Metatranscriptomic and physiological analyses of
proteorhodopsin-containing marine flavobacteria
by
sA4ETs INSWiJ
H TECHNOLOGY
OF
Hana Kim
B.S. Biology, Second Major in Oceanography
Inha University (2006)
U BAR IES
Submitted to the Department of Civil and Environmental Engineering
in Partial Fulfillment of the Requirements for the Degree of
MASTER OF SCIENCE
in Civil and Environmental Engineering
at the MASSACHUSETTS INSTITUTE OF TECHNOLOGY
September 2013
02013 Massachusetts Institute of Technology. All rights reserved.
Signature of Author:
Hana Kim
Department of Civil and Environmental Engineering
August 5, 2013
Certified by: .
dward F. DeLong
oulder Professor of
Mort-d
C '
Civil and Environmental Engineering and Biological Engineering
Thes
upervisor
Accepted by:
Heidi
epf
Chair, Departmental Committee for Graduate Students
Metatranscriptomic and physiological analyses of
proteorhodopsin-containing marine flavobacteria
By
Hana Kim
Submitted to the Department of Civil and Environmental Engineering
on August 5, 2013 in partial fulfillment of the requirements for the degree of Master of
Science in Civil and Environmental Engineering
Abstract
Proteorhodopsin (PR) is a seven-helix integral membrane protein that uses retinal as a
chromophore.
PRs transport protons from the cytoplasmic (CP) to the extracellular (EC)
side of the cell membrane utilizing the energy from light. Since PR was first discovered in
marine Gammaproteobacteria,similar types of rhodopsins have been found in all three
domains of life (archaea, bacteria, and eukaryotes). Recent studies have suggested that
some flavobacteria showed a light-dependent increase in cell yield and growth rate of
cultures grown in low carbon media. Although their function as proton pumps with
energy-yielding potential has been suggested in some strains, the photophysiological role
of proteorhodopsins remains largely unexplored. This thesis describes the functional
characterization of PR-containing flavobacteria previously identified from a (GomezConsarnau et al. 2007; Yoshizawa et al. 2012). We describe here experiments performed to
help understand how PR-containing marine flavobacteria respond to varied DOC
concentrations during light-dependent growth, using growth curve observations, inhibitor
experiments and transcriptomic analyses.
The light-dependent growth effects demonstrated a dependence on carbon concentration,
decreasing at increasing carbon concentration in all PR-harboring strains examined in this
study. Interestingly however, the inverse results were observed at high carbon
concentration (48.5 mM C) which resulted in higher cell yields when grown in the dark
than in the light. Growth experiments using 2-(4-methylphenoxy)triethylamine (MPTA) as
an inhibitor of f-carotene synthesis were performed for the representative isolates,
Dokdonia sp. MED134 and Gilvibactersp. SZ-19, at low and high concentrations of DOC.
These experiments showed that inhibition of retinal biosynthesis abolished the lightstimulated growth response at low DOC concentrations.
Transcriptomic experiments
were designed to determine the effect of DOC concentration on gene expression of PR-
2
containing MED134 under light and darkness. The results show the both PR and retinal
biosynthetic enzymes exhibit significant upregulation in the low carbon condition when
they exposed to the light. Among protein-coding transcripts of high carbon concentration,
beta-oxidation-associated proteins were expressed at significantly higher levels in the dark.
This work furthers our understanding of the details of light-enhanced growth rates and
cell yields in diverse marine flavobacterial isolates, and demonstrate proteorhodopsinassociated light-dependent growth effects at various carbon concentrations in several
different flavobacterial proteorhodopsin photosystems.
Thesis Supervisor: Edward F. DeLong
Title: Morton and Claire Goulder Professor of Civil and Environmental Engineering and
Biological Engineering
3
Acknowledgements
This thesis would not have been possible without the guidance and help of several
individuals who in one way or another contributed and extended their valuable assistance
in the preparation and completion of this study.
First and foremost, I would like to offer my sincerest gratitude to my advisor, Dr.
Edward DeLong, who has supported me throughout my thesis with his patience and
knowledge whilst allowing me the room to work in my own way. I appreciate all his
contributions of time, ideas, and funding to make my Master of Science experience
productive and stimulating. The joy and enthusiasm he has for research was motivational
for me.
I am also thankful to have joined a group of caring, fun, and hardworking colleagues in
the DeLong lab and in Parsons. I would like to especially thank Chon Martinez for her all
the advice she gave me; my fellow graduate students, Mike Valliere, and Tsultrim Palden
for their assistance in the metatranscriptomic data analysis and sharing many happy
moments working on projects together; my lab mates Oscar Sosa, Jessica Bryant and postdoctoral fellow, Dr. Scott Gifford and Dr. Kristina M Fontanez, and John Eppley, for their
insightful discussions and making my lab experience enjoyable.
