Ca. P. ubique is dependent on
exogenous HMP for growth. Exclusion of thiamine from Ca. P. ubique cultures growing
on a defined medium amended with excess carbon and sulfur resulted in lower cell
densities than cultures amended with thiamine. Cell growth was restored with picomolar
additions of HMP. We report the vertical distributions (0 – 300 meters) of dissolved
HMP and thiamine in seawater collected from the Sargasso Sea. HMP was detected in all
samples and ranged from 1-36 pM with maximum concentrations coinciding with the
deep chlorophyll maximum. Thiamine ranged from undetectable to 22.3 pM with
maximum concentrations at the surface. We provide evidence that marine
Prochlorococcus and Synechococcus bacteria are a probable source of environmental
HMP, and that HMP production may have diel periodicity. These findings are evidence
of dynamic thiamine and thiamine precursor cycling the marine euphotic zone that may
have implications in community energy and carbon metabolism over short time scales.
48
Introduction
Thiamine (vitamin B1) is a sulfur-containing coenzyme used by proteins that
catalyze crucial manipulations of carbon in all living systems. Specifically, thiamine is
required for enzymes of the TCA cycle, the non-oxidative portion of the pentosephosphate pathway, light-independent photosynthetic reactions and for the biosynthesis
of branched-chain amino acids, isoprenoids and some vitamins (79). The pathways and
enzymes utilized for de novo thiamine synthesis and salvage have been the topic of
extensive research in bacteria and eukaryotic yeasts (80), and in all organisms capable of
de novo biosynthesis, the formation of thiamine monophosphate (ThP) is the result of the
enzyme-catalyzed linkage of two separately synthesized moieties: 4-amino-5hydroxymethyl-2-methylpyrimidine
diphosphate
(HMP-PP)
and
4-methyl-5-(2-
phosphoethyl)-thiazole (THZ-P). Phosphorylation of ThP yields the active thiamine
coenzyme, thiamine diphosphate (ThPP) (Fig. 3-1; reviewed in (80)).
Research of oceanic vitamin distributions is experiencing a renaissance, largely
due to more sensitive analytical techniques and a greater overall understanding of the
factors influencing productivity. There is new evidence that oceans have complex
distribution patterns of vitamins (81, 82) and that these cofactors may act synergistically
with other vitamins or inorganic nutrients to increase bacterial and phytoplankton
productivity (83). Direct measurements of thiamine in the sea revealed that surface-water
standing stocks were low except in regions of upwelling (81, 82). To contextualize such
vitamin abundance patterns and relate them to community structure and ecosystem
function, an understanding of the community members that produce and consume
49
vitamins is vital (84). Previously, eukaryotic phytoplankton have been shown to both
produce (85) and require (86) thiamine. Although the synthesis and utilization of other Bvitamins proceeds through complex symbiotic relationships (87), very little is known
regarding thiamine metabolism of marine bacteria and archaea.
The most abundant heterotrophic bacterioplankton in the marine euphotic zone
belong to the SAR11 clade of bacteria (17). Both in situ and in vitro studies have shown
that SAR11 cells actively contribute to the cycling of carbon and sulfur in the ocean (26,
28, 37, 88). The first cultivated SAR11 bacterium, ‘Candidatus Pelagibacter ubique’
HTCC1062 (Ca. P. ubique), contains one of the smallest genomes found in free living
organisms and shows substantial evidence for genome streamlining (25). Genome
streamlining has been proposed as an explanation for the unusual combination of amino
acids, reduced organosulfur compounds and organic acids required for the growth of Ca.
P. ubique (26, 32, 37, 89). Although the macronutrient requirements of Ca. P. ubique
have been identified (89), the trace requirements for vitamins have not been investigated.
Herein we investigate the metabolism of thiamin by Ca. P. ubique and identify 4-amino5-hydroxymethyl-2-methylpyrimidine (HMP) as a required trace nutrient.
Results & Discussion
Comparative genomics of thiamine biosynthesis and regulation
Previous metabolic reconstructions using the Ca. P. ubique genome did not
identify a complete biosynthetic pathway for thiamine (25), yet multiple essential genes
encoding proteins that require thiamine were identified (Figure A2-1 in appendix 2),
50
suggesting thiamine is necessary for normal metabolism. Homologs for the HMP
synthase (thiC) and tyrosine lyase (thiH), encoding essential proteins for de novo
thiamine biosynthesis in Escherichia coli, were not identified in Ca. P. ubique (Figure 31a). Genes encoding proteins that may function to supply HMP independently of ThiC
were also not identified in the Ca. P. ubique genome. For example, NMT1 homologs,
which catalyze the formation of HMP from vitamin B6 and histidine in Saccharomyces
cerevisiae (90), were not identified. Moreover, genes commonly ascribed to thiamine
salvage (thiM, thiK, tenA, tenI) (80, 91) and transport (thiBPQ) (92) were absent from the
Ca. P. ubique genome.
Genes encoding homologs for the remaining proteins required for thiamine
biosynthesis were identified in the Ca. P. ubique genome (Figure 3-1), including: (i) the
hydroxy-(phospho)methylpyrimidine kinase (thiD); (ii) thiazole sulfur transfer protein
(thiS); (iii) thiazole biosynthesis protein (thiG); (iv) two copies of the thiamine
monophosphate synthase (thiE); and (v) thiamine monophosphate kinase (thiL). The
protein sequence of E. coli’s ThiS-adenylyltransferase (encoded by thiF; b3992) is
homologous to the translated product of SAR11_0403 (blastp E-value: 1 × 10-54, 86/237
identities), which is annotated as moeB and predicted to encode a protein involved in
molybdopterin biosynthesis. During THZ-P synthesis in E. coli, sulfur is mobilized from
cysteine to the thiazole ring by the IscS protein (encoded by iscS; b2530) (93). The best
Ca. P. ubique homolog to the E. coli IscS protein is the protein product of SAR11_0742,
annotated as a selenocysteine lyase (blastp E-value: 3 × 10-28; 99/384 identities). In B.
subtilis, a glycine oxidase (encoded by thiO; BSU11670) forms dehydroglycine instead
51
of the ThiH protein used by E. coli (94). The best Ca. P. ubique protein hit to the B.
subtilis ThiO protein is encoded by SAR11_1221, and annotated as a sarcosine
dehydrogenase (blastp E-value: 1 × 10-26 100/378 identities). However, it is possible that
the formation of dehydroglycine is not enzyme-catalyzed. Experiments in vitro showed
that ammonia and glyoxylate are in equilibrium with dehydroglycine in aqueous solution
and can serve as thiazole precursors (95). Glyoxylate was previously proposed to have a
central role in glycine synthesis in Ca. P. ubique (89), thus intracellular dehydroglycine
may arise from the non-enzymatic condensation of ammonia and glyoxylate. In E. coli,
the thiamine biosynthesis genes (thiCEFSGH and thiMD) are transcribed to polycistronic
messages, however, in Ca. P. ubique, only two thiamine biosynthesis genes were found
adjacent to one another (thiS and thiG), suggestive of co-transcription (Figure 3-1a).
The transcription of thiC and other thiamine biosynthetic and transport genes is
negatively regulated by intracellular ThPP concentrations through the action of mRNA
leader sequences called riboswitches. When ThPP is bound to ThPP-binding riboswitches
transcription and translation of the downstream coding sequence is repressed (96).
Genomic searches for ThPP-binding riboswitch motifs have been used to identify genes
putatively involved in thiamine biosynthesis and transport (97). In Ca. P. ubique, a single
predicted ThPP-activated RNA riboswitch was identified upstream of a coding sequence
annotated as a sodium:solute symporter family ‘proline uptake protein’ (SAR11_0811 –
Figure 3-1a) (98).
52
Physiology of thiamine deprivation & substitution
When Ca. P. ubique was grown in an artificial seawater medium with carbon,
reduced sulfur, but without thiamine, maximum cell densities of 3.09 ± 1.6 × 107 cells ml1
(mean ± s.d., n=6) (Figure 3-2a,b) were attained. To determine if there were traces of
thiamine in our base medium that may account for growth to 107 cells ml-1 in the absence
of thiamine, we measured the thiamine concentration in uninoculated medium and found
there to be 26 pM present (for method, see (81)). Efforts to remove this contaminating
thiamine were unsuccessful, which is consistent with previous reports describing the
difficulty of removing thiamine from growth media (99). We suspect the trace thiamine
(or other thiamine precursors) is from our reagent of the lowest purity (oxaloacetate –
98%) and is responsible for the growth of Ca. P. ubique in the absence of added thiamine.
When Ca. P. ubique was grown in the aforementioned medium amended with 1 µM
thiamine, maximum cell yields increased to 3.11 ± 0.04 × 108 cells ml-1 (mean ± s.d.,
n=6) (Figure 3-2a,b).
To support the interpretation that the reduction of cell yield in thiamine deplete
conditions (relative to thiamine replete conditions) was the result of thiamine limitation
and to determine which Ca. P. ubique genes were putatively involved in thiamine
acquisition and metabolism, we conducted DNA microarray experiments to compare
gene transcript abundances from cells grown with and without added thiamine (for
sample points see Figure A2-2 in appendix 2). As Ca. P. ubique cells approached
thiamine limitation, the putatively ThPP-regulated ‘proline uptake protein’ (SAR11_0811
- Figure 3-1a) was significantly upregulated (3-fold, q-value of ≤0.01). In thiamine-
53
limited conditions, Ca. P. ubique upregulated 52 genes (Table A2-1 in appendix 2) and
downregulated 71 genes (Table A2-2 in appendix 2) relative to thiamine replete
treatments. The only gene upregulated in thiamine-limited conditions that is predicted to
encode a thiamine biosynthetic enzyme was the hydroxy-(phospho)methylpyrimidine
kinase thiD (2.1-fold, q-value <0.01; Table A2-1 in appendix 2).
We investigated the effective substitution range of HMP, thiamine and the
thiamine degradation product 4-amino-5-aminomethyl-2-methylpyrimidine (AMP) across
eight orders of magnitude in Ca. P. ubique cultures growing in an artificial medium
(Figure 3-2b). Growth of Ca. P. ubique was stimulated at HMP concentrations of 10100 pM and reached maximal cell densities at concentrations ≥1 nM (Figure 3-2b).
Thiamine and AMP were ineffective at restoring thiamine-limited cell yields at picomolar
concentrations; each compound supported maximal cell yields when supplied at or above
a final concentration of 1.0 µM (Figure 3-2b). No other potential thiamine precursors,
including 4-methyl-5-thiazoleethanol (THZ) (100), histidine + vitamin B6 (101) or
pantothenate (102) restored growth in the absence of thiamine (Figure A2-3 in appendix
2).
Molecular phylogeny and comparative genomics of SAR11_0811
We propose that the SAR11_0811 gene annotated as a ‘putative proline
transporter’ encodes a transporter for HMP and that the predicted ThPP riboswitch
preceding its coding sequence is a functional ThPP-dependent regulatory element. The
increased expression of the SAR11_0811 gene upon thiamine-depletion is consistent with
the expected expression pattern for genes regulated by ThPP-binding riboswitches (96).
54
Maximum-likelihood phylogenetic analysis of proteins homologous to SAR11_0811
shows that it is closely related to protein sequences encoded by a diverse group of
organisms, including archaea, Gram-positive bacteria and other proteobacteria (Figure
A2-4 in appendix 2). In all but one instance, the putative HMP transporter is directly
preceded by probable ThPP-riboswitches or is in a predicted operon with thiamine
pyrimidine salvage genes (Figure A2-5 in appendix 2). In nine of ten sequenced
Pelagibacter genomes, SAR11_0811-like genes were identified (Figures A2-4 & A2-5 in
appendix 2). No SAR11_0811-like gene sequence was identified Pelagibacter sp. str.
HIMB59, however, a putative thiamine transporter (thiBPQ) (92) was identified,
suggesting thiamine or ThPP can be transported directly.
Measurements of HMP and thiamine in the environment
Previously it was suggested that thiamine pyrimidines may be relatively stable in
seawater (103), yet no in situ measurements of thiamine pyrimidines have been made. To
close this knowledge gap, we extracted and concentrated HMP and thiamine from
Sargasso Sea seawater collected on September 19th (20:00 local time) and 20th (08:00
local time), 2012, to a solid-phase and quantified them using high-performance liquid
chromatography-coupled tandem mass spectrometry (using method of (81)). We found
detectable HMP in all samples, ranging from 1.3-35.7 pM (Figure 3-3). The maximum
concentration of HMP was from samples collected at 08:00 (Figure 3-3) and coincided
with the deep chlorophyll maximum (Figure A2-6 in appendix 2). A decrease in the HMP
concentration was evident at every depth except 0 meters in samples collected at 20:00
relative to samples collected at 08:00 (Figure 3-3). Thiamine concentrations ranged from
55
undetectable (detection limit: 0.81 pM (81)) to 23 pM and was present in samples from 0
to 160 m but not detected in samples from 250 and 300 m (Figure 3-3). Thiamine
concentrations were generally highest at the surface and decreased with depth and did not
vary greatly with collection time (Figure 3-3).
We show that Ca. P. ubique requires thiamine or a thiamine pyrimidine for
growth and that HMP most effectively satisfies that growth requirement (Figure 3-2b).
Although thiamine and AMP can restore thiamine-limited growth, the amount required to
do so is four orders of magnitude higher than the amount of HMP required for the same
cell yield. No known direct measurements of thiamine in the sea have exceeded 1.0 nM
(81, 82), showing that environmental concentrations of thiamine are insufficient to elicit
growth responses in Ca. P. ubique. Instead, we show that the in situ concentration (1040 pM, Figure 3-3) of HMP is within the concentration range to which Ca. P. ubique
responds (10-100 pM, Figure 3-2b). There is additional evidence that natural microbial
communities are thiamine-stressed in the Sargasso Sea. For example, metaproteomic
analysis of the Sargasso Sea (104) detected peptides that mapped to ThPP-riboswitchregulated genes, including the Ca. P. ubique sodium:solute symporter family protein we
describe here (Figures A2-4 & A2-5 in appendix 2), and peptides mapping to the ThiC
protein of Synechococcus and Prochlorococcus. Expression of these ThPP-riboswitchregulated proteins would only be expected in cells experiencing thiamine stress (96).
A specific requirement for HMP has been noted in Plasmodium falciparum (105)
and Listeria monocytogenes (106) but is unknown in marine organisms. The thiC gene is
absent from all sequenced Pelagibacter genomes, which may be generalized of all
56
members of the SAR11 clade (29). The loss of thiC within the Pelagibacteraceae is
consistent with the predicted consequences of genome streamlining as outlined by the
‘Black Queen Hypothesis’ (BQH) proposed by Morris (41). The BQH states that in some
bacterial lineages, selection for the loss of metabolically expensive functions may be
favored if the lost function can be compensated for by other members of the community.
Gene deletions at potentially useful but non-essential loci can be fixed by genetic drift in
small bacterial populations. However, the population of SAR11 is massive and therefore
the loss of thiC is unlikely to be the result of neutral processes such as drift. This implies
that HMP production is costly for Pelagibacter to synthesize de novo and that a growth
advantage is gained by outsourcing its production to other plankton.
Oxygenic photoautotrophic bacteria are probably contributors to HMP levels in
the sea and the production of HMP may be periodic. Diel transcription of thiC was
observed in both laboratory cultures of Prochlorococcus MED4 (along with diel
translation of ThiC peptides) (107) and in environmental transcripts mapping to
Synechococcus (108). In both reports, maximum thiC transcript levels were observed in
the mid-late afternoon shortly after periods of highest light intensity. Consistent with the
hypothesis that photoautotrophs are the primary HMP producers, the highest HMP
concentrations (Figure 3-3) coincided with the deep chlorophyll maximum (Figure A2-6
in appendix 2). Although additional experiments are needed to address the causes of the
observed thiC expression patterns, an attractive hypothesis is that intracellular thiamine is
less stable in illuminated conditions because of light-induced decomposition. Thiamine is
light sensitive and UV-B induces cleavage of thiamine (resulting in AMP production)
57
(109, 110). UV-B radiation can pass though seawater to depths approaching 40 m in the
open ocean (111) and is an important factor in the decomposition of some types of
dissolved organic matter (71). Thus, following periods of high illumination,
decomposition of intracellular thiamine may prevent ThPP-repression of thiC (and ThiC),
perhaps resulting in HMP overproduction. The use of HMP (or AMP) instead of thiamine
may be an adaptation common other organisms in the euphotic zone as a way to increase
fitness in illuminated environments.
Because thiamine plays an important role in metabolic pathways pertaining to
energy production and carbon metabolism, its presence or absence may have unique
ramifications in how cells organize carbon and energy fluxes over short time scales. For
example, when thiamine is absent, the TCA cycle cannot function due to the ThPPrequirement of the 2-oxoglutarate dehydrogenase. However, most Pelagibacter genomes
encode a complete glyoxylate bypass (29, 32) that may circumvent the low flux of
carbon through the TCA cycle in times of thiamine stress while enabling the formation of
TCA-cycle-derived amino acids. Pelagibacter cells can also generate energy from the
thiamine-independent oxidation of one-carbon compounds, which may be an important
source of energy when thiamine levels are low (49). Recently, synchronization of gene
transcripts related to ribosome biogenesis and oxidative phosphorylation was observed
between wild populations of SAR11, SAR86 and marine group II Euryarcheota (108). In
our studies, gene transcripts that showed the largest expression increases when amended
with thiamine (compared to thiamine-limited cultures) (Table A2-2 in appendix 2) were
identified by Ottesen as positively correlated with ribosomal protein transcripts (108).
58
Although more research is necessary to elucidate the specific factors influencing the
transcription patterns observed in situ and in vitro, secreted or leaked compounds
essential for energy metabolism (such as thiamine, HMP and possibly other B-vitamins)
may be cues for SAR11 and other taxa to adopt a specific metabolic state.
The depth-distribution measurements of HMP offer clues to the cycling of
thiamine, and perhaps other B-vitamins in the euphotic zone of marine ecosystems and
raise interesting questions regarding the biologically active form of individual vitamins in
seawater. It is known that other B-vitamins are light sensitive or unstable in seawater
(103, 112) or play roles in light-mediated regulatory schemes (113). Furthermore, there
are a number of B-vitamins for which no environmental data exists (e.g. – niacin (B3),
pantothenate (B5) and folate (B9)). Given that B-vitamins are often required for essential
cellular functions, understanding their environmental distributions may help elucidate
succession patterns and the observed community connectivity in the environment (46,
47), making B-vitamin and precursor studies an exciting and fruitful avenue of future
research.
Methods
Organism source
Ca. P. ubique HTCC1062 was revived from 10% glycerol stocks and propagated
in artificial medium for SAR11 (AMS1) (89) amended with oxaloacetate (1 mM), glycine
(50 µM), methionine (50 µM) and FeCl3 (1 µM). Thiamine or precursors were added as
indicated in figure legends and text.
59
Chemicals
All AMS1 constituents, reagents and vitamins were of the highest available
quality (labeled ‘ultrapure’ when possible). Nutrient and vitamin stocks were prepared in
combusted glassware (450°C for 4 h) with nanopure water, 0.1 µm-filter-sterilized and
frozen in amber tubes immediately after preparation. Reasonable precautions were taken
to limit the number of freeze-thaw cycles and light exposure of each reagent. HMP was
synthesized as described in (114). AMP was synthesized as described in (115).
Cultivation details
All cultures were grown in acid-washed and autoclaved polycarbonate flasks and
incubated at 20°C with shaking at 60 RPM in the dark. Cells for counts were stained with
SYBR green I and counted with a Guava Technologies flow cytometer at 48-72 h
intervals as described elsewhere (26).
Vitamin-limitation procedure
To dilute out the vitamins routinely added to SAR11 cultures, Ca. P. ubique was
transferred to AMS1 amended with organic macronutrients, but without B-vitamins, a
minimum of 10 times (>1000 generations). After three passages (>30 generations)
truncated cell growth was consistently observed (104 cells ml-1 to 107 cells ml-1 - for
example, see Figure 3-2a).
Thiamine replacement experiments
Ca. P. ubique was grown in AMS1 with oxaloacetate (1 mM), glycine (50 µM),
methionine (50 µM) and FeCl3 (1 µM). The treatments for the effective range of
60
substitution experiments were: no thiamine addition (negative control), thiamine-HCl
added (1 pM to 10 µM), 4-amino-5-hydroxymethyl-2-methylpyrimidine added (1 pM to
10 µM), or 4-amino-5-aminomethyl-2-methylpyrimidine added (1 pM to 10 µM).
Experiments testing other potential thiamine precursors were conducted in AMS1 with
the same organic amendments, but with additions of 1.0 µM of pantothenate, 1.0 µM 4methyl-5-thiazoleethanol (THZ) or 1.0 µM each of histidine and B6 in place of thiamine.