Last but not least, I owe my deepest gratitude to my parents, Jong Ho Kim and Kyung
Ja Lee, my brother Jeong Su Kim for their love, encouragement and continual support in
all my pursuits.
4
Table of Contents
Abstract..................................................................................2
Acknowledgements
.................................................
4
List of Figures...................................................7
List of Tables....................................................10
CHAPTER 1: MICROBIAL OPSINS, AND INTRODUCTION
1. The important role of proteorhodopsin (PR) in marine ecosystem
1.1 Rhodopsins.................................................................11
1.2 Proteorhodopsins (PRs)......................................12
1.3. Abundance and diversity of PR-containing marine bacteria.......................17
CHAPTER 2.
THE INFLUENCE OF LIGHT AND CARBON ON PR-CONTAINING
FLAVOBACTERIA
2.1 Introduction.........
....................................
18
2.2. Methods and Materials
Bacterial strains and culture conditions..........................19
Preparation of starter cultures for growth experiments...
........
Culture conditions........................................
....... 23
23
Direct microscopic counting methods...........................26
Culture experiments with specific inhibitor
........................
27
2.3 Results and Discussions
Responses of various carbon concentrations on light-dependent growth of PRcontaining marine flavobacterial strains.........................................................
29
Specific growth rate of proteorhodopsin-containing marine flavobacterial
strain .....................................................................................................................
41
Culture experiments with an inhibitor of retinal biosynthetic pathway,
M PTA ...................................................................................................................
44
Growth characteristics of representative flavobacterial strains on MPTA
containing media..........................................47
CHAPTER 3. TRNASCRIPTOMIC ANALYSIS OF LOW AND HIGH CARBON
CONCENTRATIONS ON LIGHT-DEPENDENT GROWTH OF Dokdonia sp. MED134
3.1 Introduction.......................................................51
3.2. Materials and methods
5
Cultivation for transcriptomic analyses..........................57
Total RNA extraction......................................58
ribosomal RNA (rRNA) subtraction............................58
RNA amplification, complementary DNA (cDNA) synthesis
and Illumina sequencing.................................
...59
3.3 Results and Discussions
Cultivation for transcriptomic analyses...........................61
Transcriptom ic analyses..................................................................................
63
Transcriptomic survey for High carbon conditions.........................................64
Conclusion.........................................................74
References...............................................................................76
6
List of Figures
Fig. 1 Scheme of the carotenoid biosynthesis pathways from farnesyl pyrophosphate
(FPP) to retinal...................................................................................................
Fig. 2 Scheme of the all transretinal change to 13-cis retinal....................................
15
16
Fig. 3 Geological maps showing location of the sampling sites of strains tested in this
study........................................................................................................................20
Fig. 4.Phylogenetic tree based on PR amino acid sequences including strains that we
studied in this paper............................................................................................
22
Fig. 5. Specific inhibitor of Lycopene cyclization, 2-(4-Methylphenoxy) Triethylamine
(MPTA) (a) Chemical formula of MPTA. (b) The bioregulatory action of MPTA in
-carotene synthesis ..........................................................................................
28
Fig.6. Growth curves of Dokdonia sp. MED134 (a) and Persicivirgasp. S 1-08 (b)
incubated in the light (o) or in the dark (e).......................................................
32
Fig.7. Growth curves of Winogradskyella sp. PC-19 incubated in the light (o) or in dark
(0 )...........................................................................................................................33
Fig.8. Growth curves of Winogradskyella sp. PG-2 incubated in the light (o) or in dark
(0 )............................................................................................................................34
Fig.9. Growth curves of Gilvibactersp. SZ-19 incubated in the light (o) or in dark
(0 )............................................................................................................................35
Fig.10. Growth curves of Tenacibaculum sp. SZ-18 incubated in the light (o) or in dark
(0 )...........................................................................................................................36
Fig.11. Growth curves of Dokdonia sp. MED134 incubated in the light (o) or in dark
(0 )............................................................................................................................37
7
Fig. 12. Specific growth rate (p) determined by regression analysis based on the growth
experiment of strain Dokdonia sp. MED134................................................40
Fig. 13. Summary of specific growth rates (p) of Dokdonia sp. MED 134 (a) and
Winogradskyella sp. PG-2 (b).......................................................................
42
Fig. 14. Summary of specific growth rates (p) of Winogradskyella sp. PC-19 (a),
Gilvibactersp. SZ-19(b) and Tenacibaculum sp. SZ-18 (c)..........................43
Fig. 15. Colony images of Dokdonia sp. MED134 in culture experiment with a
M PTA ............................................................................................................