Microarray analysis
Biological replicates (n=3) of Ca. P. ubique were grown in AMS1 with
amendments of oxaloacetate (1 mM), glycine (50 µM), methionine (50 µM) and FeCl3
(1 µM). The thiamine-replete treatment was amended with 1.0 µM thiamin-HCl; the
thiamine-deplete treatment was not amended with thiamine. Each growth condition
sampled at points indicated in Figure A2-2 in appendix 2. At each sample point, cells
were harvested by centrifugation at 20,000 RPM for 1.0 h at 4°C. Transcription profiles
of cell pellets were immediately stabilized with RNAprotect Bacteria reagent (Qiagen –
Valencia, CA). RNA was extracted using an RNeasy Mini kit (Qiagen – Valencia, CA)
and amplified using the MessageAmp-II Bacteria RNA amplification kit (Ambion –
Carlsbad, CA) per the manufacturer’s instructions. Array chip hybridization to custom
‘Candidatus Pelagibacter ubique’ Affymetrix GeneChip arrays that contained probes for
Pelagibacterales strains: HTCC1002, HTCC1062 and HTCC7211 (Pubiquea520471f)
was performed as described elsewhere (37). A Bayesian statistical analysis was
conducted using Cyber-T (116). The estimate of variance was calculated in Cyber-T by
using window sizes of 101 and a confidence value of 10. A t-test was performed on log-
61
transformed expression values by using the Bayesian variance estimate. The program
QVALUE, was used to obtain a q-value, which accounts for multiple t-tests performed
(117). We defined genes as differentially expressed if both the q-value was ≤0.05 and the
gene was differentially regulated by ≥2.0-fold.
Seawater collection for depth distributions
Seawater for vitamin analysis was collected from Hydrostation S (32°10'N,
64°30'W) from casts at 20:00 (local time) on 19 September 2012, and 08:00 (local time)
on 20 September 2012. At time of collection, samples were filtered through nanopure
water-rinsed 0.2 µm supor filters into acid-washed amber polypropylene bottles and
frozen immediately. Temperature and fluorescence CTD data from these casts are show
in Figure A2-6 in appendix 2.
Extraction of HMP from seawater
HMP was extracted from seawater to a reverse-phase C18 silica bead solid phase
(Agilent HF-Bondesil) as described in (81). In short, 3.0 g of the C18 beads were
measured into chromatography columns. 100% methanol was added to the beads to form
a slurry. Methanol was drained and the beads were conditioned with 20 mL HPLC-grade
water. The seawater samples were thawed and allowed to equilibrate to room temperature
before their pH was adjusted to 6.5 with 1 N HCl. The pH 6.5 sample (300 mL) was
applied to the conditioned column at a flow rate of 1-2 mL min-1. The effluent was
collected in a clean amber polypropylene bottle. The pH of the sample effluent was readjusted to 2.0 with 1 N HCl. The column was re-conditioned with 20 mL pH 2.0 (1 N
62
HCl) HPLC-grade water. After adjusting pH to 2.0, samples were applied to columns
again. The column was washed with 20-30 mL pH 2.0 water to remove residual salt.
Organics were eluted from the C18 beads with 10 mL (1 mL 10 times) 100% MeOH.
Methanol was evaporated to dryness in a heated Savant Speedvac vacuum concentrator
and frozen until LCMS analysis. For quantification purposes, aged seawater collected
from Hydrostation S in 2009 was spiked with known amounts of HMP and thiamine
ranging from 0 pM to 100 pM and extracted alongside samples.
High pressure liquid chromatography tandem mass spectrometry of HMP and thiamine
Dried samples were reconstituted in 125 µL HPLC-grade water. Samples were
centrifuged to pellet insoluble matter and the supernatant was transferred to a sampling
vial. 5 µL of each sample was injected into an Applied Biosystems MDS Sciex 4000 Q
TRAP mass spectrometer coupled to a Shimadzu HPLC system. An Agilent Zorbax SBAq (2.1 × 100 mm, 3.5-micron) HPLC column was used for separation over a 10-minute
gradient flow was used with mobile phases of pH 4 (with formic acid) methanol (MeOH)
and pH 4 (with formic acid) 5 mM ammonium formate (AmF). The flow rate was 0.4 mL
min-1 and a gradient starting at 98% AmF: 2% MeOH for 1 minute changing to 75%
AmF: 25% MeOH over 3 minutes, 50% AmF: 50% MeOH over 0.2 minutes, and finally
to 10% AmF:90% MeOH over 0.8 minutes. The mass spectrometer was run in ‘Multiple
Reaction Monitoring’ (MRM) mode. The m/z of the HMP parent ion was 140.2, and ion
transitions of 81.1 and 54.1 were used for quantification and qualification, respectively.
Peaks were analyzed using the Analyst® software package v 1.5.2 (AB SCIEX; Concord,
ON, Canada). Thiamine was detected as described in (81).
63
A
E. coli K-12 MG1655
1.1 Mbp
thiL
thiM
Ca. P. ubique HTCC1062
thiE 39 kbp moeB
thiD
178 kbp
thiC
2 Mbp
thiE2
20 kbp
0811
76 kbp
thiD
HO
O
N
ThiC
N
Purine
nucleotide
biosynthesis
S thiG
csdB
15 kbp
N
-3O
HMP-PP
vitamin B6
NMT1
S
ThiM
OH
N
CO-AMP
COOH
[ThiS]
[ThiS] MoeB
CsdB
cys
N
S
OPO3-2
COSH
[ThiS]
dh-gly
NH2
O
OH
s
OP2O6-3
ThiL
OPO3-2
OH
O
dDXP
N+
N
NH2
N
ThP
HO
ThiO
ThiH
tyr gly
E. coli
N+
N
THZ-P
ThiG
degradation
OPO3-2
ThiE
THZ
HS
AMP
NH2
HMP(-P)
N
NH2
N
6P2O
S. cerevisiae
N
H2N
N
NH2
histidine
thiH
thiL
226 kbp
ThiD
N
HO
OH
AIR
S
thiF
TenA
H2N
N
thiG
120 kbp
B
H2O3PO
thiE
Dxs
s
ThPP
Active Cofactor
NH2
G3-P
pyr
glyoxylate + NH3
B. subtilis
Figure 3-1: Comparative genomics of thiamine biosynthesis. A) Comparative genomics
of genome content in Escherichia coli K12 MG1655 and ‘Ca. Pelagibacter ubique’
HTCC1062. Dashed black gene outlines: genes present in E. coli but absent in Ca. P.
ubique. Colored gene outlines: shared colors represent gene homologs, and match
biosynthetic steps depicted in (B). Red stem-loop structures represent predicted or
characterized ThPP-binding RNA riboswitches. B) Simplified illustration of the
reconstructed thiamine synthesis pathway in Ca. P. ubique. Black dashed lines: steps that
are missing in Ca. P. ubique. For detailed biochemistry of thiamine biosynthesis, see
(80). Abbreviations: AIR - aminoimidazole ribotide; HMP(-P) - 4-amino-5hydroxymethyl-2-methylpyrimidine (-phosphate); HMP-PP - 4-amino-5-hydroxymethyl2-methylpyrimidine diphosphate; AMP - 4-amino-5-aminomethyl-2-methylpyrimidine;
ThP – thiamine monophosphate; ThPP – thiamine diphosphate; THZ - 4-methyl-5thiazoleethanol; THZ-P - 4-methyl-5-(2-phosphoethyl)-thiazole; cys – cysteine; gly –
glycine; tyr – tyrosine; dDXP - 1-deoxy-D-xylulose 5-phosphate; dh-gly dehydroglycine; pyr – pyruvate; G3-P – glyceraldehyde 3-phosphate. TenA is described
in (91).
64
B 4.5
density (cells ml-1)
108
107
10
6
thiamine (1.0 μM)
no thiamine
105
0
5
10
15 20
time (days)
25
30
35
maximum density (cells ml-1 × 108)
A
4.0
3.5
AMP
Thiamine
HMP
no vitamins
3.0
2.5
2.0
1.5
1.0
0.5
0
none 1.0 10 100 1.0 10 100 1.0 10
pM pM pM nM nM nM μM μM
Figure 3-2: Growth of Ca. P. ubique under thiamine-limiting conditions and responses to
thiamine pyrimidines. a) Growth curve of Ca. P. ubique on artificial seawater with
methionine (50 µM), glycine (50 µM) and oxaloacetate (1 mM) with 1 µM thiamine (red)
or without thiamine (black). Points are the average densities of biological replicates ± s.d.
(n=3). b) Maximum densities of Ca. P. ubique cultures grown in artificial medium (as
described for (a)) with thiamine, HMP or AMP at culture concentrations spanning eight
orders of magnitude. Bar heights are the average densities of biological replicates ± s.d.
(n=3).
65
0
0
5
concentration (pM)
10 15 20 25 30
35
40
50
depth (meters)
100
150
200
250
[HMP] - 20:00 cast
[HMP] - 08:00 cast
[Thi] - 20:00 cast
[Thi] - 08:00 cast
300
Figure 3-3: Depth distribution of dissolved 4-amino-5-hydroxymethyl-2methylpyrimidine (HMP) and thiamine in the Sargasso Sea. Filled markers were
collected at 20:00 (local time) on 19 September 2012. Open markers were collected at
08:00 (local time) on 20 September 2012. Squares are thiamine concentrations. Circles
are HMP concentrations. HMP points are the average of replicate analyses for each
sample ± 1.0 s.d. (n=3). There was no replication for the thiamine measurements.
Thiamine was not detected in samples collected from 200 m at 20:00 or at 250 m and
300 m at either time. Temperature and fluorescence CTD data for each profile is shown
in Figure A2-6 in appendix 2.
66
Chapter 4
Distinct Phosphate-Acquisition Physiotypes and Adaptations for Extreme Marine
Oligotrophy in SAR11 Bacteria
Paul Carini1, Emily O. Campbell1, Angelicque E. White2 and Stephen Giovannoni1
1
2
Department of Microbiology, Oregon State University Corvallis, OR 97331
College of Oceanic and Atmospheric Sciences, Oregon State University, Corvallis, OR
97331
67
Abstract
Collectively, heterotrophs belonging to the SAR11 clade of oligotrophic marine
α-proteobacteria comprise one of the largest pools of biomass in the Earth’s oceans.
Despite the ubiquity of SAR11 cells, very little is understood about how their physiology
and transcription patterns respond to nutrient perturbation and how those responses may
affect geochemical cycles. Here we demonstrate evidence for distinct phosphorus (P)
acquisition strategies by SAR11 marine bacteria that manifest as different P-utilization
physiotypes. The SAR11 isolate ‘Candidatus Pelagibacter ubique’ HTCC1062 (Ca. P.
ubique) responded to inorganic phosphate (Pi) depletion by rapidly upregulating genes
encoding a high affinity Pi transport system (pstSCAB-phoUB). The remaining
transcriptional response indicated that a stringent-like stress response was induced, as
exemplified by increased abundance of stress response gene transcripts and simultaneous
reduction in transcripts for ribosomal proteins, RNA polymerase subunits and cell
division proteins. Of ten alternative organic and reduced P sources, only Pi effectively
satisfied the P requirement of Ca. P. ubique. Conversely, Pi-depletion caused a second
Pelagibacter isolate, Pelagibacter sp. str. HTCC7211, to upregulate a suite of genes that
encode a probable phosphate-ester/phosphite/ phosphonate/Pi transport system
(phnCDEE2), multiple C-P lyase complex genes that confer the ability to use
phosphonates as sole Pi sources and four genes probably involved in the synthesis of Pfree lipids. Experiments with cells in culture confirmed that Pelagibacter sp. str.
HTCC7211 used organic and reduced forms of P, including phosphonates, in place of Pi.
When grown on methylphosphonic acid (Mpn) as a sole P-source, the apparent cellular
68
requirement for P decreased. Moreover, cells grown on Mpn stoichiometrically released
methane, implicating Pelagibacter as a source of ocean methane, and a potentially key
player in the ocean methane paradox. These results show that Pelagibacterales
bacteria have adopted diverse strategies to compete for low levels of Pi in the sea.
Introduction
Phosphorus (P) in its +5 valence state (phosphate; Pi) is an essential component of
all living cells. It is a constituent of nucleic acids, phospholipids, phosphorylated proteins
and is intimately involved in energy metabolism and some transport functions (via ATP).
Most bacteria readily assimilate Pi to meet their P-requirement, however in oligotrophic
ocean gyres, Pi concentrations are extremely low (for example, 0.2-1.0 nM in the
Sargasso Sea (118)) and the availability of Pi can limit bacterial and primary production
(118-124). Several studies of P metabolism in primary producers that inhabit Pi-deplete
waters have uncovered microbial adaptations that enhance the competitiveness of these
lineages in low-Pi environments (125-131). However, the strategies used by major
chemoheterotrophic bacteria that cohabitate these nutrient poor environments are
relatively unexplored.
The SAR11 clade of oligotrophic α-proteobacteria are the numerically dominant
chemoheterotrophic cells in marine euphotic zones worldwide (17). Ca. P. ubique, the
first cultivated SAR11 isolate, is a small cell (volume of 0.01 µm3) and contains a
streamlined genome (1.3 Mbp) (23). Both in situ (28, 88, 132, 133) and in vitro (26, 32,
37, 49) work shows that the Pelagibacter population is active and metabolizes specific
forms of dissolved organic matter (DOM), including reduced sulfur compounds, amino
69
acids, one-carbon compounds and organic acids. In artificial media, Ca. P. ubique
requires an unusual combination of amino and organic acids, reduced sulfur and vitamins
for growth (89).
Microbes inhabiting Pi-deplete environments have evolved diverse strategies to
effectively compete for trace amounts of Pi. For example, some bacteria, including
marine phytoplankton, actively remodel membrane lipid structure to reduce phospholipid
content, resulting in a decreased cellular P-requirement (134-137). P-starvation in many
bacteria, including Escherichia coli, Sinorhizobium meliloti, and Prochlorococcus sp.
MED4, results in an increased cellular affinity for Pi, due to induction of high-affinity Pi
transport systems (128, 138, 139). Furthermore, Pi stored as endogenous polyphosphate
(140) or phospholipids (137) during periods when Pi is plentiful may be metabolized
when exogenous Pi is low. In low-Pi environments P is also obtained by degrading Pesters with broad-substrate range alkaline-phosphatases (APases; secreted or cytoplasmic
(141)), or phosphonates with a C-P lyase (126). Recently, Prochlorococcus was shown to
utilize phosphite, a reduced P source for which environmental distributions are unknown
(142). Both metagenomic and metaproteomic approaches show that gene suites
conferring both Pi and organic-P acquisition, P-storage (as polyphosphate) and
metabolism are abundant and expressed in ocean environments that are low in Pi (104,
143-145), suggesting that the maintenance of complex P-regulons in low-Pi environments
may allow microbes to maintain competitiveness and occupy specific P-acquisition
niches.
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The genome sequences of two organisms in the Ia-subclade of the
Pelagibacteraceae - Ca. P. ubique and Pelagibacter sp. str. HTCC7211 - show variation
in the gene complements associated with the acquisition, storage and metabolism of Pi
(29), implying that the two bacteria may respond differently to Pi-limitation. Ca. P.
ubique contains genes encoding a high affinity transport system and associated regulatory
genes (pstSCAB-phoUB) (genomic context is depicted in Figure 4-3). Pelagibacter sp.
str. HTCC7211 encodes, in addition to a high affinity transport system (pstSCABphoUB), genes involved in phosphonate acquisition (phnCDEE2), phosphonate
metabolism (phnFGHIJKLNM and phnX), polyphosphate metabolism (ppx and ppk) and
other genes putatively involved in Pi metabolism (genomic context is depicted in Figure
4-3). The P-acquisition and metabolism genes in Pelagibacter sp. str. HTCC7211 are
found clustered in the genome.
Although assorted ‘-omic’ investigations have highlighted the genomic potential
of Pelagibacter cells through in silico analyses, little is known about the specific types
and amounts of P Pelagibacter use or cellular adaptations that may benefit these cells in
low Pi ecosystems. To close this gap in knowledge, we undertook experiments designed
to determine i) the amount of P required for Pelagibacter growth; ii) alternate forms of P
that Pelagibacter cells can use and iii) the transcriptional responses to Pi-starvation. Two
Pelagibacter strains isolated from geochemically distinct ecosystems were used to
accomplish these goals – Ca. P. ubique (isolated from the NE Pacific Ocean off the coast
of Oregon USA (23)) and Pelagibacter sp. str. HTCC7211 (isolated from the North
Atlantic Ocean in the Sargasso Sea (22)). Experiments with both strains were conducted
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on a chemically defined, low-Pi artificial medium for SAR11 (AMS1) amended with
appropriate amino and organic acids and vitamins (89).
Methods
Organism source
Ca. P. ubique and Pelagibacter sp. str. HTCC7211 were revived from 10%
glycerol stocks and propagated in artificial medium for SAR11 (AMS1) (89) amended
with pyruvate (100 µM), glycine (5 µM), methionine (5 µM), FeCl3 (1 µM), and vitamins
(146) unless indicated otherwise.
Cultivation details
All cultures except those describing methane production (below) were grown in
acid-washed and autoclaved polycarbonate flasks. Cultures were incubated at 20°C with
shaking at 60 RPM under a 12-h/12-h light (140–180 µmole photons m-2 s-1)/dark cycle.
Cells for cell counts were stained with SYBR green I and counted with a Guava
Technologies flow cytometer at 48-72 h intervals as described elsewhere (26).
Growth conditions for phosphate replacement experiments
Pelagibacter isolates were grown in a base medium of AMS1 with no added Pi,
amended with pyruvate (100 µM), glycine (5 µM), methionine (5 µM), FeCl3 (1 µM) and
vitamins. The treatments were: no Pi addition (negative control), 1.0 µM Pi (as NaH2PO4
- positive control), 1.0 µM of deoxycytidine triphosphate (dCTP), 1.0 µM of
deoxyguanine triphosphate (dGTP), 1.0 µM of deoxyadenine triphosphate (dATP),
1.0 µM of deoxythymidine triphosphate (dTTP), 1.0 µM phosphite (as NaPHO35H2O),
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1.0 µM glucose 6-phosphate (G6P), 1.0 µM ribose 5-phosphate (R5P), 1.0 µM
methylphosphonic acid (Mpn), 1.0 µM 2-aminoethylphosphonic acid (2-AEP) or 1.0 µM
phosphoserine (P-ser). Flasks were incubated and counted as described in ‘Cultivation
details.’
Microarray growth and sampling conditions
Biological replicates (n=3) of Ca. P. ubique or Pelagibacter sp. str. HTCC7211
were grown in AMS1 amended with pyruvate (1 mM), glycine (50 µM), methionine
(50 µM), FeCl3 (1 µM), Pi (10 µM) and vitamins. In mid- to late-logarithmic growth, cells
were harvested by centrifugation (10,000 RPM for 1.0 h at 20°C). Cell pellets were
evenly split and washed twice with growth media either amended with 100 µM Pi (replete
conditions) or not amended with Pi (deplete conditions). After washing, pellets were resuspended in Pi-deplete or Pi-replete growth media. Samples were collected from cell
suspensions at t=0 h, 4 h, 20 h, and 38 h after resuspension for Ca. P. ubique, and t=0 h,
20 h, 38 h, 68 h, and 96 h after resuspension for Pelagibacter sp. str. HTCC7211.
Sampling consisted of centrifugation at 20,000 RPM for 1.0 h at 4°C followed by
resuspension of cell pellets in RNAprotect Bacteria reagent (Qiagen – Valencia, CA).
Preparation and analysis of microarrays
RNA was extracted using an RNeasy Mini kit (Qiagen – Valencia, CA) and
amplified using the MessageAmp-II Bacteria RNA amplification kit (Ambion – Carlsbad,
CA) using the manufacturer’s instructions. Array chip hybridization to custom
‘Candidatus Pelagibacter ubique’ Affymetrix GeneChip arrays that contained probes for
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Pelagibacterales strains: HTCC1002, HTCC1062 and HTCC7211 (Pubiquea520471f)
was performed as described elsewhere (37). A Bayesian statistical analysis was
conducted using Cyber-T (116). The estimate of variance was calculated in Cyber-T by
using window sizes of 101 and a confidence value of 10. A t-test was performed on logtransformed expression values by using the Bayesian variance estimate. The program
QVALUE, was used to obtain a q-value, which accounts for multiple t-tests performed
(117). A gene was defined as differentially expressed if both the q-value was ≤0.05 and
the gene was differentially regulated by ≥2.0-fold.
Methane production from Mpn
To test for methane (CH4) production from Mpn, Pelagibacter sp. str. HTCC7211
cells previously grown in media with excess Mpn as the sole P source were used to
inoculate 60 mL sterile, bovine serum albumin (BSA)-coated serum bottles containing 55
mL growth media (leaving a 5 mL headspace) amended with 10 µM Mpn as the sole P
source. The negative control consisted of Pelagibacter sp. str. HTCC7211 cells
previously grown in media with excess Pi as the sole P source as inoculum for 60 mL
sterile, BSA-coated serum bottles containing 55 mL growth media amended with 10 µM
Pi. Treatment and control bottles were capped with Viton septa, crimped with aluminum
seals and incubated horizontally as described in ‘Cultivation details.’ CH4 in the
headspace was measured by gas chromatography in a Shimadzu GC-8A gas
chromatograph (GC) equipped with a column packed with Porapak N (80/100 mesh size)
fitted with a flame ionization detector (FID). In brief, 100 µM samples of headspace from
each bottle were injected into the GC at a flow rate of 25 mL min-1. Peaks were integrated
74
on a Chromatopac data processor (Shimadzu C-R8A). Quantification was accomplished
by calibrating peak areas to a standard curve derived by injecting known amounts of CH4
into sealed serum bottles containing growth medium. Standards were equilibrated in a
shaking water bath for 24 h at 25ºC. Single bottles were sacrificed from each treatment
for cell counts over the duration of the experiment. Theoretical CH4 release from Mpngrown cells was calculated assuming an average Mpn cell-1 usage of 11.4 amol cell-1
(calculated from regression line in Figure 4-1).