45
Fig. 16. Colony images of Gilvibactersp. SZ-19 in culture experiment with a
M PTA ............................................................................................................
46
Fig. 17. Growth curves of Dokdonia sp. MED134 in culture experiments with a MPTA
inhibitor..........................................................................................................
49
Fig. 18. Growth curves of Gilvibactersp. SZ- 19 in culture experiments with a MPTA
inhibitor..........................................................................................................
50
Fig. 19 Scheme showing how the integration of results from different technological levels
of functional genomics leads to construction of a virtual
microorganism ................................................................................................
55
Fig. 20 Scheme of sample processing pipeline for the metatranscriptomic analysis;
adapted from Stewart et al (2010)..................................................................
56
Fig. 21 Determining optimal sampling time points for transcriptomic experiments, based
on growth curves of Dokdonia sp. MED134..................................................
62
Fig. 22. Plot of normalized mean versus log2 fold change for low carbon TI light sample
versus low carbon TI dark sample (a) and for low carbon T2 light sample versus
low carbon T2 dark sample (b).......................................................................
70
Fig. 23. Plot of normalized mean versus log2 fold change for high carbon TI light sample
8
versus high carbon T1 dark sample (a) and for high carbon T2 light sample versus
high carbon T2 dark sample (b)....................................................................
71
Fig. 25. Plot of normalized mean versus log2 fold change for T2 light low carbon versus
T2 light high carbon (a) and for low carbon T2 dark sample versus high carbon
T2 dark sam ple (b).........................................................................................
72
Fig. 24. Plot of normalized mean versus log2 fold change for T1 light low carbon versus
TI light high carbon (a) and for low carbon TI dark sample versus high carbon
T I dark sam ple (b).........................................................................................
9
73
List of Tables
Table 1. PR-containing Flavobacterial strains used in this work..................................
21
Table 2. Composition of agar media used for cultivation studies..................................25
Table 3. Maximum cell density (106 cells ml- 1) of PR-containing marine flavobacterial
strain s ..................................................................................................................
38
Table 4. Specific growth rate (day-) of PR-containing marine flavobacterial
strain s...................................................................................................................39
Table 5. Number of reads remaining after quality control step in the pipeline for quality
control....................................................................................................................65
Table 6. Preliminary analysis of functional enrichment of differential gene expression in
low carbon condition at TI.............................................................................
66
Table 7. Read number and significant upregulated genes in the light in low carbon
condition at T2..............................................................................................
67
Table 8. Read number and significant upregulated genes in the light in high carbon
condition at T I.............................................................................................
68
Table 9. Preliminary analysis of functional enrichment of differential gene expression in
high carbon condition at TI.............................................................................
10
69
CHAPTER 1: MICROBIAL OPSINS, AN INTRODUCTION
1. The important role of proteorhodopsin (PR) in marine ecosystem
1.1 Rhodopsins
Rhodopsins are light harvesting protein that contain retinal (Vitamin A aldehyde) as a
chromophore and have seven transmembrane alpha helices (Spudich 2006). These light
sensitive proteins absorb light and convert it into photonic energy to transport ions across
the cell membrane. More specifically, upon light illumination, the photocycle begins when
the retinal (Vitamin A aldehyde) in the protein undergoes photoisomerization causing
conformational changes of the protein and the creation of the electrochemical gradient that
facilitates ion transportation. Rhodopsins are basically classified into two protein families:
type I and type II rhodopsins. The big difference between type I and type II rhodopsin is
the
photoisomerization
mechanism
of
chromophore.
In
rhodopsin
I,
the
photoisomerization causes retinal changes from all-trans to 13-cis whereas in rhodopsin II,
changes occur from 11 -cis to all trans retinal (Jung 2007). Type II rhodopsins function as
photosensitive receptor proteins in animal eyes that are human rod and cone visual
pigments. Type I rhodopsin is an archaeal-type
rhodopsin and well studied in
Halobacterium salinarium, halophilic archaea. The first discovered rhodopsins are
bacteriorhodopsin (BR), halorhodopsin (HR), sensory rhodopsin I and II (SRI and SRII) in
H. salinarium (Spudich et al. 2000). A number of other rhodopsins have been identified as
playing a role in proton-motive force, although their mechanisms differ. Proteorhodopsins
have been found in marine bacteria,
archaea, and eukarya, Xanthorhodopsins (XRs) use
two chromophores, the carotenoids salinixanthin and retinal, to broaden the spectral range
for light harvesting (Balashov et al. 2005). Actinorhodopsins (ActRs) have been found in
11
abundance in freshwater, including actinobacteria from a hypersaline lagoon, an estuary,
and a freshwater lake (Sharma et al. 2008). Bacteriorhodopsin (BR) and halorhodopsin
(HR) are light-driven pumps that use light as a catalyst to translocate protons and chloride,
respectively. Rhodopsins may be involved in regulating ionic content and the osmotic state
(Mongodin et al. 2005). Sensory rhodopsins (SRs) function like photoreceptors rather than
ion transporters, mediating phototaxis or signal transduction (Spudich et al. 2000 ).