Transmission Electron Microscopy
To determine if the ppk gene conferred the ability to produce polyphosphate, we
qualitatively determined whether Pelagibacter sp. str. HTCC7211 contained electrondense polyphosphate-like granules under Pi-replete, Pi-starved and Mpn-replete
conditions using STEM. Pelagibacter sp. str. HTCC7211 cells were grown in a base
medium consisting of AMS1 with pyruvate (1 mM), glycine (50 µM), methionine (50
µM), FeCl3 (1 µM) and vitamins with either 10 µM Pi (Pi-replete) or 10 µM Mpn (Mpnreplete). Cells that were Pi-starved were harvested from the Pi-replete treatment and
resuspended in Pi-free medium for 96 hours to starve them. Cells were harvested for
microscopy by centrifugation and pellets were fixed in 2% glutaraldehyde and 1%
paraformaldehyde in 0.1 M sodium cacodylate buffer. Fixed cells were stained with 2%
osmium tetraoxide, rinsed and dehydrated (from 30% to 100% acetone). Dehydrated cells
were infiltrated with Spurr’s resin and polymerized (65°C overnight). Cells embedded in
resin were thin sectioned (50-70 nm) and stained with 4% Pb2+ (as lead citrate) for 2 min,
then switched to 2% uranyl acetate for 30 min before a final 4% Pb2+ stain for 4 min.
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Stained samples were examined by Scanning Transmission Electron Microscopy (STEM)
using a Titan G2 80-200 microscope (FEI; Eindhoven, Netherlands) at 80 kV, spot size 3.
Results
Basic physiology
The maximum specific growth rate of Ca. P. ubique and Pelagibacter sp. str.
HTCC7211 on AMS1 was 0.59 ± 0.02 day-1 (average ± s.d. n=24) and 0.35 ± 0.02 day-1
(average ± s.d. n=24), respectively, regardless of media Pi concentration (Table A3-1 in
appendix 3). Both Ca. P. ubique and Pelagibacter sp. str. HTCC7211 responded linearly
to Pi additions when grown on AMS1 with other nutrients in excess (Figure 4-1). The
apparent Pi requirement was calculated from Pi dose-response linear regressions and
found to be 10.2 attomoles Pi cell-1 for Ca. P. ubique and 45.7 attomoles Pi cell-1 for
Pelagibacter sp. str. HTCC7211 (Figure 4-1).
Alternate P-source utilization in Ca. P. ubique
Growth media without added Pi (negative control) supported maximal Ca. P.
ubique cell densities of 1.7 × 106 cells ml-1 (Figure 4-2). Growth medium with 1.0 µM Pi
supported densities of 3.3 × 107 cells ml-1 (Figure 4-2). Of nine alternate P-sources,
dCTP, phosphite, dATP, dGTP, dTTP and R5P supported minimal to moderate cell
density increases to 3.5-8.5 × 106 cells ml-1 (Figure 4-2). 2-AEP, Mpn, G5P and P-ser did
not alleviate Pi limitation.
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Alternate P-source utilization in Pelagibacter sp. str. HTCC7211
Growth media without added Pi (negative control) supported maximal
Pelagibacter sp. str. HTCC7211 cell densities of 7.8 × 105 cells ml-1 (Figure 4-2). Growth
media with 1.0 µM Pi (positive control) supported densities of 3.8 × 107 cells ml-1 (Figure
4-2). Pelagibacter sp. str. HTCC7211 grew to 4.0 × 107 cells ml-1 with 1.0 µM P-ser as a
P source (Figure 4-2). 1.0 µM additions of 2-AEP, Mpn, R5P, G6P or phosphite
supported densities above 1.0 × 107 cells ml-1 (Figure 4-2). Diauxic growth was observed
when Pelagibacter sp. str. HTCC7211 cells were grown with phosphite, G6P, R5P, Mpn,
2-AEP, or P-ser as sole P-sources (Figure A3-1 in appendix 3). Minimal to moderate cell
density increases were observed for dCTP, dGTP, dATP and dTTP.
Transcriptional responses to Pi-starvation in Ca. P. ubique
To better understand the underlying genetic basis for the observed P-utilization
patterns, we used DNA microarrays to monitor gene expression changes during the onset
of Pi-limitation in cell-suspension time-course experiments. When stressed for Pi, Ca. P.
ubique induced transcription of the high-affinity transport system (pstSCAB-phoUB)
(Figure 4-3; Tables A3-2 - A3-4 in appendix 3). Genes encoding proteins involved in the
SOS-response (umuD, recA, and lexA), heat shock (dnaK, dnaJ, ipbA), protein maturation
and folding (groEL and trxB), protein recycling (hflCK, ftsH and hslUV), and membrane
integrity (tolRAB) were upregulated during extended Pi starvation (select genes in Figure
4-3; Tables A3-3 & A3-4 in appendix 3). Genes encoding proteins involved in Nmetabolism (glnA, gltBD), ammonia transport (amt), regulation (a CheY-like response
regulator) and other hypothetical proteins were also upregulated in the absence of Pi
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(Tables A3-3 & A3-4 in appendix 3). We observed a concomitant reduction in transcripts
encoding for ribosomal proteins (rpsBEHNPK and rplBCFNP), translation machinery
(fusA and trmD), RNA polymerase subunits (rpoA and rpoC), cell division-related
proteins (mraZ and ftsZ) and type IV pilus assembly (pilB and pilQ) (select genes in
Figure 4-3; Tables A3-3 & A3-4 in appendix 3). Also downregulated were genes
encoding a Fur/Zur-like Fe2+/Zn2+-transcriptional regulator, periplasmic binding proteins
for probable amino acid ABC transporters (ydhW and livJ) and proteins involved in the
glyoxylate cycle (aceA) and methionine biosynthesis (metY).
Transcriptional responses to Pi-stress in Pelagibacter sp. str. HTCC7211
A transcriptional response to Pi-limitation in Pelagibacter sp. str. HTCC7211 was
not observed 0 h, 20 h, or 38 h after resuspension. At 68 h, a probable phosphate-ester/
phosphite/phosphonate/Pi ABC transporter (phnCDEE2), multiple components of the C-P
lyase (phnGHIJKLM), the high-affinity Pi transport system (pstSCABphoU), two genes
annotated as putative hemolysins, a probable glycosyl transferase (rfaG-like), a
calcineurin-like metallophosphoesterase (plsC) and an HD-domain-hydrolase were
significantly upregulated along with genes of unknown function (Figure 4-3; Table A3-5
in appendix 3). After 96 h of Pi starvation, a probable operon consisting of a
glutaredoxin-arsenate reductase (arsC) and putative glpF-family transporter (aqpS) were
also significantly elevated (Figure 4-3; Table A3-6 in appendix 3), as were genes found
elsewhere in the genome, including a putative alginate lyase and a cytoplasmic
hydroxyglutathionine hydrolase (gloB) (Table A3-6 in appendix 3).
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Methane release and change in apparent cellular P-requirement in Pelagibacter sp. str.
HTCC7211 when grown on Mpn
When grown with Mpn as the sole P source, Pelagibacter sp. str. HTCC7211
produced methane concomitantly with cell growth (Figure 4-4). Methane was not
produced when cells were grown with Pi additions (Figure 4-4). Pelagibacter sp. str.
HTCC7211 responded linearly to Mpn additions in the absence of Pi (Figure 4-1) and
growth rate was not dependent on the amount of Mpn in the media (Table A3-1 in
appendix 3). Using the slope of the regression line in Figure 4-1, the apparent amount of
Mpn required per cell was calculated to be 11.4 attomoles Mpn cell-1 (34.3 attomoles
cell-1 less P than when grown on Pi (Figure 4-1)).
Polyphosphate production in Pelagibacter sp. str. HTCC7211
Electron-dense granules were observed in Pelagibacter sp. str. HTCC7211 cells
that were grown on Mpn as a sole P-source. Similar granules were not observed in cells
grown with Pi as the sole Pi-source, nor were they present in Pi-starved cells (Figure A3-6
in appendix 3).
Discussion
One of the objectives of this study was to determine the amount of P Pelagibacter
cells need for growth. We found that when grown on AMS1 with excess organic
amendments, Ca. P. ubique required 10.2 amol Pi cell-1 (calculated from regression line in
Figure 4-1). 81% of the Pi-requirement could be accounted for by estimating the P
contained in various cellular constituents: DNA (one chromosome cell-1) = 4.3 amol;
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rRNA (200 ribosomes cell-1) = 1.5 amol; and phospholipids (134) 2.5 amol cell-1. The
remaining 1.9 amol of P are conceivably contained in nucleotide pools (for nucleic acid
synthesis and energy), mRNA transcripts, phosphorylated proteins and other biosynthetic
intermediates. In contrast to Ca. P. ubique, we observed a larger apparent Pi-requirement
(that is, a lower cell yield per nmol Pi) for Pelagibacter sp. str. HTCC7211 when grown
on Pi (45.7 amol cell-1; calculated from the regression line in Figure 4-1).
To test whether the relatively high amount of Pi required by Pelagibacter sp. str.
HTCC7211 was due to polyphosphate accumulation, we assayed for polyphosphate using
a non-quantitative scanning transmission electron microscopy (STEM) assay (similar to
(147)). Polyphosphate readily stains with Pb2+ giving rise to electron-dense granules
when imaged by STEM (148). Pelagibacter sp. str. HTCC7211 cells grown in Pi-replete
conditions or cells starved for Pi did not contain electron-dense granules, suggesting that
long polyphosphate molecules are not synthesized in either condition (Figure A3-6 in
appendix 3). Although polyphosphate accumulates in E. coli grown with excess Pi under
some growth conditions, and when Pi-starved (149-151), it does not accumulate in E. coli
cells grown in amino acid and Pi-replete conditions (149). In E. coli, polyphosphate
accumulation is dependent on the PhoB response regulator which is expressed during Pistress (149). Pelagibacter sp. str. HTCC7211 did not differentially regulate phoB under
Pi-limiting conditions (Tables A3-5 & A3-6 in appendix 3), hinting that phoB
transcription and polyphosphate accumulation might be controlled by alternate factors.
In addition to determining the cellular requirement for Pi, we sought to gain an
understanding of the transcriptional responses to Pi-limitation in each organism. The pho-
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regulon typically includes genes encoding a high affinity Pi transport system
(pstSCABphoU) and, as many organisms do (125, 127, 152, 153), Ca. P. ubique quickly
upregulated the high affinity Pi-transport system when P-limited (Figure 4-3; Tables A3-2
& A3-4 in appendix 3). The remaining transcriptional responses in Ca. P. ubique (of both
genes upregulated and downregulated) were reminiscent of transcription profiles
observed in E. coli cells in which the “stringent response” was induced (154, 155). The
stringent response is a bacterial stress response that results from the intracellular
accumulation of the guanosine 3’,5’-bispyrophosphate (ppGpp) nucleotide (reviewed in
(156)). ppGpp is produced by Pi-starved E. coli cells (157) and is involved in regulating
transcription of the pho regulon regulator, PhoB (149, 158). When the stringent response
is artificially induced in E. coli, in addition to increased transcription of pstS and phoB,
induction of multiple SOS-response genes is evident, including lexA, recA and umuD
(154), similar to what we observe here for Ca. P. ubique (Figure 4-3; Tables A3-3 & A34 in appendix 3). The accumulation of ppGpp also represses the synthesis of tRNAs and
rRNAs, and results in reduced transcription of ribosomal protein genes (reviewed in
(159)). Reduction of ribosomal protein transcripts was also observed in Pi-stressed Ca. P.
ubique cultures (Figure 4-3; Tables A3-3 & A3-4 in appendix 3). Activation of E. coli’s
stringent response represses some temperature shock proteins and chaperones, while
inducing others (154, 160). Similar regulatory patterns were previously described for Pilimited marine Prochlorococcus (125) and Synechococcus (127) bacteria, in which
ribosomal protein and chaperone transcripts were differentially regulated under Pi-deplete
conditions. Although the specific chaperone and heat shock proteins that are differentially
81
transcribed in Ca. P. ubique are not identical to those in other organisms, a conserved
theme of stress-induced proteome re-configuration is evident, perhaps to prepare the cell
for prolonged nutrient deprivation.
A stringent-like transcriptional response was conspicuously absent in
Pelagibacter sp. str. HTCC7211. Instead, an extended pho regulon was induced, akin to
the canonical pho- regulon of E. coli, including genes for alternate P-source transport
(phnCDEE2 and pstSCABphoU), utilization (phnGHIJKLM) and other functions that are
presumably advantageous in Pi-limited conditions. Similar to pho-regulon expression
patterns of the phnCDE operon in E. coli (161), Pelagibacter sp. str. HTCC7211
upregulated the phnCDEE2 operon to a greater extent (20.3-fold mean across 4 genes)
than the pstSCABphoU operon (3.2-fold mean across 5 genes) when Pi -stressed (Figure
4-3, Tables A3-5 & A3-6 in appendix 3).
Although it is unclear if the stringent-like response observed in Ca. P. ubique is a
bona fide stringent response (i.e.- intracellular ppGpp accumulates), the absence of a
stringent response-like gene expression pattern in Pelagibacter sp. str. HTCC7211 was
unexpected given that nearly all of the differentially regulated genes in Ca. P. ubique are
present in similar genomic context in Pelagibacter sp. str. HTCC7211. In E. coli, the
stringent response alarmone, ppGpp, is kept in dynamic balance through synthesis and
degradation by the paralogous proteins RelA and SpoT, respectively (159, 162).
However, most bacteria only contain SpoT (including Pelagibacter) (163), which
catalyzes both the synthesis and degradation of ppGpp (159). The absence of a stringentlike response and phoB expression (which requires ppGpp (158)) in Pelagibacter sp. str.
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HTCC7211 suggests that intracellular ppGpp is not accumulating. SpoT hydrolysis of
ppGpp (to pp + Gpp) occurs in a HD-hydrolase domain of the SpoT protein (159, 164).
Interestingly, a gene predicted to encode a HD-hydrolase was significantly upregulated
under Pi-limitation in Pelagibacter sp. str. HTCC7211 (Figure 4-3, Tables A3-5 & A3-6
in appendix 3). We propose that Pelagibacter sp. str. HTCC7211 may have recruited this
HD-hydrolase to its pho-regulon to combat elevated ppGpp levels that would normally
trigger the stringent response, thus enabling cell proliferation under Pi-deficient
conditions (provided an alternate P source is available).
Consistent with the induction of the phn genes in Pelagibacter sp. str.
HTCC7211, inorganic phosphite and organophosphorus sources were utilized in place of
Pi (Figure 4-2). In E. coli, genetic evidence indicated that the phnCDE operon encodes for
a promiscuous ABC transporter that is able to transport phosphate-esters, phosphite, Pi
and phosphonates and that the phnGHIJKLMN gene products confer the ability to
metabolize phosphonates and phosphite for use as P-sources (165, 166). The phn operons
are part of the pho-regulon in E. coli and are induced during Pi-stress (152). When
Pelagibacter sp. str. HTCC7211 is starved for Pi, multiple phn genes are induced (Figure
4-3, Tables A3-5 & A3-6 in appendix 3) and this induction is presumably responsible for
the observed diauxic nature of growth when switching to growth on phosphonates,
phosphite, or phosphate-esters as primary P-sources (Figure A3-1 in appendix 3).
Although Pelagibacter sp. str. HTCC7211 utilized the phosphate-esters G6P, R5P and Pser for growth, the mechanism of utilization is unclear and might involve a novel
phosphatase. Pelagibacter sp. str. HTCC7211 does not encode for known APases (phoA,
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phoD and phoX homologs are absent) typically associated with phosphate-ester
metabolism (167). There are two differentially regulated genes that might exhibit
phosphatase activity and may explain the use of organic phosphates. The HD-hydrolase
previously described in the context of ppGpp metabolism may also have phosphatase
activity on phosphate esters (164). A second gene, divergently transcribed downstream of
phnE2 and annotated as a ‘hypothetical protein’ (PB7211_828; Figure 4-3, Tables A3-5
& A3-6), was upregulated 12 to 15-fold in Pi-deplete conditions. PB7211_828 is unique
only to Pelagibacter sp. str. HTCC7211 (a so-called ORFan (168)), and given its
genomic position adjacent to an organophosphate transporter (phnCDEE2) and strong
upregulation under Pi-deplete conditions, may be involved in phosphate-ester
metabolism.
The utilization of Mpn for P by Pelagibacter sp. str. HTCC7211 may have an
effect on atmospheric concentrations of the potent greenhouse gas methane (CH4). The
open ocean’s surface is supersaturated with CH4 relative to the atmosphere in a
phenomenon coined the ‘oceanic methane paradox.’ Aerobic metabolism of Mpn for use
as a P-source results in the stoichiometric production of methane in Pseudomonas
testosteroni (169) and has been linked to the phnCDE transport system and C-P lyase
activity in E. coli (165). CH4 is released from Mpn-amended surface seawater samples
containing native bacteria, showing that utilization of Mpn for P is a plausible source of
the observed CH4 supersaturation (170). Previously, Trichodesmium erythraeum IMS101
was shown to utilize phosphonates and produce CH4 from Mpn (126, 171). We show that
Pelagibacter sp. str. HTCC7211 used Mpn in place of Pi (Figures 4-1 & 4-2) and when
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doing so, released CH4 stoichiometrically (Figure 4-4). Published metagenomic analyses
of multiple oceanic environments show that the Pelagibacter-like phn gene clusters are
not universally distributed, but instead are more common in Pi-deplete waters (143, 144).
Recently, a novel Mpn biosynthetic pathway was discovered and characterized in
Nitrosopumilus maritimus, a member of the abundant Marine Group I ammoniaoxidizing archaea (172). Marine group I archaea are ubiquitous, have a large global
population size and in marine ecosystems, can account for up to 30% of all cells below
the ocean’s thermocline (18, 173), suggesting that archaeal-derived Mpn may be an
important phosphorus source in the sea. Given the abundance of Pelagibacter in Pi and
nutrient deplete waters such as the Sargasso Sea, Pelagibacter sp. str. HTCC7211-like
cells may play a central role in the metabolism of archaeal-derived Mpn and the release
of aerobically produced CH4 from Pi-deplete ocean systems.
In addition to releasing CH4, the physiology of Pelagibacter sp. str. HTCC7211
changed when it was grown on Mpn in ways that implied Mpn was utilized more
efficiently than Pi. For example, the apparent cellular P-demand for Pelagibacter sp. str.
HTCC7211 decreased from 45.7 amol cell-1 (when grown on Pi) to 11.4 amol cell-1 when
cells were grown on Mpn (calculated from the regression lines in Figure 4-1). This
implies that Pelagibacter sp. str. HTCC7211 may decrease their cellular P-requirement
when growing on Mpn. Some P-starved bacteria reduce their cellular P-requirement in
low- Pi environments by replacing the P-containing polar head groups on lipids with nonP containing moieties such as sulphoquinovosyldiacylglycerol (SQDG) (134), glycolipids
(174) or amino acid lipids (175).
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Previous metabolic reconstructions did not find support for the hypothesis that
Pelagibacter sp. str. HTCC7211 can modify their lipid polar head groups; canonical
genes encoding proteins used in the synthesis of P-free betaine lipids, SQDG-lipids or
glycolipids were absent. However, a four-gene cluster containing two genes annotated as
putative hemolysins, a probable glycosyltransferase and a calcineurin-like
metallophosphoesterase, were differentially regulated under Pi-stress in Pelagibacter sp.
str. HTCC7211 and might be involved in lipid polar head group remodeling (Figure 4-3;
Tables A3-5 & A3-6 in appendix 3). The first step of lipid remodeling in the αproteobacteria Sinorhizobium meliloti, is to cleave phosphatidylcholine and
phosphatidylethanolamine from phospholipids with phospholipase C (PlcP) to form
diacylglycerol plus phosphocholine or phosphoethanolamine, respectively (137). A
search of the Pelagibacter sp. str. HTCC7211 genome for homologs to S. meliloti’s PlcP,
revealed that the calcineurin-like metallophosphoesterase gene that was upregulated
under Pi-stress had a high percent identity (118/251 identities; blastp e-value: 2 × 10-87),
suggestive of similar function. Immediately downstream of the putative plcP gene in a
probable operon, is a group 1 glycosyltransferase (COG438; rfaG). Glycosyltransferases
transfer saccharide groups from nucleotide sugars to a variety of acceptor molecules. For
example, during the synthesis of SQDG-lipids, a glycosyltransferase is responsible for
the transfer of a modified saccharide (sulphoquinovose) to diacylglycerol (176). Given
the genomic context of the Pelagibacter sp. str. HTCC7211 glycosyltransferase adjacent
to the probable phospholipase C gene, we predict that it may be involved in the
glycosylation of the diacylglycerol resulting from PlcP-mediated cleavage of
86
phospholipids. A putative alginate lyase is also upregulated during Pi stress (Table A3-6
in appendix 3), hinting that the saccharide used by this glycosyltransferase may be an
alginate derivative. Two divergently transcribed genes, immediately downstream of the
glycosyltransferase, annotated as putative hemolysins, were also upregulated during Pi
stress (Figure 4-3). These genes belong to the same cluster of orthologous groups (COG)
as homologs to OlsB from S. meliloti (COG3176), which catalyze the first step in the
synthesis of Pi-free ornithine lipids in Pi-stressed cultures (175, 177). We propose that
this four-gene cluster acts to remodel the lipid composition of Pelagibacter sp. str.