Recently, type I rhodopsins have been found also in other lineages such as, Eubacteria,
and unicellular Eukarya (Bieszke et al. 1999; Sineshchekov et al. 2002; Jung et al. 2003).
That is the reason why type I rhodopsin is also called microbial rhodopsin.
Archaeal-type rhodopsins, type I rhodopsins, serve as a model for proteorhodopsin
photosystems (PRPS) (Kawanabe et al. 2006). In reaction to a light signal, they establish a
chemical gradient from light energy and change their conformation to facilitate the
translocation of protons. In this study, I am focused on proteorhodopsin (PR), which
belongs to the type I rhodopsin family.
1.2 Proteorhodopsins (PRs)
Proteorhodopsins (PRs), a member of the microbrial rhodopsin superfamily of proteins,
are retinal-binding transmembrane proteins that play a key role in light-activated proton
efflux (Beja et al. 2000; Beja et al. 2001; Martinez et al. 2007).
Earlier studies showed microbial rhodopsins to be associated with Archaea but later
studies found PRs in an uncultured marine gammaproteobacterial SAR86 group (Morris et
al. 2002; Boichenko et al. 2006). PR-containing bacteria are found in a minimum of 13%
of all microorganisms in the photic zone, the layer of the ocean that is exposed of sunlight
12
(Stingl et al. 2007).
Planktonic Bacteria, Archaea and Eukarya reside and compete for light in the photic
zone of the ocean (McCarren and DeLong 2007). The PR-bearing marine microbes use
light energy for the acquisition of adenosine triphosphate (ATP) by proteorhodopsin
(Frigaard et al. 2006). The proton motive force (PMF) is generated by an electrochemical
gradient that transports protons across an energy-transducing
membrane. Through
oxidative phosphorylation, bacteria use the PMF to synthesize ATP, drive chemiosmotic
reactions, and power the rotary flagellar motor (Kashket 1985).
The light energy
absorbed by rhodopsins is used to translocated protons outside the cell, thereby generating
the protonmotive force. Proteorhodopsin have been identified in the world's oceans
making this light-transducing protein a key source of solar energy absorption in
photosystems.
Protoeorhodopsins are composed of the opsin protein and the chromophore trans-retinal
(Bielawski et al. 2004; Giovannoni et al. 2005).
Retinal is formed through the
conversion of the carotenoid -carotene (Sabehi et al. 2005; Walter et al. 2007). Many but
not all marine bacteria contain the genes required for retinal biosynthesis, or genes that
enable the conversion of the precursor beta carotene to retinal(Peck et al. 2001).
The 15, 15' C=C bond is located at the central cleavage of p-carotene to form all-trans
retinal. The conversion of the isoprenoid precursors into 0-carotene is catalyzed by four
ci genes: crtE, crtB, crtI, and crtY (Fig.1) (Martinez et al. 2007; McCarren and DeLong
2007). One molecule of
p-carotene
produces two retinal molecules. The rhodopsin
chromophore retinal produces the oxidative cleavage of 0-carotene.
This enzyme produced by cleaving the beta-carotene into two retinals is called 15, 15'0-carotene-dioxgenase. Some proteobacteria can also synthesize f-carotene and convert it
to retinal by using the enzyme encoded by the blh gene to produce functionally active PRs
13
(Kim et al. 2008).
Proteorhodopsin is all a photoactive retinylidene protein that functions as a heptahelical
proton pump (de la Torre et al. 2003). It is simple heptahelical proton pumps containing a
retinal chromophore covalently bound via a lysine Schiff's base to helix G A single
molecule of all-trans retinal is binded by key residues lining the inner surface of the
channel. Ion pumping is then induced by a corresponding change in protein conformation
which causes ion transport accessory effector proteins to interact with the sensory
rhodopsin family of proteins (Fig2) (Ottolenghi and Sheves 1989; Birge 1990).
Abundant evidence exists of PRs function as a transmembrane proton pump, including
the light-mediated transport of protons in right-side-out PRs vesicles. Also, genetic
community surveys suggested that proteorhodoopsin-harboring
microorganisms are
common in the different phylogenetic groups of the oceans by lateral gene transfer,
indicating a fitness advantage (de la Torre et al. 2003; Frigaard et al. 2006; Martinez et al.