HTCC7211 cells during Pi-stress to replace phosphorus-containing polar head groups
with saccharides and/or ornithine (or other amino acids) in order to reduce the cellular Prequirement (Figure A3-5 in appendix 3). Previously, it was estimated that Pelagibacter
cells grown in Pi-replete conditions contain 2.5 amoles P cell-1 in their lipids (134), thus
replacement of P-lipids with non-P lipids may save up to 25% of their total P-requirement
(assuming 10-11 amol P cell-1).
Polyphosphate is a linear polymer of Pi-residues that has been associated with
many cellular activities including P-storage, stationary-phase survival, pathogenicity and
DNA uptake (reviewed in (151)). The precise physiological role of polyphosphate in
marine bacterioplankton is unclear, however metagenomic analysis of polyphosphate
genes in the GOS database suggests that polyphosphate may be important to microbial
survival in low-P environments (143, 145). When Pelagibacter sp. str. HTCC7211 cells
previously grown with Mpn as a sole P-source were transferred into P-free medium, cell
yields were 30 times higher than cells previously grown on Pi, suggestive of potential P
87
stores in the Mpn-grown cells. Indeed, electron-dense granules characteristic of
polyphosphate were observed in Mpn-grown cells (Figure A3-6 in appendix 3) (147).
Assuming insignificant external sources or carryover of P, the internal stores of
phosphate in the cells used to inoculate the flasks was calculated to be ~660 amol P cell-1.
Although the polyphosphate synthesis and utilization genes (ppx and ppk, respectively)
were not differentially expressed under Pi-limiting conditions, we did not explore gene
regulation when growing on organic P-sources. Synthesis of polyphosphate from organic
P-sources has not been intensely investigated, although growth experiments in the
diazotroph Trichodesmium IMS101 suggested that P from P-esters could be used to form
polyphosphate (178).
We undertook these experiments to gain an understanding of how the genetic
differences related to P metabolism are manifested physiologically in two similar
organisms and how Pelagibacter may participate in oceanic P-cycling. Our results show
that despite the high degree of phylogenetic similarity between Ca. P. ubique and
Pelagibacter sp. str. HTCC7211 by both 16S ribosomal sequence comparisons (22) and
whole genome analysis (29, 179), non-conserved strategies resulting in distinct Putilization physiotypes were evident. Ca. P. ubique exhibits a physiotype that is
characterized by primary reliance on Pi for P nutrition. This interpretation is highlighted
by the observation that Ca. P. ubique does not use organic P-esters or phosphonates
effectively (Figure 4-2). Further support for this interpretation can be linked to gene
expression changes under Pi-limiting conditions that show a rapid and strong
upregulation of the high-affinity Pi transport system followed by expression patterns
88
indicative of a stringent-like stress response, suggesting that Pi-depletion is recognized as
a global stress inducer (Figure 4-3, Tables A3-2 – A3-4 in appendix 3). In contrast to this,
Pelagibacter sp. str. HTCC7211’s physiotype is characterized by the effective and broad
use of organic phosphorus sources, including phosphonates, when Pi is limiting (Figures
4-1 & 4-2). Gene expression differences support this interpretation; during Pi-starvation,
genes encoding proteins conferring the use of alternate P-sources showed the greatest
degree of induction (Figure 4-3, Tables A3-5 & A3-6 in appendix 3). Differential
regulation of genes involved in the stringent response in Pelagibacter sp. str. HTCC7211
was absent.
The different physiotypes we describe probably reflect cellular adaptations
pertinent to the environments from which each organism was isolated. For example, Ca.
P. ubique was isolated from the northeast Pacific Ocean off the coast of Oregon, USA
(23) where Pi concentrations are usually sufficient to support the native plankton
populations and rarely drop below 100 nM. The high-affinity Pi transporter encoded by
Ca. P. ubique may be transiently advantageous in situations where modulation of Piuptake increases competitiveness, possibly due to Pi-depletion by other plankton.
However, genome sequences of multiple Pelagibacter isolates from the northeast Pacific
Ocean show that the high affinity Pi transport system is not required for environmental
persistence or for growth in vitro (23). Although we are able to grow one such isolate in
AMS1, (‘Ca. Pelagibacter ubique’ str. HTCC1002) the organic growth requirements have
not been optimized to the extent required to practically conduct the in vitro experiments
described here. Interestingly, all of the Pelagibacter genomes lacking a pstSCAB-phoUB
89
operon contain homologs to the Ca. P. ubique gene SAR11_1181 (Figure 4-3).
Metagenomic analyses show that Pelagibacter genes homologous to SAR11_1181 are
enriched in the Pacific Ocean compared to the Atlantic Ocean (143). Analysis of the
SAR11_1181 protein sequence with the RaptorX protein structure prediction web server
(180) revealed a domain with distant similarity (2.70 × 10-6 across amino acid residues
21-273) to the PHO4-family of Pi-cation symporters in Neurospora crassa (181). We
observed significant upregulation of SAR11_1181 in Pi-deplete conditions, which is also
suggestive of a role in Pi-acquisition (Figure 4-3; Tables A3-3 & A3-4 in appendix 3).
We suspect that SAR11_1181 encodes a Pi-symporter that may co-transport cations with
Pi, similar to the PitA transporter in E. coli (182) and may explain the absence of
identifiable Pi transporters in multiple Pelagibacter genomes (29).
Organisms that thrive in the Sargasso Sea should be adapted to low Pi-levels and
high concentrations of dissolved organic-P (DOP) and arsenate. The results presented
here suggest that Pelagibacter sp. str. HTCC7211 is adapted to life in the Sargasso Sea.
Pelagibacter sp. str. HTCC7211 upregulated genes required for DOP utilization (Figure
4-3, Tables A3-5 & A3-6 in appendix 3) and effectively used DOP in place of Pi (Figure
4-2). DOP is more abundant than Pi in many oligotrophic environments, including the
Sargasso Sea (122, 183-185), thus our in vitro experimental results showing efficient use
of DOP are compatible with the relative availability of Pi and DOP in Pelagibacter sp.
str. HTCC7211’s native habitat. In the surface waters of the Sargasso Sea, arsenic
concentrations often exceed those of Pi (186). Arsenate is a Pi-analog and commonly
enters cells through Pi-transport systems. During Pi-limitation, we observed upregulation
90
of a two-gene operon consisting of genes encoding an arsenate reductase (ArsC) and a
glpF-like aquaglyceroporin (AqpS). In S. meliloti, ArsC and AqpS were shown to
facilitate arsenate reduction to arsenite (ArsC) and passive efflux from the cell (AqpS) as
a mechanism of arsenate detoxification (187). We suspect that these genes function
similarly in Pelagibacter sp. str. HTCC7211 and are induced to combat elevated levels of
arsenate transported into the cell by the promiscuous phnCDEE2 transporter in low Pi
environments.
Previous metaproteomic analysis of peptides isolated from native Sargasso Sea
bacterioplankton identified a number of peptides that mapped to genes that were
differentially expressed in our studies, implying that our in vitro experimental results
have relevance in situ. For example, peptide spectra from the Sargasso Sea mapped to
translations of Pelagibacter sp. str. HTCC7211’s pstS, pstA, pstB, phoU, phnC, phnD,
phnE, PB7211_828 and PB7211_757 genes and other genes that were differentially
expressed in Ca. P. ubique (104). Although the composition of DOP in the environment
has not been fully chemically defined, we show that Pelagibacter sp. str. HTCC7211
efficiently used a broad range of organic-P compounds for P-nutrition. Previously, it was
suggested that the range of phosphonate compounds that can be utilized by E. coli was
limited by the specificity of transport by the phnCDE transporter not the activity C-P
lyase (188, 189). If true, P-utilization niche specificity in Pelagibacter, and other
organisms encoding the phnCDE transporter, might vary depending on the evolution of
transport specificity of the phnCDE transporter.
91
The term ‘ecotype’ is commonly used to describe groups of closely related
bacteria within which ecologically distinct populations are observed. Separate ecotypes
are assumed to differ in traits conferring niche specificity. Previously, the presence of Putilization gene suites was described to be incongruent with phylogenetic placement in
Prochlorococcus ecotypes (125, 143). Physiological assessments of P-utilization patterns
in Prochlorococcus isolates also showed that different P-utilization physiotypes were
evident (128, 190).
The Ia-subclade of the Pelagibacteraceae has been described as an ecotype based
on 16S rRNA sequence analysis and tRFLP patterns that clearly show reproducible and
coherent spatiotemporal distributions in the Sargasso Sea (191, 192). The data we present
here show that within the SAR11 Ia-subclade ecotype there is substantial physiological
and transcriptional variation related to P-utilization. Although we describe two different
physiotypes, it is not clear how many different P-utilization physiotypes exist in the
environment and whether the diversification of P-utilization physiotypes is adaptive or
neutral. Metagenomic analyses of the Sargasso Sea show higher abundances of pstSCABphoU genes relative to C-P lyase and phnCDEE2 genes, suggesting that not all
Pelagibacter cells in the Sargasso Sea contain the extended pho-regulon that we describe
here and implying that different P-utilization physiotypes co-exist in nature (143, 144).
Analysis of multiple Pelagibacter genomes suggests that there are additional P-utilization
physiotypes that may vary in the scope of phosphonate utilization (29). Thus, further
characterization of Pelagibacter P-utilization physiotypes may unveil interesting and
92
geochemically relevant findings pertinent to the global P cycle and help to further refine
the concept of an ecotype.
93
Pelagibacter sp. str. HTCC7211 + Mpn
Pelagibacter sp. str. HTCC7211 + Pi
Ca. P. ubique HTCC1062 + Pi
y=87,542x + 1,980,400; R2=0.995
y=21,900x + 7,883; R2=0.991
y=97,669x + 365,626; R2=0.999
maximum cell density (× 106 cells ml-1)
12
10
8
6
4
2
0
0
10
20 30 40 50 60 70 80 90 100
concentration of P-source (nmol L-1)
Figure 4-1: Linear dose responses of Ca. P. ubique and Pelagibacter sp. str. HTCC7211
to P-sources. Each point is the mean of the maximum density achieved for biological
replicates ± 1.0 s.d. (n=3). Linear regression through P-source additions of 1.0 nM,
3.3 nM, 10 nM, 33.3 nM and 100 nM are shown. Abbreviations: Mpn –
methylphosphonic acid; Pi – inorganic phosphate. Full growth curves are presented in
Figures A3-2 - A3-4 in appendix 3.
94
Ca. P. ubique HTCC1062
Pelagibacter sp. str HTCC7211
4
3
2
1
hi
te
co
se
-5
R
P
ib
os
e5P
M
pn
2AE
P- P
se
rin
+P
e
i (p
os
ct
r)
TP
G
lu
os
p
TP
dG
TP
dA
TP
dC
Ph
i
-P
(n
eg
ct
r)
0
dT
max cell density (× 107 cells ml-1)
5
Figure 4-2: P-source utilization by Ca. P. ubique and Pelagibacter sp. str. HTCC7211.
Bar height is the mean of the maximum density achieved for biological replicates ± 1.0
s.d. (n=3). denotes diauxic growth pattern (diauxy shown in Figure A3-1 in appendix
3). Growth medium was not amended with Pi in the negative control. All other treatments
had 1.0 µM of potential P source added.
95
A Ca. Pelagibacter ubique HTCC1062
ftsZ
mraZ ipbA
trmD rpsP
umuD
recA
lexA 0964 0965
rpoA
rpsK
rpsS
rplB
fusA
1164
4h
20 h
38 h
4h
20 h
38 h
phoB phoU
pstB
pstA
pstC
phoR
pstS
-4.0
1181
high-affinty phosphate transport
B Pelagibacter sp. str. HTCC7211
lipid modification
LCB5
plsC
rfaG
hemolysins
phoR
high-affinty Pi transport
78
pstS
pstC
pstA
pstB
-1.5 1.5
>30.0
amount regulated (fold)
poly-P metabolism
phoU phoB sixA
ppk
ppx
NUDIX 913
72 h
96 h
72 h
96 h
phnX
aqpS arsC HD-hyd
araJ
828
phnE2
phnE
phnD
phnC
organic-P transport
phnF
G phnH
phnI
phnJ
phnK
phnL phnN
764
phnM
1178
C-P lyase
Figure 4-3: Comparative genomics of gene expression during Pi-starvation in Ca. P.
ubique and Pelagibacter sp. str. HTCC7211. Time elapsed since resuspension is listed to
the left of each gene row. A) Select genes differentially regulated in Ca. P. ubique. Gene
names, annotations and actual expression values are listed in Tables A3-2 – A3-4 in
appendix 3. B) Select genes differentially regulated in Pelagibacter sp. str. HTCC7211.
Gene names, annotations and actual expression values are listed in Tables A3-5 & A3-6
in appendix 3. Vertical black lines represent a lack of genomic synteny. Genes shaded
grey are not differentially expressed.
96
1.6
+ Pi
+ Mpn
Predicted CH4
B
- Pi
- Mpn
- No Pi
total CH4 evolved (μmol L-1)
1.4
A
1.2
1.0
0.8
0.6
0.4
0.2
0
cell density (cells ml-1 × 107)
8.0
6.0
4.0
2.0
0
0
5
10
15 20 25
time (days)
30
35
40
Figure 4-4: Methane (CH4) production by Pelagibacter sp. str. HTCC7211 utilizing Mpn
as a sole P-source. A) CH4 production by Pelagibacter sp. str. HTCC7211 cultures grown
with Mpn (open triangles) or Pi (open circles). Open circles and triangles are the mean
amount of CH4 measured from biological replicates ± 1.0 s.d. (n=3). Theoretical CH4
production based on cell densities in (B) assuming 11.4 amol Mpn cell-1 (inverted filled
triangles and dashed line). B) Cell densities from single bottles sacrificed for cell density
measurements for Pelagibacter sp. str. HTCC7211 cultures grown with no Pi (×), Mpn
(filled triangles) or Pi (filled circles).
97
Chapter 5
Analysis of the Vitamin B5, B6, B7 And B12 Requirements of ‘Candidatus
Pelagibacter ubique’ HTCC1062: Evidence for B7 Auxotrophy.
Paul Carini1, Emily O. Campbell1, Sergio Sañudo-Wilhelmy2 and Stephen Giovannoni1
1
2
Department of Microbiology, Oregon State University Corvallis, OR 97331
Department of Biological Sciences, Marine Environmental Biology, and Earth Science,
University of Southern California, Los Angeles, CA 90089
98
Abstract
‘Candidatus Pelagibacter ubique’ strain HTCC1062 is a member of the globally
distributed and ecologically important SAR11 clade of marine bacteria. Previously, in
metabolic reconstructions of the Ca. P. ubique genome it was noted that the biosynthetic
pathways for the essential B-vitamins pantothenate (B5), pyridoxine (B6), biotin (B7) and
cobalamin (B12) were absent or incomplete. Despite the absence of genes encoding
proteins required in these biosynthetic pathways, genes coding for proteins that utilize
vitamin B5 (or its derivative, coenzyme A), B6 and B7 were identified in the Ca. P. ubique
genome, suggesting that these vitamins are required for normal cellular metabolism.
Although four of the five genes encoding proteins required for vitamin B7 synthesis are
absent, a probable transporter for B7 was identified, hinting that Ca. P. ubique obtains B7
from the environment. We confirmed this experimentally by limiting the growth of Ca. P.
ubique by excluding vitamin B7 in a defined growth medium and showing that picomolar
additions of vitamin B7 restore cell yields. Because most, but not all, enzymes required
for the biosynthesis of vitamin B5 were identified, we hypothesized that Ca. P. ubique
was either auxotrophic for vitamin B5 or utilized novel enzymatic reactions to complete
B5 synthesis. To test the hypothesis that Ca. P. ubique is auxotrophic for vitamin B5, we
attempted to limit the growth of Ca. P. ubique cultures by excluding vitamin B5. We were
unable to limit Ca. P. ubique growth with vitamin B5, however, we observed a significant
growth rate reduction in the absence of vitamin B5. These results are consistent with the
conclusion that Ca. P. ubique is bradytrophic for vitamin B5, suggesting synthesis is
possible at the expense of more rapid growth. Re-analysis of the Ca. P. ubique genome
99
shows that an alternate vitamin B5 pathway is likely present. Recent work in Escherichia
coli uncovered a novel biosynthetic route for de novo vitamin B6 synthesis from the
glycolytic/gluconeogenic intermediate 3-phosphoglycerate. Re-analysis of the Ca. P.
ubique genome identified this new vitamin B6 biosynthetic pathway, suggesting de novo
synthesis of vitamin B6 is possible. Although only two of the 18+ genes required for
vitamin B12 synthesis were identified in the Ca. P. ubique genome, no genes encoding
B12-dependent enzymes were identified, implying this vitamin is neither produced nor
used by Ca. P. ubique. We propose that reactions commonly catalyzed by B12-dependent
enzymes are instead performed by B12-independent analogs encoded within the Ca. P.
ubique genome. These studies are an important step in understanding the unusual
organization of metabolic pathways that result in the unique oligotrophic physiology of
Ca. P. ubique. Additionally, the discovery of vitamin B7 auxotrophy suggests that
environmental distributions of vitamin B7 have the potential to affect SAR11 population
sizes.
Introduction
Patterns of B-vitamin production and consumption in nature are poorly
understood, as are the metabolic ramifications of vitamin deficiency, at both the cellular
and ecosystem levels. Traditionally, measurements of vitamins in the sea are conducted
using bioassay techniques that are relatively insensitive and subject to errors caused by
organismal responses to vitamin analogs or degradation products, in addition to the intact
B-vitamins themselves (193-195). Sensitive and accurate direct measurements of Bvitamins extracted from seawater have been determined by high performance liquid
100
chromatography (HPLC) coupled to either a UV-vis detector (196, 197) or tandem mass
spectrometer (81). These measurements showed that some B-vitamins have complex
distribution patterns in the sea. An understanding of the vitamin requirements and
vitamin production by microbes in the environment is necessary to contextualize the
unusual distribution patterns (84).
SAR11 chemoheterotrophic marine bacteria are found worldwide and make up a
large percentage of cells in marine photic zones (17), where they are important in the
cycling of carbon and sulfur (26, 28, 32, 37, 88). ‘Candidatus Pelagibacter ubique’ str.
HTCC1062 (Ca. P. ubique) was the first member of the SAR11 clade to have its
complete genome sequenced (25). Metabolic reconstruction of the Ca. P. ubique genome
found that B-vitamin synthesis pathways for B5, B6, B7 and B12 are incomplete. Despite
the incompleteness of these pathways, genes coding for proteins that require B5, B6 and
B7 were identified, suggesting that these vitamins are necessary for normal metabolism in
Ca. P. ubique (Figure 5-1). Knowledge of the vitamin requirements of Ca. P. ubique is
potentially important to help understand SAR11 population sizes in nature, which may be
controlled by vitamin supply, and for further refinement of current models of Ca. P.
ubique metabolism.
Biosynthetic pathways of vitamin synthesis can be difficult to predict from in
silico metabolic models derived from genome sequences. Most knowledge of vitamin
synthesis pathways is based on data from a small number of model organisms and
divergence from these canonical synthesis pathways at one or multiple reaction steps is
not uncommon (198). Vitamin biosynthetic pathways can be complex (for example
101
vitamin B12; see Figure 5-5) and often utilize unusual and distinct chemistries during their
synthesis (198). Enzymes involved in vitamin biosynthesis may have multiple cellular
functions, including roles in other metabolic pathways, multiple roles in the synthesis of a
single vitamin, or in the synthesis of other vitamins, and in the case of vitamin B1, the
synthesis of itself (198).
Vitamin B5 biosynthesis
Vitamin B5 (pantothenate) is required for the synthesis of coenzyme A, an acyl
carrier that, among other reactions, transfers pyruvate-derived carbon to the tricarboxylic
acid cycle, where it can be oxidized for energy. Vitamin B5 is also the precursor to
phosphopantetheine, a prosthetic group on acyl carrier proteins used in the synthesis of
fatty acids. Vitamin B5 is formed from the linkage of pantoate and β-alanine. Pantoate is
formed from two pyruvate molecules in five enzymatic steps, four of which are shared
with the initial stages of valine and leucine biosynthesis. The divergence between these
pathways occurs at the metabolic intermediate 2-oxioisovalerate (Figure 5-2), thus the
first committed step in pantoate biosynthesis is the hydroxymethylation of 2oxioisovalerate to dehydropantoate (199). Dehydropantoate is reduced to pantoate via
PanE in E. coli, however some organisms utilize IlvC to exclusively perform this reaction
(200, 201). In E. coli, β-alanine is formed the decarboxylation of aspartate by PanD
(202). In other organisms, β-alanine can be derived from a number of compounds
including spermine (203), propionate (204) and uracil (205).
102
Vitamin B6 biosynthesis
Vitamin B6 (pyridoxine) is an important cofactor in amino acid aminotransferase
reactions, but is also used by some amino acid racemases and sulfinate desulfinases.