2007)
14
IPP 6-isomerase
0
6-
0
o
-p-O-P-O0
, (idl)
-P-o-P-O.
-*
O
0-
6-
Isopentenyl iphosphate
(IPP)
Dimethylallyl diphosphate
(DMAPP)
Ipp
I P
FPP synthase
(ispA)
OPP FPP
GGPP synthase
PP
(crtE)
OPP GGPP
Phytoene synthase
GGPP
(citB)
Phytoene
Phytoenedehydrogenase
4
(ct/)
Lycopene
Lycopene cyclase
Q -Carotene
15,15'-p-carotene dioxygenase
OH
(b/h)
6(-"b
OH
Retinal
Fig. 1 Scheme of the carotenoid biosynthesis pathways from farnesyl pyrophosphate
(FPP) to retinal. (FPP: farnesyl pyrophosphate, IPP: isopentenyl pysophosphte
(diphosphte), CrtE: GGPP synthase, GGPP: geranlygeranyl pyrophosphate, CrtB: phytone
synthase, CrtI: phytoene desaturase, CrtY: Lycopene cyclase, BIh: Bacteriorhodopsinrelated-protein-like-homolog protein)
15
H
Retinal
all irma isomer
Protonated Schiff base
H+
H+N
Light induced
isomnerization
Spontaneous
isomedzation
H+N
H
Retinal
13-cis isomer
Protonated Schiff base
Fig. 2 Scheme of the all trans retinal change to 13-cis retinal.
16
1.3. Abundance and diversity of PR-containing marine bacteria
Oceanic picoplankton play a role in driving biogeochemical cycles. The following
bacteria have been found to be the most prevalent in oceanic picoplankton: phyla
Proteobacteria (63%), Bacteroidetes (13%), Cyanobacteria (7.9%), Firmicutes (7.5%), and
Actinobacteria (4.6%) (Venter et al. 2004).
Following the discovery of PR in uncultured marine gamma proteobacterial SAR86
clade (Beja et al. 2001; Sabehi et al. 2004; Rusch et al. 2007), over 4,000 variants have
been identified (Beja et al. 2001; Dioumaev et al. 2002; Spudich 2006) and several
variants have been cloned into Escherichia coli.
PR families have been found in
Monterey Bay (Eastern Pacific Ocean), Hawaii Ocean Time (HOT, Central North Pacific
Ocean), Palmer station (Beja et al. 2001), Mediterranean Sea, Red Sea (Sabehi et al. 2003),
Sargasso Sea (Venter et al. 2004) and Pacific Ocean (Rusch et al. 2007). Most of the
variants of PR fall under one of two groups depending on their photochemical properties:
green-absorbing Proteorhodopsin (GPR) and blue-absorbing Proteorhodopsin (BPR)
(Kralj et al. 2008). GPR has higher absorption efficiency. GPR absorbs light with a
absorption maxima of k.x 525 nm (green) whereas BPR has a maxima of kmx 490nm
(blue) (Man et al. 2003). Green-absorbing pigments predominately function in surface
water while 'blue-absorbing' pigments function in deeper waters depending on light
availability (Beja et al. 2001).
Recently, it has been suggested that there exist PR-like genes in CFB (CytophygaFlavobacteria-Bacteroides)subdivision as well as Proteobacteria (Venter et al. 2004). In
the Northwest Atlantic, among metagenome fragments, the Global Ocean Sampling
(GOS) expedition found that the taxa of the PR gene was predominantly present in two
assembled flavobacterial genomes (Rusch et al. 2007; Woyke et al. 2009). Flavobacteria,
17
which belong to the phylum Bacteroidetes previously called by the CytophagaFlavobacterium-Bacteroides.
(CFB), and Alpha- and Gammaproteobacteria are major
carriers of photometabolic genes, including the microbial rhodopsin proteorhodopsin in
marine environments (Giovannoni et al. 2005; Rusch et al. 2007). Flavobacteria play a
role in maintaining the earth's energy balance in the biogeochemical cycle of marine
systems (Kirchman 2002; Abell and Bowman 2005; Alonso et al. 2007)
CHAPTER 2.
THE INFLUENCE OF LIGHT AND CARBON ON PR-CONTAINING
FLAVOBACTERIA
2.1 Introduction
Research in recent years has revealed new information about the role and diversity of
PR in proteobacteria; however less attention has focused on the function and diversity of
PR in Bacteroidetes (Zhao et al. 2009). Flavobacteria itself was found both free-living and
attached to organic aggregates and are considered as major mineralizers of organic matter.