Vitamin B6 is synthesized by joining deoxy-xylulose-5-phosphate (dXP) with 3-amino-2oxypropyl-phosphate (Figure 5-3). dXP is derived from pyruvate and glyceraldehyde5’phosphate by the deoxyxylulose-phosphate synthase (Dxp). 3-amino-2-oxypropylphosphate is derived from O-phospho-4-hydroxythreonine (4HT-P). In the canonical
vitamin B6 biosynthetic pathway in E. coli, 4HT-P is formed from D-erythrose-4phosphate, a pentose pathway intermediate (Figure 5-3), however in some pyridoxine
synthesis mutants other compounds can substitute (206-208).
Vitamin B7 biosynthesis
Vitamin B7 (biotin) is a sulfur-containing coenzyme that is universally required
for the carboxylation of acetyl-CoA to malonyl-CoA in the first committed step of fatty
acid biosynthesis and other (de)carboxylation reactions. Canonical microbial synthesis of
vitamin B7 starts with pimeloyl-CoA and proceeds through four enzymatic reactions that
require inputs of alanine, S-adenosylmethionine and ATP (reviewed in (209)) (Figure 54). In bacteria, the genes involved in vitamin B7 biosynthesis are co-transcribed in an
operon that is under the control of the bi-functional BirA protein. BirA activates vitamin
B7 with ATP to form biotinyl-5’-adenylate and catalyzes the addition of vitamin B7 to
biotinylated proteins (e.g. - the acetyl-CoA carboxylase). When levels of biotinyl-5’adenylate are elevated, BirA binds biotinyl-5’-adenylate and acts as a repressor of
vitamin B7 synthesis (210-215).
103
Vitamin B12 biosynthesis
Vitamin B12 (cobalamin) is a soluble organic molecule containing a single cobalt
ion chelated within a tetrapyrrole structure. Vitamin B12 can be a part of many cellular
processes, including methionine biosynthesis, ribonucleotide reduction and some
methyltransferase and isomerase (mutase) reactions (216). The de novo biosynthesis of
vitamin B12 in bacteria involves ~20 enzymes to convert the heme biosynthetic
intermediate uroporphyrogen –III (uro’-III) to the active vitamin (217-220) (Figure 5-5).
In short, the corrin ring is initiated by the methylation of uro’-III to precorrin-2. There are
two paths by which vitamin B12 corrin rings can be biosynthesized from precorrin-2: the
oxygen-dependent (aerobic) and oxygen independent (anaerobic) pathways. In the
aerobic pathway, corrin rings are formed prior to the insertion of cobalt to form
cob(II)yrinic acid a,c diamide (Figure 5-5). In the anaerobic pathway, cobalt is initially
inserted into the precorrin-2 molecule and the corrin rings are formed subsequently to
form cob(II)yrinic acid a,c diamide (Figure 5-5). After the formation of cob(II)yrinic acid
a,c diamide, the aerobic and anaerobic pathways share the same biosynthetic processes to
the form the active vitamin (Figure 5-5).
B-vitamin biosynthesis in Pelagibacter
Metabolic reconstruction of the Ca. P. ubique genome found that the biosynthetic
machinery for de novo vitamin B5, B6, B7 and B12 synthesis was incomplete (25). We
investigated these predicted B-vitamin deficiencies by using a combination of metabolic
reconstruction and growth experiments on a defined artificial medium. We show that Ca.
P. ubique is auxotrophic for vitamin B7 and bradytrophic for vitamin B5. We identified a
104
probable vitamin B7 transporter and propose a complete biosynthetic pathway for B5. We
identified a recently characterized (in E. coli) vitamin B6 biosynthetic pathway, and did
not observe growth defects upon vitamin B6 exclusion. Finally, we discuss the lack of
vitamin B12 metabolism and identify B12-independent enzymes that probably catalyze
reactions that usually employ vitamin B12.
Methods
Organism source
Ca. P. ubique HTCC1062 was revived from 10% glycerol stocks and propagated
in artificial medium for SAR11 (AMS1) (89) amended with oxaloacetate (1 mM), glycine
(50 µM), methionine (50 µM) and FeCl3 (1 µM). B-vitamins were added as indicated in
figure legends and text.
Chemicals
All AMS1 constituents, reagents and vitamins were of the highest available
quality (labeled ‘ultrapure’ when possible). Nutrient and vitamin stocks were prepared in
combusted glassware (450°C for 4 h) with nanopure water, 0.1 µm-filter-sterilized and
frozen in amber tubes immediately after preparation. Reasonable precautions were taken
to limit the number of freeze-thaw cycles and light exposure of each reagent.
Cultivation details
All cultures were grown in acid-washed and autoclaved polycarbonate flasks and
incubated at 20°C with shaking at 60 RPM in the dark. Cells for counts were stained with
105
SYBR green I and counted with a Guava Technologies flow cytometer at 48-72 h
intervals as described elsewhere (26).
Vitamin-limitation procedure
To dilute out the vitamins routinely added to SAR11 cultures, Ca. P. ubique was
transferred to AMS1 amended with nutrients, but without B-vitamins, a minimum of 10
times (>1000 generations). After three passages (>30 generations) truncated cell growth
was consistently observed (104 cells ml-1 to 107 cells ml-1. For example, see Figure 5-6).
This truncation was attributed to thiamine-limitation in chapter 3.
Trace vitamin measurements
Vitamins were measured in un-inoculated medium using the solid-phase
extraction plus HPLC tandem mass spectrometry method described in (81).
Vitamin B7-scrubbing
Trace contaminants of vitamin B7 were removed from growth medium by adding
streptavidin-coated magnetic beads (0.5 µL beads L-1; rinsed 3X with Tris-EDTA) (New
England BioLabs, Ipswich, MA) and incubating at 16°C for 24 hours with gentle
shaking. After incubation, beads were centrifuged out of the medium and sequestered at
the bottom of the centrifuge tube with a magnet while the supernatant (the scrubbed
growth medium) was transferred into a new container for use in experiments.
106
Results
Metabolic reconstruction of vitamin B5 biosynthesis
Genes encoding homologs to all but one gene (PanE) used for the synthesis of
pantoate in E. coli were identified in the Ca. P. ubique genome (Figure 5-2). Specifically,
genes encoding homologs for the i) acetolactate synthase complex (IlvH – small subunit
& IlvB – large subunit); ii) ketol-acid reductoisomerase (IlvC); dihydroxy-acid
dehydratase (IlvD) and 3-methyl-2-oxobutanoate hydroxymethyltransferase (PanB) were
identified (Figure 5-2). Although homologs to PanE were not identified in Ca. P. ubique,
IlvC has been shown to have a promiscuous activity that can replace the function of PanE
(200, 201). Ca. P. ubique encodes for IlvC, therefore we propose that IlvC is functional
ketopantoate reductase. In E. coli, β -alanine formation results from PanD-catalyzed
decarboxylation of aspartate. Ca. P. ubique lacks PanD homologs, thus the vitamin B5
biosynthetic pathway appears to be incomplete because of a deficiency in β-alanine
synthesis (Figure 5-2).
Metabolic reconstruction of vitamin B6 biosynthesis
Ca. P. ubique is missing the canonical route for 4HT-P synthesis via the pentosephosphate pathway, intermediate D-erythrose-4-phosphate (by action of the D-erythrose4-phosphate dehydrogenase (Epd), erythronate-4-phosphate dehydrogenase (PdxB) and
phosphoserine aminotransferase (SerC); Figure 5-3). A recently discovered (in E. coli
(206)) alternate 4HT-P synthesis pathway from glycolaldehyde and glycine appears to be
present in Ca. P. ubique. Enzymes involved in the alternate pathway include the L-
107
threonine aldolase (LtaE) and homoserine kinase (ThrB) (Figure 5-3). In E. coli,
glycolaldehyde formation may have multiple routes, including one starting from 3phosphoglycerate (Figure 5-3) (206). Ca. P. ubique is missing a gene encoding YeaB, a
protein to derive glycolaldehyde from 3-phosphoglycerate, but encodes probable
homologs for LtaE and ThrB (Figure 5-3).
Ca. P. ubique encodes for the remaining proteins necessary to form vitamin B6
from 4HT-P. Genes encoding homologs for the 4HT-P dehydrogenase (PdxA);
pyridozine-5-phosphate synthase (PdxJ) and the pyridoxamine-5-phosphate oxidase
(PdxH) were identified (Figure 5-3).
Metabolic reconstruction of vitamin B7 biosynthesis
Ca. P. ubique encodes for only one of the five genes involved in vitamin B7
biosynthesis in E. coli: the adenosylmethionine-8-amino-7-oxononanoate transaminase
(BioA). The remaining genes encoding required vitamin B7 biosynthetic proteins in E.
coli – i) biotin synthase (BioB); ii) the predicted methyltransferase of biotin synthesis
(BioC); iii) dethiobiotin synthase (BioD); and iv) 8-amino-7-oxononanoate synthase
(BioF) were not identified in Ca. P. ubique (Figure 5-4). A gene encoding a probable
vitamin B7 transporter (bioY, SAR11_0202) was identified in Ca. P. ubique (Figure 5-4).
Metabolic reconstruction of vitamin B12 biosynthesis
Only two of the more than 18 genes that encode proteins required for vitamin B12
synthesis were identified in the Ca. P. ubique genome - cobS and cobT, encoding cobalt
chelatase subunits (Figure 5-5). No genes predicted to encode vitamin B12 transporters
108
were identified in the Ca. P. ubique genome. Further investigation of the Ca. P. ubique
genome did not find any genes coding for enzymes predicted to require vitamin B12 as a
cofactor (Table 5-1). In some instances, we identified vitamin B12-independent isozymes
that may perform equivalent functions in vivo (Table 5-1).
Measurements of B-vitamins in growth medium
To determine if there were trace B-vitamins in our ‘vitamin-free’ growth medium
we measured the B-vitamin concentrations of vitamins B6, B7 and B12 using the method
described in (81) (no assay for B5 in seawater has been developed). There were
contaminating amounts of vitamin B6 (30.921 ± 1.817 pM; average ± s.d., n=3) and B7
(96.535 ± 10.08 pM; average ± s.d., n=3). Vitamin B12 was not detected (limit of
detection 0.18 pM (81)).
Growth responses to vitamin exclusions
When 1.0 µM each of vitamin B5 and B7 were added to the growth medium,
maximal cell densities of 1.22 ± 0.51 × 107 cells ml-1 (herein presented as mean ± s.d.,
n=3) were attained and cells grew at a specific growth rate of 0.52 ± 0.01 day-1 (herein
presented as mean ± s.d., n=3) (Figure 5-6). When 1.0 µM of vitamin B7 alone was added
to the growth medium, maximal cell densities of 1.06 ± 0.52 × 107 cells ml-1 were
attained and cells grew at a specific growth rate of 0.34 ± 0.03 day-1 (Figure 5-6). When
1.0 µM of vitamin B5 alone was added to the growth medium, maximal cell densities of
4.75 ± 1.25 × 106 cells ml-1 were attained and cells grew at a specific growth rate of 0.37
± 0.04 day-1 (Figure 5-6). When no B-vitamins were added to the growth medium,
109
maximal cell densities of 3.59 ± 1.15 × 106 cells ml-1 were attained and cells grew at a
specific growth rate of 0.39 ± 0.01 day-1 (Figure 5-6).
Biotin limitation of Ca. P. ubique
To reliably demonstrate a vitamin B7 requirement, it was necessary to scrub
contaminating vitamin B7 from the growth medium (see methods for details). When
vitamin B7 was added back to the scrubbed medium, growth resumed, indicating that the
scrubbing procedure was not detrimental to the growth of Ca. P. ubique (Figure 5-7a).
When vitamin B7 was excluded from vitamin B7-scrubbed medium amended with organic
carbon, sulfur and thiamine, cell yields of 1.87 × 106 ± 0.07 cells ml-1 were attained.
When the media was amended with 1 µM vitamin B7, cell yields increased to
2.64 ± 0.17 × 108 cells ml-1 (mean ± s.d., n=3) (Figure 5-7b). Cell yields responded
incrementally to picomolar additions of vitamin B7 to the scrubbed medium (Figure 57b).
Identification of vitamin B12-requiring enzymes and B12-independent analogs
No genes encoding vitamin B12-requiring enzymes were identified in the Ca. P.
ubique genome (Table 5-1). Instead, we identified genes encoding vitamin B12independent enzymes that catalyze reactions that are typically vitamin B12-dependent
(Table 5-1).
Conclusions
The data presented here are one of the final steps in the elucidation of the organic
nutrient requirements of Ca. P. ubique, and help resolve the last of the metabolic
110
anomalies first presented with the Ca. P. ubique genome publication in 2005 (25). In
addition to the unusual requirement for the thiamine precursor HMP presented in chapter
3, we show that Ca. P. ubique has an absolute requirement for vitamin B7 (Figure 5-7a,b).
When vitamin B7 is present, addition of vitamin B5 increases growth rate (Figure 5-6),
suggesting that Ca. P. ubique is bradytrophic for vitamin B5 in a vitamin B7-dependent
manner. We have identified the probable transporter for vitamin B7 (bioY; Figure 5-4). In
Rhodobacter capsulatus, BioY was shown to be a high capacity biotin transporter (221).
Biosynthesis of vitamin B5 appears to be dependent on a source of vitamin B7.
When vitamin B5 is added together with vitamin B7, growth rates nearly double (Figure
5-6), suggesting that vitamin B7 availability may play a role in vitamin B5 synthesis. We
propose that the synthesis of β-alanine is dependent on malonyl-CoA, which requires
vitamin B7 for synthesis (Figures 5-1 & 5-2). MmsA is a methylmalonate-semialdehyde
dehydrogenase that can act on malonyl-CoA together with NADPH and H+, to form 3oxopropanoate (222). BioA is a β-alanine:pyruvate transaminase that transfers the amino
group from alanine to a variety of acceptor molecules, including 3-oxopropanoate,
forming pyruvate and β-alanine in the process (222). Because malonyl-CoA production is
vitamin B7-dependent, a shortage of vitamin B7 may cause a biosynthetic bottleneck
resulting in reduced growth rate as shown in Figure 5-6.
The finding that Ca. P. ubique is missing vitamin B12-based metabolism is
unusual and surprising, but may confer an advantage to SAR11 cells by reducing their
Co2+-requirement. There may be intense competition for limiting amounts of the cobalt
ions required for vitamin B12 biosynthesis in marine surface waters. Cobalt is rare in
111
ocean surface waters and displays an uncommon nutrient distribution, being more
concentrated in the euphotic zone and less concentrated below (223). The marine
cyanobacteria Prochlorococcus and Synechococcus bacillaris have an absolute
requirement for cobalt atoms (224, 225), and physiological and genomic evidence
suggest that their cobalt requirement may be primarily used for vitamin B12 biosynthesis
(50, 226, 227). Despite the apparent inability to produce vitamin B12, we predict that trace
amounts of Co2+ are still required by Ca. P. ubique for use by proteins that require the
divalent ion as a cofactor. For example the 3-dehydroquinate synthase involved in
tryptophan biosynthesis requires divalent cobalt ions. We predict that retention of two
cobalt chelatase subunits, encoded by cobST, serves to ensure this trace requirement for
cobalt is met in Ca. P. ubique.
The initial predicted requirement for vitamin B6 was based on the best metabolic
reconstruction information available at the time (ca. 2005). Since then new experimental
data has emerged that suggests Ca. P. ubique encodes the machinery to synthesize
vitamin B6 from glycolaldehyde and dXP (Figure 5-3)(206). We did not conduct growth
experiments with vitamin B6, however vitamin B6 was excluded from all vitamin
experiments without detriment. Because Ca. P. ubique is missing a homolog to YeaB,
glycolaldehyde is likely synthesized from an alternate precursor. We propose that the
glycolaldehyde required for vitamin B6 is formed during the synthesis of folate (as a
byproduct of the dihydroneopterin aldolase). The presence of this recently described
vitamin B6 synthesis pathway in Ca. P. ubique highlights the importance for revisions in
metabolic models over time. As new science expands our knowledge at the intersection
112
of genetics and metabolism, insight based on metabolic reconstruction can be improved
by reinterpretation.
From the limited environmental distribution data available, vitamin B7, like other
B-vitamins, has unusual distribution patterns in the sea (81, 193-195, 228, 229). The
finding that Ca. P. ubique requires B7 raises interesting questions about the sources of
this vitamin in the environment. The current paradigm pertaining to vitamin metabolism
in the sea is that prokaryotes are able to synthesize their own B-vitamins. Our data
implies that the numerically dominant heterotrophs in the euphotic zone do not fit into
this theoretical framework and that Ca. P. ubique may compete for limited stocks of
vitamin B7. Currently, vitamin research in marine systems is experiencing a resurgence as
technological advances provide more sensitive vitamin assays and oceanographers seek a
more sophisticated understanding of factors influencing microbial diversity and nutrient
flux in plankton communities. The data we present suggest that B7 concentrations in the
sea could impact population sizes of the important chemoheterotroph Ca. P. ubique,
underscoring the potential importance of vitamin cycling for understanding microbial
community dynamics.
113
B1 (thiamin) required
B5 (pantothenate) required
B6 (pyridoxine) required
B7 (biotin) required
glucose-6P
Pentose-Phosphate Pathway
fructose-6P
Non-mevalonate
isoprenoid biosynthesis
deoxy-D-xylulose-5P
dxs
GA-3P
retinol
undecaprenyl-PP
PRPP
nucleotides
α-KG
isocitrate
ubiquinone
ribose-5P
sedohep-P
tktBCN
PEP
B6 & B1
ribulose-5P
xylulose-5P
ery-4P
pdhD
sucAB
CO2
CO2
HCO3-
B5
fatty acid
biosynthesis
cys
CO2
succinyl-CoA
pyruvate
pdhD
aceEF
TCA Cycle
ilvBH
acetyl-CoA
acetolactate
valine
leucine
histidine
tryptophan
accB
malonyl-CoA
OAA
aatA
succinate
malate
asp
argD
lysA
lys
asp-4-SA
cysK glyA
thrC thrC
ser
gly thr
homoserine
Figure 5-1: Simplified illustration of B-vitamin-requiring reactions in Ca. P. ubique.
Gene abbreviations are listed as annotated in the Ca. P. ubique genome. Abbreviations:
PEP: phosphoenolpyruvate; GA-3P: glyceraldehyde-3-phosphate; OAA: oxaloacetate; αKG: α-ketoglutarate; PRPP: phosphoribosyl pyrophosphate; asp-4-SA: aspartate-4semialdehyde; ery-4P: erythrose 4-phosphate; sedohep-P: sedoheptulose 7-phosphate.
Amino acids are listed with standard three-letter abbreviations.
114
A
Escherichia coli K12 MG1655
1.3
D
panC
panB
293
3400
panE
ilvH
100
ilvI
2.6
ilvD
ilvC
‘Candidatus Pelagibacter ubique’ HTCC1062
ilvC
ilvH
239
ilvB
395
mmsA
panB
144
bioA
161
107
ilvD
panC
B
pyruvate
O
OH
O
IlvBH
O
IlvC
O
IlvD
OH
OH
PanB
O
OH
OH
HO
2-acetolactate
O
OH
O
2-oxoisovalerate
MmsA
OH
O
O
pyruvate
HO
O
S-CoA
malonyl-CoA
IlvC
2-dehydropantoate
pantoate
3-oxopropanoate
OH
HO
O
H
N
OH
O
pantothenate
O
HO
O
OH
OH
BioA
O
HO
PanC
O
HO
OH
O
O
2,3-dihydroxyisovalerate
PanE
O
HO
NH2
β-alanine
alanine pyruvate
PanD
Proposed β-alanine synthesis pathway in Ca. P. ubique
O
HO
NH2
O
OH
aspartate
Figure 5-2: Comparative genomics of vitamin B5 biosynthesis in Ca. P. ubique. A)
Genomic orientation of vitamin B5 biosynthetic genes in E. coli and Ca. P. ubique. B)
Illustration of canonical and proposed vitamin B5 biosynthesis. Genes depicted in (A) are
colored by the reaction they catalyze in (B). Dashed lines are reactions predicted to be
absent in Ca. P. ubique, but present in E. coli. Proposed route of β-alanine is
encapsulated in dashed box. Gene abbreviations are listed as they appear in the Ca. P.
ubique or E. coli genome, respectively. D: PanD. Numbers between syntenic fragments is
the distance between genes in kilobasepairs.
115
H2O3PO
A
HO
B
OH
O
E. coli K-12 MG1655
3-phosphoglycerate
canonical
H2O3PO
serC
O OH
O
3-phosphohydroxypyruvate
thrB
HO
epd
600 kbp
900 kbp
ltaE
1 Mbp yeaB 1.2 Mbp
serA
OH
OH
O
OPO3H2
O OH
3-hydroxypyruvate
D-erythrose-4-phosphate
non-enzymatic
Folate biosynthesis
pdxB
alternate
YeaB
O
1.5 Mbp
Ca. P. ubique HTCC1062
Epd
HO
thrB
540 kbp
0689
620 kbp
serA
OH
HO
OPO3H2
O OH
O
glycolaldehyde
4-phosphoerythronate
NH2
O
OH
PdxB
glycine
O
HO
OPO3H2
O OH
NH2
HO
OH
O OH
2-oxo-3-hydroxy4-phosphobutanoate
4-hydroxythreonine
NH2
HO
OPO3H2
O OH
SerC
O-phospho-4-hydroxythreonine
H2N
O
OPO3H2
C
E. coli K-12 MG1655
pdxA
120 kbp
pdxH
2.5 Mbp
pdxJ
3-amino-2-oxopropyl phosphate
Ca. P. ubique HTCC1062
OPO3H2
HO
OH
pdxH
O
deoxy-D-xylulose 5-phosphate
93 kbp
pdxA
330 kbp
pdxJ
OH
OH
OPO3H2
N
pyridoxine phosphate
O
OH
OPO3H2
N
pyridoxal phosphate
Figure 5-3: Comparative genomics of vitamin B6 biosynthesis in Ca. P. ubique. A)
Illustration of canonical and alternate vitamin B6 biosynthetic routes. B) Genomic
orientation of phospho-4-hydroxythreonine (4HT-P) biosynthesis genes in E. coli and Ca.
P. ubique. C) Genomic orientation of vitamin B6 biosynthesis genes (from 4HT-P) in E.
coli and Ca. P. ubique. Genes depicted in (B&C) are colored by the reaction they catalyze
in (A). Dashed lines and genes are reactions and genes predicted to be absent in Ca. P.
ubique, but present in E. coli. Gene abbreviations are listed as they appear in the Ca. P.
ubique or E. coli genome, respectively. 0689: SAR11_0689 gene identifier in the Ca. P.
ubique genome.
116
E. coli K-12 MG1655
A
bioA
bioB
bioF
bioC
bioD
Ca. P. ubique HTCC1062
bioA
B
O
HO
~720 kbp
bioY
CoA + CO2
O
S-CoA
pimeloyl-CoA
BioF
SA-4-me-thio-2-OB
8-amino7-oxononanoate
ala
BioA
7,8-diaminononanoate
SAM
O
NH
O
NH
HO
biotin
S
(sulfur donor)
+ SAM
BioB
ATP
+ CO2
BioD
ADP
+ Pi
dethiobiotin
met + 5’ dA
Figure 5-4: Comparative genomics of vitamin B7 biosynthesis in Ca. P. ubique. A)
Genomic orientation of vitamin B7 biosynthetic genes in E. coli and Ca. P. ubique. B)
Illustration of canonical vitamin B7 biosynthesis in Gram negative organisms. Genes
depicted in (A) are colored by the reaction they catalyze in (B). Dashed lines are
reactions predicted to be absent in Ca. P. ubique, but present in E. coli. Gene
abbreviations are listed as they appear in the Ca. P. ubique or E. coli genome,
respectively. bioY encodes a probable biotin transport protein. Abbreviations: ala:
alanine; met: methionine; SAM: S-adenosyl-methionine; SA-4-me-thio-2-OB: Sadenosyl-4-methylthio-2-oxobutanoate; 5’dA: 5’-deoxyadenosine. The sulfur
incorporation into dethiobiotin to form biotin is derived from an Fe -S cluster contained
within BioB and is indicated by (sulfur donor).
117
OH
OH
O
HO
OH
O
O
5-AL
HO
HN
NH
HN
OH
O
OH
Figure 5-5: Routes of
canonical vitamin B12
biosynthesis. Red gene
abbreviations depict genes
absent in Ca. P. ubique.
CbiK Co2+
Green gene abbreviations
CbiX
depict genes present in Ca.
P. ubique. Abbreviations:
Co2+-precorrin-2
5-AL: 5-aminolevulinic
CbiL
acid; UroCo2+-precorrin-3
III:Uroporphyrinogen III;
CbiH/G 2-AP: 2-aminopropanol.
Co2+-precorrin-4
heme biosynthesis
O
O
CobA
CysG
NH
HO
Uro-III
O
precorrin-2
CobI
precorrin-3a
CobG
precorrin-3b
CobJ
CobF
CobK
precorrin-5
precorrin-6x
precorrin-6y
CobL
precorrin-8x
Aerobic pathway
precorrin-4
CobM
Anaerobic pathway
O
CbiF
Co2+-precorrin-5
CbiD
Co2+-precorrin-6x
CbiJ
Co2+-precorrin-6y
Co -precorrin-8x
CbiC
CobH
cobyrinic acid
hydrogenobryinic acid
CobB
CobNST
hydrogenobryinic
acid-a,c-diamide
NH2
H2N
NH2
O
cob(I)yrinic acida,c-diamide
CobO
BtuR
NH2
O
N
N
Co+
N
H2N
cob(II)yrinic acida,c-diamide
O
N
O
N
HO
O
HN
H2N
O
O
OH
adenosylcobyrinic acida,c-diamide 2-AP
CobQ
NH2
N
N
N
CbiP
adenosylcobyrinic acid
CobD
CbiB
N
-O
O
P
O
O
HO
CbiA
CobW
Co2+
O
CbiET
2+
N
OH
adenosylcobinamide
CobP
CobU
O
adenosylcobalamin
adenosyl-GDP-cobinamide
CobV
CobS CobC
α-ribazole
α-ribazole-5P
118
density (cells ml-1)
107
106
105
No Vitamins
Pantothenate
Biotin
Pantothenate + Biotin
104
0
10
20
30
time (days)
Figure 5-6: Ca. P. ubique is bradytrophic for vitamin B5 (pantothenate) in the presence of
vitamin B7 (biotin). Base medium was AMS1 amended with oxaloacetate (1 mM),
glycine (50 µM), methionine (50 µM) and FeCl3 (1 µM). Vitamin B5 and B7 were added
at a final concentration of 1 µM, where indicated. Points represent the mean of biological
replicates ± s.d. (n=3).
119
A
B
density (cells ml-1)
density (cells ml-1)
108
107
10
6
scrubbed no biotin
scrubbed + biotin
un-scrubbed no biotin
un-scrubbed + biotin
105
0
10
20
time (days)
30
10
8
10
7
+0 biotin
+5 pM biotin
+10 pM biotin
10
6
0
5
10
15
20
time (days)
Figure 5-7: Vitamin B7-scrubbed medium allows for demonstration of vitamin B7requirement in Ca. P. ubique. Base medium was AMS1 amended with oxaloacetate
(1 mM), glycine (50 µM), methionine (50 µM), FeCl3 (1 µM) and thiamine (1 µM). A)
Vitamin B7 scrubbing was necessary to remove traces of vitamin B7 and not detrimental
to growth. Vitamin B7 was at a final concentration of 1 µM when added. B) Titrations of
vitamin B7 into vitamin B7-scrubbed medium. Points represent the mean of biological
replicates ± s.d. (n=3).
120
Table 5-1: Common vitamin B12-requiring enzymes are absent in Ca. P. ubique.
vitamin B12requiring enzyme
methionine
synthase
methylmalonylCoA mutase
ribonucleotide
reductase
gene
metH
mutAB
nrdD
D-α-lysine mutase
methyleneglutarate
mutase
β-lysine mutase
ornithine
aminomutase
isobutyryl-CoA
mutase
leucine 2,3aminomutase
methylaspartate
mutase
propanediol
dehydratase
ethanolamine
ammonia-lyase
glycerol
dehydratase
kamED
mutES
pduCDE
etuBC
dhaBCE
reaction catalyzed
Homocysteine + CH4 →
Methionine
Methylmalonyl-CoA →
Succinyl-CoA
ribonucleotide +
thioredoxin→deoxyribonucleotide + thioredoxin
disulfide
D-lysine→2,5diaminohexanoate
2-methyleneglutarate→2methylene-3methylsuccinate
3,6-diaminohexanoate
→3,5-diaminohexanoate
ornithine→(2R,4S)-2,4diaminopentanoate
2-methylpropanoylCoA→butanoyl-CoA
(2S)-alpha-leucine→
(3R)-beta-leucine
L-threo-3-methylaspartate →L-glutamate
glycerol→3hydroxypropionaldehyde
Ethanolamine→acetaldehyde + ammonia
Glycerol→3Hydroxypropanal + H2O
in Ca. P.
ubique?
absent
Ca. P. ubique
vitamin B12independednt
isozyme
MmuM, BhmT
absent
N/A
absent
NrdAB,
SAR11_0723§
absent
N/A
absent
N/A
absent
N/A
absent
N/A
absent
N/A
absent
N/A
absent
N/A
absent
N/A
absent
N/A
absent
N/A
§
N/A-Reaction not predicted to be involved in Ca. P. ubique metabolism.
§
Part of a B12-dependent nucleotide reductase was identified, however it appears to be
truncated, missing the catalytic cysteine residues characteristic of a vitamin B12-dependent
nucleotide reductase.
121
Chapter 6
Conclusions
Ca. P. ubique was originally isolated in 2002 on autoclaved seawater where
natural, unknown dissolved organic compounds provided substrates for growth.
Sequencing of the Ca. P. ubique genome provided information about specific metabolic
pathways that allow these cells to thrive in the ocean environment. Direct interactions
between Ca. P. ubique and native DOM are challenging to observe. Thus, we developed
laboratory-based methods based on interpretations genome-scale metabolic
reconstructions to understand how Ca. P. ubique impacts geochemical cycles in nature.
Metabolic pathways were reconstructed to identify complete pathways and also to
identify gaps that may be indicative of a nutrient requirement. Subsequent laboratory
testing of these metabolic predictions has uncovered a number of unusual metabolic
features in Ca. P. ubique. Although cultivation is time consuming, we show that this
methodology works and have inferred minimal nutrient requirements for growth of Ca. P.
ubique from its genome sequence. These minimal requirements include sources of
glycine, reduced sulfur, pyruvate, vitamin B7 and 4-amino-5-hydroxymethyl-2methylpyrimidine (HMP).
Interestingly we found that Ca. P. ubique grew best with a glycine:pyruvate molar
ratio of 1:4 and when starved for pyruvate has unusual cell division patterns.
Additionally, we show the new finding that Ca. P. ubique is auxotrophic for thiamine
because of the inability to synthesize HMP de novo. We then measured HMP in the
Sargasso Sea and found that there was sufficient HMP to support Pelagibacter
122
populations. Moreover, we show that within the Ia-subclade of the Pelagibacterales,
distinct phosphate acquisition ecotypes are apparent and may play an important role in
the aerobic production of methane in the sea.
These findings reinforce the hypothesis that Ca. P. ubique is adapted to the low
but continuous flux of DOM in the ocean. The finding that Ca. P. ubique has multiple,
simultaneous nutrient requirements is unusual when compared to the physiology of other
cultivated marine bacteria. However, we suspect that this ‘decentralized’ mode of
metabolism may be a feature other marine oligotrophs with streamlined genomes.
Things that are still unknown about the physiology of Ca. P. ubique relate to
specific metabolic abilities and nuances related to the actual metabolic fluxes through Ca.
P. ubique. Uncommon gene arrangements of genes encoding proteins involved in the
TCA cycle, the glyoxylate bypass and glycine/serine metabolism suggest that the fluxes
through these essential pathways are not canonical. A combination of cultivation-based
experimentation and targeted metabolomic experiments may help resolve the flow of
carbon and energy through these pathways, leading to further refinement of Ca. P. ubique
metabolic models. Such experiments are more easily conducted and interpreted on a
defined medium devoid of excess carbon (as is the case in natural seawater).
Moreover, this work has the potential to serve as a blueprint for the cultivation of
other marine microbes. Currently, genomes from single cells are being sequenced (230),
with the result of shedding light on the metabolic abilities of organisms that are not
cultivated. We hope that the preceding chapters are helpful in guiding other researchers
during their attempts to deduce the nutrient requirements of other marine organisms.
123
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APPENDICES
140
The appendices herein contain documentation submitted (or are intended to be
submitted) as supplemental information documents with manuscripts for publication.
Appendix 1 is published as a supplement to: doi:10.1038/ismej.2012.122. Appendix 2
will accompany the submission of Chapter 3 and appendix 3 will accompany the
submission of Chapter 4.
Appendix 1
cell density (cells ml-1)
107
106
105
104
0
2
4
6
8
10
12
time (days)
Figure A1- 1: Transfer of Ca. P. ubique batch cultures from natural seawater to AMS1.
▲: Ca. P. ubique transferred from natural seawater amended with pyruvate (50 µM),
glycine (1 µM), methionine (1 µM) and vitamins to identically amended natural seawater
medium. ●: Ca. P. ubique transferred from natural seawater amended with pyruvate (50
µM), glycine (1 µM), methionine (1 µM) and vitamins to identically amended AMS1.
Points are the mean of triplicate cell density measurements. Error bars: ± 1.0 s.d., n=3.
When error bars are not visible, they are smaller than the size of the symbols.
141
†
‡
A
B
C
D
E
A
B
C
D
E
F
G
H
I
J
F
G
H
I
J
Figure A1- 2: Flow cytometry profiles and corresponding microscopic images at different
pyruvate:glycine molar ratios. All cultures were grown to stationary-phase in AMS1
containing methionine (10 µM) with the following glycine and pyruvate concentrations:
A) 0 µM pyruvate, 50 µM glycine; B) 0.1 µM pyruvate, 50 µM glycine; C) 0.9 µM
pyruvate, 50 µM glycine; D) 5 µM pyruvate, 50 µM glycine; E) 25 µM pyruvate, 50 µM
glycine; F) 50 µM pyruvate, 0 µM glycine; G) 50 µM pyruvate, 0.1 µM glycine; H)
50 µM pyruvate, 0.9 µM glycine; I) 50 µM pyruvate, 5 µM glycine; J) 50 µM pyruvate,
25 µM glycine. For each treatment, the upper panel shows relative DNA fluorescence
profiles of SYBR Green I-stained cells; the lower panel shows microscopic images of
SYBR green I-stained cells. In (A), †: the relative DNA fluorescence of single cells; ‡:
the relative DNA fluorescence of doublets
142
time after inoculation (days)
14
15
17
19
2000 nM
500 nM
250 nM
125 nM
0 nM
2000 nM
500 nM
250 nM
125 nM
0 nM
cell density (x 106 cells ml-1)
10
4
2
0
alanine concentration
Figure A1- 3: DNA fluorescence profiles and maximum cell densities in response to
alanine additions. Top panel: Relative DNA fluorescence time course of batch cultures
grown in AMS1 amended with limiting pyruvate (0.5 µM) glycine (1 µM), methionine
(1 µM) and different L-alanine concentrations, as indicated. Bottom panel: Maximum cell
densities of cultures for which relative DNA fluorescence values are shown in the top
panel.
143
1.60E+06 maximum density (cells ml-­‐1) 1.40E+06 1.20E+06 1.00E+06 8.00E+05 6.00E+05 4.00E+05 2.00E+05 0.00E+00 0 2 4 6 8 10 12 Dose of vitamin mix added (fold) Figure A1- 4: Effect of vitamin additions on Ca. P. ubique growth. Ca. P. ubique grown
in AMS1 without added organic carbon and different doses of freshly prepared vitamins
(as listed in Table 1). Points show the maximum density achieved of single flasks
(triplicates for each dose are shown).
Discussion of Figure A1-4 in appendix 1(above): Upon exclusion of the vitamin
mixture and in the absence of added organic carbon, Ca. P. ubique grew to densities of
ca. 2.0 ×105 cells ml-1. Higher densities, exceeding 1.2 ×106 cells ml-1, were observed, in
a dose-dependent manner, when a freshly prepared vitamin solution was added (Figure
A1-4, above). We speculate that Pelagibacter makes efficient use of organic carbon that
enters the cultures as reagent contaminants (for example, in the vitamin stocks), or by
144
diffusion from plastic culture ware and the air. Previously Ca. P. ubique has been shown
to use a variety of volatile organic carbon compounds as sources of electrons for
respiration (49). Also in support of this interpretation, we note that the growth of bacteria
in water purification systems is not uncommon (231). Collectively, we conclude that
unintentional trace levels of nutrients entering our experiments, coupled with the low
amounts of carbon required by Ca. P. ubique, is a probable explanation for the observed
growth without added carbon.
145
Appendix 2
D-glucose-6P
D-fructose-6P
Pentose-phosphate pathway
TktB,C,N
D-ery-4P
Non-mevalonate
isoprenoid bioynthesis
deoxy-D-xylulose-5P
B1
TktB,C,N
Dxs
GA-3P
B6
retinol
pantothenate
D-sedohep-P
isocitrate
ubi
D-ribulose-5P
D-ribose-5P
PRPP
nucleotides
α-KG
PEP
undecaprenyl-PP
valine
leucine
D-xylulose-5P
SucA
SucB
PdhD
CO2
succinyl-CoA
pyruvate
IlvB
CO
IlvH 2
acetolactate
AceE
CO2 AceF
PdhD
TCA cycle
succinate
acetyl-CoA
OAA
malate
Figure A2- 1: Simplified illustration of Ca. P. ubique’s central metabolism, highlighting
thiamine-requiring biosynthetic reactions. Red lines and enzyme abbreviations depict
thiamine-dependent biosynthetic steps and enzymes, respectively. Gene abbreviations are
listed as they are annotated in the Ca. P. ubique genome: Dxs: 1-D-deoxyxylulose 5phosphate synthase; AceE: pyruvate dehydrogenase (lipoamide) E1 component; AceF:
dihydrolipoamide S-acetyltransferase; PdhD: dihydrolipoyl dehydrogenase; IlvB:
acetolactate synthase large subunit (ALS III); IlvH: acetolactate synthase small subunit;
TktB: transketolase; TktC: transketolase-family protein; TktN: transketolase; SucA: 2oxoglutarate dehydrogenase E1 component; SucB: 2-oxoglutarate dehydrogenase
complex E2 component. Compound abbreviations: OAA: oxaloacetate; α-KG: αketoglutarate; PRPP: phosphoribosyl pyrophosphate; D-ery-4P: D-erythrose 4-phosphate;
D-sedohep-P: D-sedoheptulose 7-phosphate; GA-3P: glyceraldehyde 3-phosphate; PEP:
phosphoenolpyruvate; ubi: ubiquinone; B1: thiamine; B6: pyridoxine. TCA cycle:
Tricarboxylic acid cycle.
146
1 μM thiamine
no added thiamine
cell density (cells ml-1)
108
-thiamineT2
+thiamineT2
+thiamineT1
10
7
-thiamineT1
10
6
105
0
10
20
30
time (days)
Figure A2- 2: Reproduction of Figure 3-2a in main text with microarray sample points
labeled.
maximum density (cells ml-1 × 108)
3.5
2.5
1.5
0.5
0
no
thi
H+B6 pant THZ
thi
Figure A2- 3: Growth responses of Ca. P. ubique to potential thiamine precursors. Bar
heights are the average maximum densities achieved of biological replicates ± 1.0 s.d.
(n=3). Potential precursors were added at a final concentration of 1.0 µM, as described in
methods. Abbreviations: H: Histidine; B6: pyridoxine (vitamin B6); pant: pantothenate;
THZ: 4-methyl-5-thiazoleethanol; thi: thiamine.
147
Figure A2- 4: Maximum-likelihood phylogenetic reconstruction of the SAR11_0811
amino acid sequence. The tree is rooted at its midpoint. The node labels are bootstrap
values based on 1,000 replicates. Branches are labeled with the gene locus identifier and
organism name. Branches in red are further described in Figure A2-5 in appendix 2. See
supplementary text in appendix 2 for details of tree construction.
148
Figure A2-4: Maximum-likelihood phylogenetic reconstruction of the SAR11_0811
amino acid sequence.
149
AAG G TGTCGTTAACATGACTGAGATTA AC CTAAGTA C TGATCCCAG TA TGTCATCAGAAAG GAGAAACAA
C
TA G
C GGAA G
A
G GT
G
TC
T C TA
TT
G G
A
TT
A
G
C TG
CGG
A
T
GC
TG
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
CG
*
AAGGGGTGTCGTAACTGACTGAGAATAAACCCTAATAACCTGTCC--GGTAAT--TCAGAAAGGGAGAACA
AAGGGGTGTCGTAACTGACTGAGATTAAACCCTAATAACCTGTCC--GGTAAT--TCAGAAAGGGAGAACA
AAGGGGTGTCGTAACTGACTGAGAATAAACCCTAATAACCTGTCC--GGTAAT--TCAGAAAGGGAGAACA
AAGGGGTGTCGTAACAGACTGAGATTAAACCCTAAGAACCTGTCC--GGTAAT--TCAGAAAGGGAGAATA
AAGGGGTGTCGTAAAAGACTGAGATTAAACCCTTAGAACCTGTCC--GGTAAT--TCAGAAAGGGAAAAAA
AGGGGGTGTCTTTACAGACTGAGATTAAACCCCAGGAACCTGGCT--GGTAAT--TCAGTAAGGGAATAAA
AAGGGGTGCCTTTAATGGCTGAGAGTAAACTCTAGGAACCTGACT--GGTAAT--TCAGTGAGGGAATAGG
CAGGGGTGCGAAAAC--GCTGAGATTATACCCTACGAACCTGATG--TTTAATTACAACGAAGGGAGAACC
CAGGGGCGGCTTAGC---CTGAGA-TGAACCCTTTAAACCTGATCCAGATAATGCTGGCGGAGGAAGAAAA
TGTGATTCCTGGGGGCGCCATTGCGATGGCTGAGACTCCGCACTTTGTGCGGTAACCCTTCGAACCTGATCCGGGTAATGCCGGCGAAGGAAGGATAAAGCCTGCTTT
AAAAATTGTAAGGGGTGTCGTAACTGACTGAGAATAAACCCTAATAACCTGTCCGGTAATTCAGAAAGGGAGAACAATGGATAAAACAT
*
*
thiD
thiM thiE
tenA
tenA
tenA
tenA
tenA
tenA
tenA
tenA
tenA
Putative HMP transporter
Putative HMP transporter
Putative HMP transporter
Putative HMP transporter
Putative HMP transporter
Putative HMP transporter
Figure A2- 5: Illustration of predicted thiamine regulatory elements or thiamine
pyrimidine salvage genes genetically associated with the putative HMP transport protein
coding sequence. Phylogeny is reproduced from Figure A2-4 in appendix 2, with
modifications for clarity. Nucleotide sequences are located immediately upstream (5’) of
the putative HMP transport protein. Sequences that are marked with (*) were predicted to
contain ThPP-binding motifs by rfam (http://rfam.sanger.ac.uk/). Dashed box
encapsulates sequences from 9 of 10 sequenced Pelagibacter genomes and their
consensus sequence is illustrated.