(DeLong et al. 1993; Abell and Bowman 2005; Cottrell and Kirchman 2009; Zhao et al.
2009). Among them, PR in Flavobacteria (Dokdonia sp. MED134) was revealed to
enhance the cell yields in the presence of light compared to in dark so that PR fulfils a
phototrophic function in marine bacteria (Gomez-Consarnau et al. 2007). Also, in a PRcontaining marine flavobacterial suspension, light-driven proton transport activity was
sufficient for ATP generation was demonstrated (Yoshizawa et al. 2012). The effect of
light on cell yields has been little studied in cultivated marine alpha proteobacterium, and
primarily limited to the SAR11 strain HTCC1062 Pelagibacterubique (Giovannoni et al.
2005) and gamma proteobacterium SAR92 strain HTCC2207. Therefore, it is expected
18
that flavobacteria will give us information about in vivo function of PR as well as the
relationship between light and cell growth (Stingl et aL 2007).
This study will focus on the discovery of the effect of a light-enhanced growth yield in
proteorhodopsin-containing flavobacterial strains. To see the physiological characteristics
of PR-containing Flavobacteria, we performed growth experiment with various carbon
concentrations in the light or in the darkness. Also, we explored the effect of retinal
biosynthesis inhibitor, MPTA, on light-enhancement growth. To our knowledge, this is the
first time a light-dependent growth yield effect under low and high carbon DOC
concentrations has been demonstrated in native cells. The chapter will conclude with
discussion of the significance of the findings and future directions.
2.2. Methods and Materials
Bacterial strains and culture conditions
Dr. Kazuhiro Kogure (The University of Tokyo, Japan) kindly provide us PR-containing
marine Flavobacteria isolates from sea ice collected in Saroma-ko Lagoon and from the
surface seawater collected at Sagami Bay Station P and western North Pacific Station S
(Fig. 3.).
The bacterial strains used are listed in Table 1. The Flavobacteria strains were routinely
grown in Marine Agar 2216 (Difco Laboratories, Detroit, MI, USA) at 22 'C for 48 h.
19
Fig. 3. Geological maps showing location of the sampling sites of strains tested in this
study.
20
Table 1. PR-containing Flavobacterial strains used in this work
GenBank Accession Number
Organism
Reference or Source
16S rRNA
PR
Dokdonia sp. Strain
DQ481462
Gonzilez et al., 2011
MED134
Winogradskyella sp. PC-19
AB557530
AB557551
Yoshizawa et al., 2007
Winogradskyella sp. PG-2
AB557522
AB557552
Yoshizawa et al., 2007
Gilvibactersp.
AB557542
AB557573
Yoshizawa et al., 2007
Persicivirgasp. S 1-08
AB602426
Not yet
Yoshizawa et al., 2007
Tenacibaculum sp. SZ-18
AB557536
AB557572
Yoshizawa et al., 2007
SZ-19
21
WnogradsA yana
sp
WInogradsyeft
so PG-3 (AB557553)
PC-O
(AI35575W)
-
(AS567555)
110ogodkyelasoPCIS AB5575) *1
~4n
RaGrouprmsk
Wifoogradsktyefia so PG-10
Winogradskyete so PG-20 (AS557559)
Wnogradskyofia
so P2-1 I (AS565650)
GopW
W2
waogodskeyla so PC-8 (AR55549) ~ Group W3
Uncultured bactedum clone A-04-6(3) (EU663483)
Polsittwoor so, PG-S 4ASS57564)
100 Pcaa.boctar sp PGA19 (AB57558)
Group P2
Ufedbacter su PGo2 (A557556) *
w
Group T1
Group T2
00000000)
M 024-3C(EA
num
l bacter
Uncuturgo
o*dona
d=r
a
onVsae MEM134 (AAM2DOOO(05)
POeyRdbtor sp. MED1 52 MED152 (AAA00005000)
Paolbrecfer
aeriuM
234- (AAG04000M
00)
1o0
1
arbacter
o
SA4-10
m p(A557575) a
tbecter sp SA4-47 (AB55753)
G
7G
pP
Uncultured bacteium dcone W1-4 (EU(A83550)
Uncultured bectedum clone W-50-2 (EU683584)
Uncultured bacterium cone WW1-2 (ELMUM49)
Uncultured bacterium clone A140-3 (EU683491)
sp .
-ordia
Winogradkye
s. PG-2
10 WUncraistu
abacePuclne
S.-5iS2>
(A9557551)
Group W1
(A757557)
e
I
eigonie douq train
mnsis
n PRHC
O6
(F427093)
84romwsp PZ- 3 (AS557561)
PSYChAWOMeUS ftfrgds ATCC 700755 A APR00000000)
Wogradsmyea
4
PG-16
so
50
PAthosate
sd S n atoo
su
bogr adapte bo
foed
(AAin00
s
SA4-32S(AB55758I)*
Floolacteiou nBsp
PsF''awobactedumso.SA415 (ASS57576)
wiay). ctedum sp.SA4-23 (Ao57579)
Rbstitutiduo
s. SA4-4
s
(AB57585)
Flawobactehumso, SA4-8 (AS557584)
'00 oareu So. SA4-44 (AB557582)
sFlembcrehum so. SA4-20 (ASS57578)
Flatobactenumop. SA4-10 (ASS57577)
at Flemabscewrrurso, SA4-3W (AB557580)
54Uncultured bacterium olonm WI-13 (E~MOMS
Uncultured bacterium ARCTIC02-G-04 (EU?95231)
bacterium dlone A1-0-7(3) (EL*83481)
Uncultured bacterium dlone W6.30-8 (ELM683MI)
96Uncultured bacledurn clone WO-0-10 (EULS3M72)
-SpirosameMoguerp DSM 74 (CP001769)
-P&Wgirbmtertfi
e strain HTWc1062 (EF64091 1)
Uncultured
Fig. 4.Phylogenetic tree based
on PR amnino acid sequences including strains that we studied in
this paper. Figure adapted from Yoshizawa et al., 2007. Red boxes indicate strains examined in this
study. Strains are color coded to indicate site of isolation (Blue, Saromako-Lagoon;
western North Pacific, Green, Sagami Bay). Bootstrap
substitutions per nucleotide position.
22
Orange,
values are shown at each node. Bar, 0. 1
Preparation of starter cultures for growth experiments
Starter cultures for the growth experiment at various carbon concentrations were grown
in acid-washed borosilicate glass culture bottles (1 L, VWR), with 500 mL of artificial
seawater (ASW, 15 practical salinity units, Sigma CAS No: 7647-14-5) supplemented
with a full strength media (FSM, 1.1 mM C; Table 2). The ASW was filtered through 0.2
mm-pore-size bottle-top vacuum filter system (Nalgene, NY, USA) and autoclaved. A
colony grown on marine agar plate streaked from frozen stock was inoculated in ASW
amended with 1.1 mM C FSM media. The starter culture was covered with aluminum foil
(Reynolds) and grown in the dark at 21'C - 22*C until near the middle of the exponential
phase, which took from 3 to 5 days, depending on the strain, the conditions of growth, and
the initial cell density. Cell density of the starter culture was determined during lateexponential phase by the direct cell counting method. Then, the starter culture was stored
in a 4C refrigerator and used to inoculate further growth experiments. And then all
cultures were inoculated with a known number of cells (1000 cells/ml).
Culture conditions
Experiments to determine the effect of DOC concentration on light-dependent
physiological effect of the PR-containing Flavobacterial strains were inoculated from a
stationary phase of starter culture grown on 1.1 mM DOC in a dark condition. Inoculation
was taken from this stationary phase culture stored in 4
C. The culture density was
followed (cells/mL) up to the time of inoculation and was used to determine the dilution of
the starter culture into the DOC concentration experiment cultures. And then all cultures
were inoculated with a known number of cells (1000 cells/ml). A common culture was
23
inoculated for all experiment conditions in ASW amended with FSM. Thorough mixing
was provided at each step through rigorous vortexing.
Cultures of PR-containing flavobacterial strains were grown in artificial seawater (ASW,
15 practical salinity units, Sigma CAS No: 7647-14-5) supplemented with a dissolved
organic carbon (DOC) at the indicated concentration (Table 2; full strength FSM contains
1.1 mM DOC). The ASW was filter-sterilized through 0.2 mm-pore-size bottle-top
vacuum filter system (Nalgene, Rochester, NY, USA) and autoclaved. Then, 250 ml
aliquots of ASW (containing a background concentration of 0.05 mM C of DOC) were
partially supplemented with full strength medium (FSM). Concentrations of DOC in the
ASW media tested were: 0.05 mM, 0.14 mM, 0.39 mM, 1.1 mM, and 48.5 mM. To avoid
inorganic nitrogen and phosphate limitation, the media were also amended with 225 Im
NH 4Cl (Sigma CAS No: 12125-02-9) and 44.7 pm Na 2HPO 4 -12H 20 (Sigma CAS No:
10039-32-4). Cultures were grown in 50 mL glass tubes. Tubes for the dark condition
were wrapped with aluminum foil and tubes for the light condition were left unwrapped.