Supplementary methods construction of phylogenetic trees:
Construction of phylogenetic tree in Figure A2-4 & A2-5 in appendix 2: To
determine genomic context of ‘proline uptake protein’ (SAR11_0811) homologs in other
organisms, SAR11_0811 was used to construct a gene tree in MicrobesOnline
(http://microbesonline.org/). The pre-computed tree for SAR11_0811 using COG591
(amino acid residues 13-446) demonstrated conserved features in the genomes of
Methylobacillus flagellatus KT, Marinobacter sp. ELB17, Clostridium sp. OhILAs,
Haloquadratum walsbyi DSM 16790, Haloarcula marismortui ATCC 43049,
Halorhabdus utahensis DSM 12940, Haloferax volcanii DS2 and Halogeometricum
borinquense PR3, DSM 11551.
150
Analysis of the putative ‘proline uptake protein’ transporter SAR11_0811 using
Hal (232) revealed homologous sequences in eight other SAR11 genomes - HTCC1002,
HTCC9565, HTCC7211, HIMB5, AAA240-E13, AAA288-G21, HIMB114, and
IMCC9063. The nine SAR11 and eight additional homologs from Gram-positive
bacteria, Archaea, and proteobacteria were searched against the Hidden Markov Model
(HMM) database of over 430,000 protein families, the so-called “Sifting Families”
(SFam) (233), using hmmscan on default settings as part of the hmmer 3.0 package (234):
$ hmmscan --domtblout txport.tab -o txport.hsc Sifted_Families/SFam_HMMs_all
txport_starters.faa
All 17 genes used in the initial search had best hits to one of two SFam HMMs,
346434 or 31377. Inspection of the members of these SFams revealed that 346434
contained several thousand bacterial and archaeal members with various transport
annotations, whereas 31377 contained 23 genes exclusively from fungal taxa annotated
predominantly as hypothetical proteins. A Prokaryotic tree was constructed using BLAST
to select sequences from the 346434 SFam. The fasta file for the SFam was generated
using the tree_parse.py script:
$ python tree_parse.py --model 346434 | sed 's/^/lcl|/g' | blastdbcmd -entry_batch - -db
img_v350_prot > 346434_v350.faa
This file was used to generate a database for BLASTP searches:
$ makeblastdb -in 346434_v350.faa -out 346434_SEQS -parse_seqids -hash_index
$ blastp -query halo_SAR_starters.faa -db 346434_SEQS -out 346434_like_SAR11 outfmt 7 -max_target_seqs 50" -r SAR11_346434_blastp
Unique hits with E-values > 1e-30 were selected for construction of the tree:
151
$ cat 346434_like_SAR11 | grep -v # | awk '$11<1e-30' | cut -f 2 | sort | uniq | xargs -i++
blastdbcmd -entry "lcl|++" -db 346434_SEQS >> 346434_like_SAR11.faa
These were parsed and run through a bash script (protPipeline3) automating alignment
with MUSCLE (235), curation with GBlocks (236), amino acid substitution modeling
with ProtTest (237), and maximum-likelihood tree construction with RAxML (238):
$ cat 346434_like_SAR11.faa halo_SAR_starters.faa > transport_group6.faa
$ cat transport_group6.faa | sed 's/\ .*//g' | sed 's/lcl|//g' > transport_group6_cln.faa
$ bash protPipeline3 transport_group6_cln.faa
protPipeline3:
#!/bin/bash
filename=$1
muscle -in $1 -out $filename.al
cat $filename.al | fasta2phy | phyToFasta.pl > $filename.tmp
rm -f $filename.al
mv $filename.tmp $filename.al
seqs=`grep -c ">" $filename.al`
Gblocks $filename.al -t=p -p=n -e=.fst -b1=$(((seqs/2)+1)) -b2=$(((seqs/2)+1)) b3=$(((seqs/2))) -b4=2 -b5=h
mv $filename.al.fst $filename.gb
cat $filename.gb | fasta2phy > $filename.phy
java -Xmx250m -classpath /local/cluster/spatafora/ProtTest/ProtTest.jar
prottest.ProtTest -i $filename.phy -o $filename.model -sort A -S 0 -t1 T -t2 T
model=`grep "Best model according to" $filename.model | awk '{print$6}' | awk -F+
'{print $1'} | tr "[:lower:]" "[:upper:]"`
raxmlHPC -f a -m PROTCAT$model -n $filename -N 1000 -s $filename.phy -p 1234 -x
1234
phyTofasta.pl:
#!/usr/bin/perl –w
# $Header$
# $Log$
# Revision 1.1 2006/08/07 19:09:38 reevesj
# converts a phylip file to fasta format. can read/write standard in/out for pipelining.
#
152
#
use Getopt::Std;
use Bio::AlignIO;
use Bio::SeqIO;
my %opt;
getopts('i:o:h', \%opt);
checkOpts(\%opt);
my ($inaln, $aln, $outobj, $seq);
if(defined($opt{i})){
close(STDIN);
open(STDIN, $opt{i});
}
if(defined($opt{o})){
close(STDOUT);
open(STDOUT, ">$opt{o}");
}
$inaln = Bio::AlignIO->newFh(-format => 'phylip', -fh => \*STDIN);
warn("reading from stdin\nphyToFasta.pl -h for more help\n");
$outobj = Bio::SeqIO->newFh(-format => 'fasta');
$aln = <$inaln>;
foreach $seq ($aln->each_seq()){
print($outobj $seq);
}
exit(0);
sub checkOpts{
$opt = shift();
if($$opt{h}){ opterr(); }
return;
}
sub opterr{
#getopts('b:g:d:m:h');
print <<__HELP__;
Usage: $0 [-ioh]
-h
Print this help message.
-i <input phylip file>
Phylip format alignment file.
If unspecified, read from standard in.
Optional.
-o <output fasta file>
File where converted fasta file will be written.
If unspecified, write to standard out.
Optional.
153
__HELP__
exit(1);
}
fasta2phy :
#!/usr/bin/perl -w
# $Id: fasta2phy 905 2010-05-14 22:11:23Z alexeb112 $
#
# Copyright 2005,2006,2007 John Reeves and Ryan Yoder
<Ryan.Yoder@science.oregonstate.edu>
#
# This program is free software; you can redistribute it and/or modify
# it under the terms of the GNU General Public License as published by
# the Free Software Foundation; either version 3 of the License, or
# (at your option) any later version.
#
# This program is distributed in the hope that it will be useful,
# but WITHOUT ANY WARRANTY; without even the implied warranty of
# MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the
# GNU General Public License for more details.
#
# You should have received a copy of the GNU General Public License
# along with this program. If not, see <http://www.gnu.org/licenses/>.
use Getopt::Std;
use File::Spec;
my (undef,undef, $SCRIPT_NAME ) = File::Spec->splitpath( $0 );
my %opt;
getopts('i:o:h', \%opt);
checkOpts(\%opt);
my (%aln, @anames, $cname);
if(defined($opt{i})){
close(STDIN);
open(STDIN, $opt{i});
}
while(<STDIN>){
chomp();
if($_ =~ /^\s*$/){
next;
}
$_ =~ s/\s+//g;
if(substr($_, 0, 1) eq '>'){
$cname = substr($_, 1);
154
$aln{$cname} = '';
push(@anames, $cname);
}else{
$aln{$cname} .= $_;
}
}
my $l = length($aln{$cname});
#make sure the alignments are actually the same length.
foreach $name (@anames){
if(length($aln{$name}) != $l){
die("Error: not all alignments are the same length.\n");
}
}
if(defined($opt{o})){
close(STDOUT);
open(STDOUT, ">$opt{o}");
}
print scalar(@anames). ' '. length($aln{$cname}). "\n";
$i=0;
my $donames = 1;
my $tmpseq='';
my $tmpname='';
while($i<$l){
if($donames){
$donames = 0;
foreach $name (@anames){
$tmpseq = substr($aln{$name}, $i, 50);
$tmpseq =~ s/(.{10})/$1 /g;
$tmpname=$name;
$tmpname =~ s/,.*$//g; #remove commas from name, some programs hate them.
print substr(sprintf('%-10s', $tmpname), 0, 10). ' ';
print "$tmpseq\n";
}
}else{
foreach $name (@anames){
$tmpseq = substr($aln{$name}, $i, 50);
$tmpseq =~ s/(.{10})/$1 /g;
printf("%11s%s\n", ' ', $tmpseq);
}
}
print "\n";
$i += 50;
}
exit(0);
155
sub checkOpts{
$opt = shift();
if($$opt{h}){ opterr(); }
return;
}
sub opterr{
print <<__HELP__;
fasta2phy: convert a fasta formatted file into interleaved phylip format. Does an
in place conversion if -o is given and the input is the same file as -o.
Usage: $SCRIPT_NAME [-ioh]
Convert a fasta formatted file into interleaved phylip format.
Command line options:
-h
Print this help message.
-i <input fasta file>
Fasta format alignment file.
If unspecified, read from standard in.
Optional.
-o <output phylip file>
File to which converted alignment will be written.
If unspecified, write to standard out.
Optional.
__HELP__
exit(1);
}
The initial tree contained duplicate sequences, which were removed via RAxML. The
trimmed alignment was re-run with RAxML using the following settings:
$ raxmlHPC-PTHREADS -x 1234 -T 2 -f a -s transport_group6_cln.red.phy -n 6_red -m
PROTCATCPREV -\# 1000
156
A
B
Figure A2- 6: Profiles of in situ temperature and chlorophyll a fluorescence at
Hydrostation S in the Sargasso Sea during water collection for vitamin analysis. Panel A:
Cruise number AE1224, cast 2 at 20:00 (local time), 19 September 2012. Panel B: Cruise
number AE1224, cast 4 at 08:00 (local time), 20 September 2012.
157
Table A2- 1: Gene transcripts more abundant in thiamine-limited conditions
ORF name
Gene
Annotation
SAR11_0004
miaA
SAR11_0015
SAR11_0021
pepP
ddlB
SAR11_0022
murB
SAR11_0023
murC
SAR11_0028
murE
SAR11_0029
ftsI
tRNA isopentenyltransferase
[EC:2.5.1.8]
Xaa-Pro aminopeptidase [EC:3.4.11.9]
D-alanine--D-alanine ligase B
[EC:6.3.2.4]
UDP-Nacetylenolpyruvoylglucosamine
reductase [EC:1.1.1.158]
UDP-N-acetylmuramate--L-alanine
ligase [EC:6.3.2.8]
UDP-N-acetylmuramoylalanyl-Dglutamate [EC:6.3.2.10, 6.3.2.13]
cell division protein FtsI
[EC:2.4.1.129]
probable short-chain dehydrogenase
hypothetical protein
A/G-specific adenine glycosylase
[EC:3.2.2.-]
phytoene synthase [EC:2.5.1.32]
3-deoxy-D-manno-octulosonic-acid
transferase
lipoprotein signal peptidase
[EC:3.4.23.36]
riboflavin biosynthesis protein
[EC:2.7.7.2, 2.7.1.26]
two-component system response
regulator
DNA polymerase I [EC:2.7.7.7]
arginase [EC:3.5.3.1]
hydroxyacylglutathione hydrolase
cytoplasmic [EC:3.1.2.6]
glutamate 5-kinase [EC:2.7.2.11]
probable primosomal protein N
3-isopropylmalate dehydratase
[EC:4.2.1.33]
ribulose-phosphate 3-epimerase
[EC:5.1.3.1]
hypothetical protein
SAR11_0082
SAR11_0093
SAR11_0111
mutY
SAR11_0121
SAR11_0148
crtB
kdtA
SAR11_0157
lspA
SAR11_0159
ribF
SAR11_0199
chvI
SAR11_0200
SAR11_0206
SAR11_0219
polA
speB
gloB
SAR11_0223
SAR11_0234
SAR11_0251
proB
priA
leuD
SAR11_0317
rpe
SAR11_0318
fold
change
q-value
2.28
≤0.005
2.11
2.74
≤0.005
≤0.005
2.92
≤0.005
2.30
≤0.005
2.11
≤0.005
2.67
≤0.005
2.33
2.04
2.65
≤0.005
≤0.005
≤0.005
2.35
2.26
≤0.005
≤0.005
2.26
≤0.005
2.17
≤0.005
2.29
≤0.005
2.04
2.01
2.33
≤0.005
≤0.005
≤0.005
2.60
2.04
2.27
≤0.005
≤0.005
≤0.005
2.04
≤0.005
2.36
≤0.005
158
Table A2-1 (continued)
SAR11_0343
SAR11_0362
SAR11_0365
nth
SAR11_0408
mutM
SAR11_0443
pheT
SAR11_0457
fmt
SAR11_0494
aroA
SAR11_0590
SAR11_0602
folP
SAR11_0604
thiD
SAR11_0610
SAR11_0634
SAR11_0636
SAR11_0638
ispA
umuC
SAR11_0684
mobA
SAR11_0712
SAR11_0818
SAR11_0831
surA
amtB
pabB
SAR11_0920
gltX
SAR11_0931
SAR11_0993
recG
SAR11_0996
mltB
SAR11_1010
recJ
SAR11_1019
xerD
hypothetical protein
hypothetical protein
probable endonuclease III
[EC:4.2.99.18]
formamidopyrimidine-DNA
glycosylase [EC:3.2.2.23]
phenylalanine-tRNA ligase
[EC:6.1.1.20]
methionyl-tRNA formyltransferase
[EC:2.1.2.9]
3-phosphoshikimate 1carboxyvinyltransferase [EC:2.5.1.19]
5-formyltetrahydrofolate cyclo-ligase
[EC:6.3.3.2]
dihydropteroate synthase-like protein
[EC:2.5.1.15]
phosphomethylpyrimidine kinase
[EC:2.7.4.7]
geranyltranstransferase [EC:2.5.1.10]
DNA polymerase IV [EC:2.7.7.7]
hypothetical protein
phosphoribulokinase/uridine kinase
family protein
molybdopterin-guanine dinucleotide
biosynthesis protein A
SurA-like protein [EC:5.2.1.8]
ammonium transporter
para-aminobenzoate synthase
component I [EC:6.3.5.8]
glutamyl-tRNA synthetase
[EC:6.1.1.17]
putative transmembrane protein
ATP-dependent DNA helicase RecG
[EC:3.6.1.-]
membrane-bound lytic transglycolaserelated protein
single-stranded-DNA-specific
exonuclease
integrase/recombinase XerD-like
protein
2.20
2.22
2.76
≤0.005
≤0.005
≤0.005
2.05
≤0.005
2.15
≤0.005
2.01
≤0.005
2.05
≤0.005
2.15
≤0.005
2.48
≤0.005
2.06
≤0.005
2.06
2.21
2.35
2.37
≤0.005
≤0.005
≤0.005
≤0.005
2.12
≤0.005
2.38
2.42
2.89
≤0.005
≤0.005
≤0.005
2.07
≤0.005
2.07
2.41
≤0.005
≤0.005
2.22
≤0.005
2.01
≤0.005
2.04
≤0.005
159
Table A2-1 (continued)
SAR11_1173
SAR11_1248
SAR11_1330
SAR11_1331
SAR11_1342
bhmT
ppiA
betaine-homocysteine Smethyltransferase [EC:2.1.1.5]
winged helix DNA-binding
putative lipoprotein
peptidylprolyl cis-trans isomerase
precursor [EC:5.2.1.8]
alpha/beta hydrolase fold
2.17
≤0.005
2.09
2.23
2.26
≤0.005
≤0.005
≤0.005
2.37
≤0.005
160
Table A2- 2: Gene transcripts less abundant in thiamine-limited conditions
ORF name
Gene
Annotation
SAR11_0001
ilvC
SAR11_0007
hflC
SAR11_0008
hflK
SAR11_0060
SAR11_0098
pilQ
fbcH
SAR11_0099
petB
SAR11_0100
petA
SAR11_0117
atpE
SAR11_0130
SAR11_0134
SAR11_0135
coxC
cox1
coxB
SAR11_0162
SAR11_0183
groE
SAR11_0197
ahcY
SAR11_0221
SAR11_0229
rpmA
atpC
SAR11_0230
atpD
SAR11_0232
atpA
SAR11_0348
SAR11_0423
SAR11_0500
SAR11_0504
rho
carD
ketol-acid reductoisomerase
[EC:1.1.1.86]
probable integral membrane
proteinase
probable integral membrane
proteinase [EC:3.4.-.-]
type II secretion PilQ
ubiquinol-cytochrome-c reductase
[EC:1.10.2.2]
ubiquinol-cytochrome-c reductase
[EC:1.10.2.2]
cytochrome b6-f complex iron-sulfur
subunit [EC:1.10.2.2]
H+-transporting two-sector ATPase
(subunit C) [EC:3.6.3.14]
cytochrome-c oxidase [EC:1.9.3.1]
cytochrome-c oxidase [EC:1.9.3.1]
probable cytochrome c oxidase
polypeptide II (cytochrome aa3
subunit 2) [EC:1.9.3.1]
60 kDa chaperonin
xanthine/uracil/vitamin C permease
family protein
S-adenosyl-L-homocysteine hydrolase
[EC:3.3.1.1]
50S ribosomal protein L27
ATP synthase epsilon chain (ATP
synthase F1 sector epsilon subunit)
[EC:3.6.3.14]
F1-ATP synthase beta chain
[EC:3.6.3.14]
H+-transporting two-sector ATPase
(F0F1-type ATP synthase) alpha chain
[EC:3.6.3.14]
transcription termination factor rho
hypothetical transcriptional regulator
hypothetical protein
cytochrome c
cycM
fold
change
q-value
2.56
≤0.005
2.50
≤0.005
2.21
≤0.005
2.04
3.26
≤0.005
≤0.005
2.36
≤0.005
2.45
≤0.005
2.49
≤0.005
2.71
2.16
2.08
≤0.005
≤0.005
≤0.005
2.01
2.52
≤0.005
≤0.005
2.47
≤0.005
2.72
2.62
≤0.005
≤0.005
3.29
≤0.005
2.60
≤0.005
2.01
2.04
2.38
2.31
≤0.005
≤0.005
≤0.005
≤0.005
161
Table A2-2 (continued)
SAR11_0510
SAR11_0630
glcB
SAR11_0644
icdA
SAR11_0708
SAR11_0717
acpP
ndk
SAR11_0766
gabD
SAR11_0801
hisD2
SAR11_0808
bioA
SAR11_0809
SAR11_0835
ald
aprM
SAR11_0836
aprB
SAR11_0837
aprA
SAR11_0842
garD
SAR11_0843
SAR11_0844
SAR11_0861
SAR11_0865
SAR11_0866
SAR11_0953
yhdW
SAR11_0954
yhdX
malate synthase [EC:2.3.3.9]
transcriptional regulators, TraR/DksA
family
isocitrate dehydrogenase
[EC:1.1.1.42]
acyl carrier protein (ACP)
nucleoside diphosphate kinase
[EC:2.7.4.6]
succinate-semialdehyde
dehydrogenase (NAD(P))
[EC:1.2.1.16]
histidinol dehydrogenase
[EC:1.1.1.23]
adenosylmethionine-8-amino-7oxononanoate aminotransferase
[EC:2.6.1.62]
alanine dehydrogenase [EC:1.4.1.1]
adenylylsulfate reductase membrane
anchor
adenylyl-sulfate reductase chain B
[EC:1.8.99.2]
adenylyl-sulfate reductase chain A
[EC:1.8.99.2]
probable galactarate dehydratase
[EC:4.2.1.42]
D-galactarate dehydratase/altronate
[EC:4.2.1.42]; K01708 galactarate
dehydratase
hypothetical protein
short chain dehydrogenase, unknown
specificity
TRAP-type mannitol/chloroaromatic
compound transport system
mannitol transporter
ABC transporter - general L-amino
acid transport system substratebinding protein
ABC transporter - general L-amino
acid transport system permease
protein
2.14
2.33
≤0.005
≤0.005
2.13
≤0.005
2.76
2.09
≤0.005
≤0.005
2.32
≤0.005
2.22
≤0.005
2.04
≤0.005
2.53
3.65
≤0.005
≤0.005
3.12
≤0.005
3.85
≤0.005
2.57
≤0.005
2.66
≤0.005
2.71
2.12
≤0.005
≤0.005
3.01
≤0.005
2.67
2.27
≤0.005
≤0.005
2.12
≤0.005
162
Table A2-2 (continued)
SAR11_0957
yhdZ
SAR11_0979
SAR11_0980
yajC
secD
SAR11_0987
SAR11_1033
SAR11_1037
SAR11_1040
ppiB
rpmG
rpmF
hppA
SAR11_1043
SAR11_1048
ribH
glyA
SAR11_1093
rpoA
SAR11_1097
SAR11_1103
SAR11_1119
secY
rpsH
fusA
SAR11_1122
rpoC
SAR11_1123
rpoB
SAR11_1124
SAR11_1129
SAR11_1130
rplL
SAR11_1143
SAR11_1150
grlA
dhs
SAR11_1181
SAR11_1203
SAR11_1210
tctC
occT
SAR11_1211
speE
SAR11_1245
folE
tufB
general L-amino acid transport ATPbinding protein
preprotein translocase
protein-export membrane protein
SecD
peptidylprolyl isomerase [EC:5.2.1.8]
ribosomal protein L33
50S ribosomal protein L32
membrane-bound protontranslocating pyrophosphatase
[EC:3.6.1.1]
riboflavin synthase
glycine hydroxymethyltransferase
[EC:2.1.2.1]
DNA-directed RNA polymerase
[EC:2.7.7.6]
preprotein translocase SecY subunit
30S ribosomal protein S8
translation elongation factor EF-G
[EC:3.6.5.3]
DNA-directed RNA polymerase beta
prime chain [EC:2.7.7.6]
DNA-directed RNA polymerase
[EC:2.7.7.6]
probable 50S ribosomal protein L31
hypothetical protein
translation elongation factor EF-Tu
[EC:3.6.5.3]
glutaredoxin
2-dehydro-3-deoxy-phosphoheptonate
aldolase [EC:2.5.1.54]
hypothetical protein
putative tricarboxylic transport
ABC transporter - octopine/nopaline
transport system substrate-binding
protein
spermine/spermidine synthase
[EC:2.5.1.16, 4.1.1.50]
GTP cyclohydrolase I [EC:3.5.4.16]
2.02
≤0.005
2.19
2.81
≤0.005
≤0.005
2.49
2.69
3.06
4.75
≤0.005
≤0.005
≤0.005
≤0.005
2.02
2.80
≤0.005
≤0.005
2.82
≤0.005
2.36
2.26
3.33
≤0.005
≤0.005
≤0.005
2.73
≤0.005
2.48
≤0.005
2.28
4.42
4.19
≤0.005
≤0.005
≤0.005
2.43
2.35
≤0.005
≤0.005
2.15
2.01
2.94
≤0.005
≤0.005
≤0.005
3.05
≤0.005
2.38
≤0.005
163
Table A2-2 (continued)
SAR11_1266
SAR11_1274
cspL
SAR11_1274
cspL
SAR11_1336
potD
membrane protein of unknown
function (DUF1430)
cold shock DNA-binding domain
protein
cold shock DNA-binding domain
protein
spermidine/putrescine-binding
periplasmic protein
2.07
≤0.005
3.82
≤0.005
2.03
≤0.005
2.02
≤0.005
164
Appendix 3
density (cells ml-1)
107
106
105
No PO4
PO4
PO3
Me-PO3
2-AEP
dATP
104
5
10
15
20
25
time (days)
dTTP
dGTP
dCTP vs
Ribose-5P
Glucose-6P
P-serine
30
35
Figure A3- 1: Growth curves of Pelagibacter sp. str. HTCC7211 on alternate phosphorus
sources showing diauxic growth pattern. Growth medium was AMS1 with amendments
of pyruvate (50 µM), glycine (5 µM), methionine (5 µM), FeCl3 (1 µM) and vitamins.
Each alternate phosphorus compound was supplied at a final concentration of 1.0 µM.
Points are the mean density of biological replicates ± 1.0 s.d. (n=3).
165
108
1 nM
3.3nM
10 nM
33 nM
100 nM
1000 nM
3000 nM
cell density (cells ml-1)
107
106
105
104
103
0
5
10
15
time (days)
20
25
30
Figure A3- 2: Growth curves for linear dose responses of Pelagibacter sp. str.
HTCC7211 grown on Pi. Points are the mean density of biological replicates ± 1.0 s.d.
(n=3).
166
1 nM
3.3 nM
10 nM
33 nM
100 nM
1000 nM
3000 nM
cell density (cells ml-1)
107
106
105
104
0
5
10
15
20
25
time (days)
Figure A3- 3: Growth curves for linear dose responses of Ca. P. ubique grown on Pi.
Points are the mean density of biological replicates ± 1.0 s.d. (n=3).
167
cell density (cells ml-1)
107
106
1 nM
3.3 nM
10 nM
33.3 nM
100 nM
3000 nM
105
5
10
15
20
time (days)
Figure A3- 4: Growth curves for linear dose responses of Pelagibacter sp. str.
HTCC7211 grown on Mpn. Points are the mean density of biological replicates ± 1.0 s.d.
(n=3).
168
Figure A3-5: Simplified illustration of phospholipid biosynthetic routes and predicted
reactions involved in lipid remodeling in Pelagibacter sp. str. HTCC7211. Red lines:
Canonical lipid biosynthetic routes for phosphatidylethanolamine and
phosphatidylglycerol as identified in both Ca. P. ubique and Pelagibacter sp. str.
HTCC7211. Green lines: Predicted reactions involved in lipid remodeling in
Pelagibacter sp. str. HTCC7211. See text for detailed rationale for predicted functions.
169
gluconeogenesis
OPO3H2
HO
glycerone-P
O
HO
OPO3H2
glycerol-P
OH
Ca. P. ubique and
Pelagibacter sp. str. HTCC7211
O
R
S-CoA
O
Pelagibacter sp. str. HTCC7211 only
R
LCB5
O
Pi
OPO3H2
R
O
O
phosphatidate
CTP
serine
glycerol-P
CDP-diacyl-glycerol
O
O
O
R
O
OPO2HO
OH
R
R
O
OPO2HO
PO4H2
NH2
O
R
O
OH
O
O
CO2
NH2
R
O
R
OPO2HO
R
Pi
O
O
O
OPO2HO
R
O
O
OH
OH
O
O
phosphatidylethanolamine
phosphatidylglycerol
plcP
NH2
PO4
PO4
OH
OH
O
R
O
OH
R
O
O
HOH
HO
HO
HO
H
OH
OH
H
H
O
NH2
HO
NH2
ornithine
NDP-hexose
rfaG-like
glycosyltransferase
hemolysins
COG3176
O
O
HO H
O
OH
H
R
O
R
HO
O
OH
OH
H
H
NH2
HO
R
NH
O
R
O
O
O
Figure A3- 5: Simplified illustration of phospholipid biosynthetic routes and predicted
reactions involved in lipid remodeling in Pelagibacter sp. str. HTCC7211.
170
+Pi
Pi-starved
+Mpn
Figure A3- 6: Scanning transmission electron microscopy images of Pelagibacter sp. str.
HTCC7211 cells grown under different P-regimes. +Pi: cells grown with excess Pi
(10 µM). Pi-starved: cells grown with excess Pi (10 µM), washed and resuspended in Pifree medium for 96 hours. +Mpn: cells grown with excess Mpn (10 µM) and no Pi. Cells
for the +P and +Mpn treatments were harvested for imaging in mid-logarithmic growth
phase. Cells were stained with osmium and lead as described in methods. Arrowheads in
“+Mpn” indicate putative polyphosphate granules.
171
Table A3- 1: Specific growth rates1 of Ca. P. ubique & Pelagibacter sp. str. HTCC7211
in AMS1 with different amounts of Pi or Mpn
specific growth rate (day-1)
amount added (nM)
HTCC1062 HTCC7211+Pi HTCC7211+Mpn
0
0.24 ± 0.03
0.58 ± 0.01
0.41 ± 0.01
1
0.29 ± 0.05
0.59 ± 0.03
0.42 ± 0.01
3.3
0.31 ± 0.05
0.59 ± 0.02
0.45 ± 0.03
10
0.35 ± 0.04
0.58 ± 0.01
0.43 ± 0.02
33
0.41 ± 0.02
0.6 ± 0.01
0.45 ± 0.00
100
0.41 ± 0.02
0.57 ± 0.02
0.44 ± 0.02
1000
0.39 ± 0.02
0.60 ± 0.01
0.39 ± 0.07
3000
0.39 ± 0.02
0.58 ± 0.00
0.45 ± 0.02
Average (n=24)
0.35 ± 0.07
0.59 ± 0.02
0.43 ± 0.03
1
presented as mean of biological replicates ± 1.0 s.d. (n=3)
172
Table A3- 2: Genes differentially regulated in P-limited Ca. P. ubique 4 hours after resuspension.
ORF name
Gene
SAR11_1176 pstB
SAR11_1177
SAR11_1178
pstA
pstC
SAR11_1179
pstS
Fold
qAnnotation Change value
transport system ATP-binding protein
2.37 ≤0.005
transport system permease protein
phosphate transport system permease
protein
transport system substrate-binding
protein
2.92 ≤0.005
3.74 ≤0.005
3.58 ≤0.005
173
Table A3- 3: Genes differentially regulated in P-limited Ca. P. ubique 20 hours after resuspension.
ORF name
SAR11_0032
SAR11_0033
SAR11_0059
SAR11_0060
SAR11_0181
SAR11_0254
Gene
ftsZ
mraZ
pilB
pilQ
ibpA
trmD
SAR11_0635 umuD
SAR11_0641 recA
SAR11_0747 glnA
SAR11_0921
lexA
SAR11_0953 yhdW
SAR11_0964
SAR11_0965
SAR11_1093
rpoA
SAR11_1100
SAR11_1102
SAR11_1103
SAR11_1104
SAR11_1107
SAR11_1110
SAR11_1114
SAR11_1119
SAR11_1122
rpsE
rplF
rpsH
rpsN
rplN
rplP
rplB
fusA
rpoC
SAR11_1164
SAR11_1174
SAR11_1175
SAR11_1176
phoB
phoU
pstB
SAR11_1177
SAR11_1178
pstA
pstC
Annotation
cell division protein FtsZ
cell division protein MraZ
type IV pilus assembly protein PilB
type IV pilus assembly protein PilQ
heat shock protein A
tRNA (guanine-N(1)-)methyltransferase
DNA polymerase V subunit
recombination protein RecA
glutamine synthetase I
LexA repressor
general L-amino acid transport system
substrate-binding protein
transcriptional regulator, Fur family
unknown protein
DNA-directed RNA polymerase
subunit alpha
ribosomal protein S5
ribosomal protein L6
30S ribosomal protein S8
30S ribosomal protein S14
50S ribosomal protein L14
50S ribosomal protein L16
50S ribosomal protein L2 RplB
translation elongation factor EF-G
DNA-directed RNA polymerase beta
prime chain
hypothetical protein
regulatory protein
transport regulon regulator PhoU
transport system ATP-binding protein
transport system permease protein
phosphate transport system permease
protein
Fold
Change
-2.16
-2.05
-2.00
-2.01
2.70
-2.15
qvalue
≤0.005
≤0.005
≤0.005
≤0.005
≤0.005
≤0.005
2.41
3.43
2.29
4.48
-2.01
≤0.005
≤0.005
≤0.005
≤0.005
≤0.005
-2.80 ≤0.005
-4.17 ≤0.005
-2.35 ≤0.005
-2.05
-2.31
-2.16
-2.42
-2.50
-2.32
-3.10
-2.27
-2.74
≤0.005
≤0.005
≤0.005
≤0.005
≤0.005
≤0.005
≤0.005
≤0.005
≤0.005
2.25
3.30
3.84
9.13
≤0.005
≤0.005
≤0.005
≤0.005
10.58 ≤0.005
10.68 ≤0.005
174
Table A3-3 (continued)
SAR11_1179
SAR11_1181
pstS
transport system substrate-binding
protein
hypothetical protein
40.50 ≤0.005
2.84 ≤0.005
175
Table A3- 4: Genes differentially regulated in P-limited Ca. P. ubique 38 hours after resuspension.
ORF name
Gene
SAR11_0007
hflC
SAR11_0008
hflK
SAR11_0033 mraZ
SAR11_0077
trxB
SAR11_0162 groEL
SAR11_0181
ibpA
SAR11_0203
SAR11_0254 trmD
SAR11_0255
SAR11_0266
rpsP
SAR11_0333
hslV
SAR11_0334
hslU
SAR11_0367
SAR11_0368
SAR11_0399
SAR11_0433
SAR11_0434
SAR11_0520
SAR11_0595
SAR11_0596
SAR11_0597
SAR11_0601
SAR11_0641
SAR11_0747
SAR11_0765
SAR11_0788
SAR11_0818
SAR11_0841
SAR11_0861
SAR11_0907
dnaK
rbr
gltD
gltB
tolR
tolA
tolB
ftsH
recA
glnA
amtB
yeiH
rpsB
Annotation
probable integral membrane proteinase
probable integral membrane proteinase
cell division protein MraZ
thioredoxin reductase
chaperonin GroEL
heat shock protein A
response regulator
tRNA (guanine-N(1)-)methyltransferase
ribosomal protein S16
TRAP dicarboxylate transporter - DctP
subunit
ATP-dependent HslUV protease,
peptidase subunit
ATP-dependent HslUV protease ATPbinding subunit
molecular chaperone DnaJ
molecular chaperone DnaK
Rubrerythrin
glutamate synthase (NADPH) beta
chain
glutamate synthase large subunit
hypothetical protein
TolR transport protein
TolA protein
TolB protein
metalloprotease FtsH
recombination protein RecA
glutamine synthetase I
hypothetical protein
Possible transmembrane protein
ammonium transporter
putative integral membrane protein
short chain dehydrogenase, unknown
specificity
ribosomal protein S2
Fold
Change
2.87
2.84
-2.29
2.69
6.33
6.95
4.64
-2.27
qvalue
≤0.005
≤0.005
≤0.005
≤0.005
≤0.005
≤0.005
≤0.005
≤0.005
-2.13 ≤0.005
2.29 ≤0.005
3.41 ≤0.005
2.44 ≤0.005
3.40
3.39
2.15
2.68
≤0.005
≤0.005
0.0050
≤0.005
2.65
-2.89
2.29
2.20
2.05
2.77
2.37
2.51
3.26
3.26
2.20
-2.11
-2.00
≤0.005
≤0.005
≤0.005
≤0.005
≤0.005
≤0.005
0.0270
≤0.005
≤0.005
≤0.005
≤0.005
0.0110
≤0.005
-2.20 ≤0.005
176
Table A3-4 (continued)
SAR11_0921
SAR11_0964
SAR11_1019
lexA
xerD
SAR11_1030
SAR11_1093
metY
rpoA
SAR11_1094
SAR11_1113
SAR11_1114
SAR11_1117
SAR11_1119
SAR11_1151
rpsK
rpsS
rplB
rplC
fusA
glmS
SAR11_1163
SAR11_1164
SAR11_1174
SAR11_1175
SAR11_1176
phoB
phoU
pstB
SAR11_1177
SAR11_1178
pstA
pstC
SAR11_1179
pstS
SAR11_1181
SAR11_1240
SAR11_1346
aceA
livJ
LexA repressor
transcriptional regulator, Fur family
integrase/recombinase XerD-like
protein
O-acetylhomoserine (thiol)-lyase
DNA-directed RNA polymerase
subunit alpha
ribosomal protein S11
ribosomal protein S19
ribosomal protein L2
ribosomal protein L3
elongation factor EF-G
glutamine-fructose-6-phosphate
transaminase (isomerizing)
unknown
hypothetical protein
regulatory protein
transport regulon regulator PhoU
transport system ATP-binding protein
2.92 ≤0.005
-2.23 ≤0.005
-3.30 ≤0.005
transport system permease protein
phosphate transport system permease
protein
transport system substrate-binding
protein
hypothetical protein
isocitrate lyase
branched-chain amino acid transport
substrate-binding protein
11.52 ≤0.005
16.37 ≤0.005
-2.04 0.0280
-2.96 ≤0.005
-2.36
-2.12
-2.22
-2.05
-2.26
-2.09
≤0.005
≤0.005
0.0080
0.0060
0.0080
0.0060
2.63
2.17
3.94
5.00
9.17
≤0.005
≤0.005
≤0.005
≤0.005
≤0.005
27.92 ≤0.005
2.24 ≤0.005
-2.00 ≤0.005
-2.08 0.0070
177
Table A3- 5: Genes differentially regulated in P-limited Pelagibacter sp. str. HTCC7211
68 hours after re-suspension.
ORF name
Gene
PB7211_757
PB7211_1178
PB7211_503
phnM
PB7211_450
phnL
N/A
phnK
PB7211_943
PB7211_192
PB7211_797
phnJ
phnI
phnH
PB7211_1322
phnG
PB7211_620
phnC
PB7211_926
phnD
PB7211_232
phnE
PB7211_725
phnE2
PB7211_828
PB7211_545
PB7211_679
PB7211_412
PB7211_586
phoU
pstB
pstA
PB7211_733
pstC
PB7211_1190
pstS
PB7211_635
PB7211_1302
PB7211_960
PB7211_983
Annotation
Fold
qChange value
GYD domain superfamily
2.02 ≤0.005
conserved hypothetical protein
2.23 ≤0.005
alkylphosphonate utilization protein
7.26 ≤0.005
PhnM
phosphonate C-P lyase system protein
2.05 ≤0.005
PhnL
alkylphosphonate utilization protein
2.21 ≤0.005
PhnK
phosphonate metabolism PhnJ
3.21 ≤0.005
phosphonate metabolism PhnI
4.39 ≤0.005
alkylphosphonate utilization protein
3.78 ≤0.005
PhnH
alkylphosphonate utilization protein
2.92 ≤0.005
PhnG
phosphonate ABC transporter, ATP21.76 ≤0.005
binding protein
phosphonate ABC transporter,
17.79 ≤0.005
periplasmic binding protein
probable phosphonate ABC transporter,
9.60 ≤0.005
permease protein
phosphonate ABC transporter,
7.29 ≤0.005
permease protein
hypothetical protein
12.01 ≤0.005
probable HD-hydrolase
2.74 ≤0.005
transport regulon regulator PhoU
2.14 ≤0.005
ABC transporter
3.22 ≤0.005
ABC transporter; transport system
3.46 ≤0.005
permease protein
ABC transporter; transport system
3.47 ≤0.005
permease protein
transport system substrate-binding
3.74 ≤0.005
protein
Hemolysin (COG3176)
5.17 ≤0.005
Hemolysin (COG3176)
2.92 ≤0.005
probable glycosyl transferase
2.74 ≤0.005
calcineurin-like metallophosphoesterase
2.68 ≤0.005
178
Table A3- 6: Genes differentially regulated in P-limited Pelagibacter sp. str. HTCC7211
96 hours after re-suspension.
ORF name
Gene
PB7211_613
gloB
PB7211_351
PB7211_503
phnM
PB7211_764
N/A
PB7211_450
phnN
phnL
N/A
phnK
PB7211_943
PB7211_192
PB7211_797
phnJ
phnI
phnH
PB7211_1322
phnG
PB7211_620
phnC
PB7211_926
phnD
PB7211_232
phnE
PB7211_725
phnE2
PB7211_828
PB7211_545
PB7211_619
PB7211_98
arsC
glpF
PB7211_412
PB7211_586
pstB
pstA
PB7211_733
pstC
PB7211_1190
pstS
Annotation
Fold
qChange value
cytoplasmic hydroxyacylglutathione
2.21 ≤0.005
hydrolase
putative alginate lyase
3.13 ≤0.005
alkylphosphonate utilization protein
9.87 ≤0.005
PhnM
conserved hypothetical protein
2.13 ≤0.005
phosphonate metabolism protein phnN
2.46 ≤0.005
phnL; phosphonate C-P lyase system
3.29 ≤0.005
protein PhnL
phnK; alkylphosphonate utilization
3.68 ≤0.005
protein PhnK
phosphonate metabolism PhnJ
5.23 ≤0.005
phosphonate metabolism PhnI
6.02 ≤0.005
alkylphosphonate utilization protein
4.77 ≤0.005
PhnH
alkylphosphonate utilization protein
4.13 ≤0.005
PhnG
phosphonate ABC transporter, ATP30.55 ≤0.005
binding protein
phosphonate ABC transporter,
21.11 ≤0.005
periplasmic binding protein
probable phosphonate ABC transporter,
17.75 ≤0.005
permease protein
phosphonate ABC transporter,
11.93 ≤0.005
permease protein
hypothetical protein
15.89 ≤0.005
probable HD-hydrolase
2.71 ≤0.005
probable arsenate reductase
2.30 ≤0.005
putative transport transmembrane
2.07 ≤0.005
protein
ABC transporter
2.86 ≤0.005
ABC transporter; transport system
3.12 ≤0.005
permease protein
ABC transporter; transport system
3.11 ≤0.005
permease protein
transport system substrate-binding
3.66 ≤0.005
protein
179
Table A3-6 (continued)
PB7211_635
PB7211_1302
PB7211_960
PB7211_983
PB7211_1423
PB7211_870
PB7211_1410
PB7211_497
Hemolysin (COG3176)
Hemolysin (COG3176)
probable glycosyl transferase
calcineurin-like metallophosphoesterase
ycfV
ABC transporter ATP-binding protein
homoserine O-acetyltransferase
conserved hypothetical protein
unknown major facilitator family
permease
8.17
2.75
4.07
3.61
2.11
2.20
2.17
2.03
≤0.005
≤0.005
≤0.005
≤0.005
≤0.005
≤0.005
≤0.005
≤0.005