Cultures were incubated in a walk-in light room incubator. The temperature of the
incubator is maintained at 21 C - 22 C.
To avoid DOC contamination in the growth experiment, it was extremely important to
clean all glassware used in the analysis of growth characteristics very carefully. All
glassware was acid-washed with 10% HC1 (Macron Chemicals CAS No: 7647-01-0) with
an overnight contact time and then thoroughly rinsed with distilled water. Acid-washed
glassware was autoclaved before use.
24
Table 2. Composition of agar media used for cultivation studies.
Marine Agar 2216
Full Strength Medium
(g/L)
(g/L)
Peptone
5
Peptone
5
Yeast extract
1
Yeast extract
1
Ferric citrate
0.1
Artificial sea water (ASW),
1000
ml
Sodium chloride
19.45
Magnesium chloride
5.9
Magnesium sulfate
3.24
Calcium chloride
1.8
Potassium chloride
0.55
Sodium bicarbonate
0.16
Potassium bromide
0.08
Strontium chloride
34 mg
Boric acid
22 mg
Sodium silicate
4 mg
Sodium Fluoride
2.4 mg
Ammonium nitrate
1.6 mg
Disodium phosphate
8 mg
Agar
DDW, ml
15
1000
25
Direct microscopic counting methods
Total cell numbers were determined by direct microscopic count according to the
method described previously (Kimura et al, 2011). The appropriate volume of culture was
diluted using ASW and stained with 100 pL of lOOX SYBR Green (Molecular
Probes/Invitrogen) for 15 minutes under darkness. Then, the solution was vacuum filtered
with a 25 mm diameter, 15-mL funnel filter apparatus (VWR) onto a black 0.22 pm
isopore membrane (Millipore, #GTBP02500). Microscope slides were made using a glass
slide (Gold Seal Microslides #3010) with immersion oil (Zeiss Immersol 518) and
coverslip (Thermoscientific mSeries Lifterslip 22x251-2-4816). Finally, cellular growth
was determined using an epifluorescence microscope (Zeiss Axioskop 2 microscope) with
a 100 X magnification objective (Zeiss Plan-NEOFLUAR). Bacterial cell densities with
the proper dilution were counted at least 10 separate fields of view across the filter. Total
cell densities of samples were calculated using the following equation (from Wetzel and
Likens, 1991).
Bacteria ml' = (membrane conversion factor * ND)
Membrane conversion factor = Filtration area/area of micrometer field
N = Total number of bacteria counted/number of micrometer fields counted
D = Dilution factor; volume of sample stained/total volume of sample available
26
Culture experiments with specific inhibitors
Kimura et al. (2011) proposed that retinal-bound PR has a important role in the lightstimulated growth by culture experiment with MPTA inhibitor. MPTA act as a inhibitor of
lycopene cyclization in retinal biosynthesis pathway (Cunningham et al., 1994; Armstrong,
1999).
First, to confirm the effect of MPTA inhibitor on PR-containing flavobacterial strains,
we cultured strains on Marine Agar 2216 amended with MPTA at a final concentration of
300 mM.
The agar plates were incubated at room temperature for 3 days. When colonies were
clearly visible, we determined the effect of MPTA against flavobacterial strains on the
inhibition of 0-carotene synthesis as indicated by colony pigmentation. Next, to see the
PR-induced light enhancement in growth, Dokdonia sp. MED134 and Gilvibacter sp. SZ-
19 were grown in ASW slightly enriched with FSM (0.14mM C and 48.5mM C) and
amended with MPTA. MPTA was dissolved in methanol and added to ASW at a final
concentration of 100 mM. The same volume of methanol was added to negative control
cultures without MPTA. These cultures were incubated at 21- 22 C under continuous
white light (approximately 150 mmol of photons m-2 s-1) or in the darkness in both carbon
concentrations. The culture experiments with MPTA were performed in triplicate.
Bacterial cell density was measured every day by the direct counting method with
epifluorescence microscope described above.
27
(a)I
N
CH
3
CH 3
2-(4-Methylphenoxy)Triethylamine (MPTA)
(b)
Lycopene
M PTA
Lycopene cycla
(cit)
+-Carotene
15,15'- -carotene dioxygenase
OH
(b/h)
6"%)%COH
Retinal
Fig. 5. Specific inhibitor of Lycopene cyclization, 2-(4-Methylphenoxy)Triethylamine (MPTA) (a)
Chemical formula of MPTA. (b) The bioregulatory action of MPTA in 0-carotene synthesis.
28
2.3 Results and Discussions
Responses of various carbon concentrations on light-dependent growth of PRcontaining marine flavobacterial strains
Several research studies have been shown that the light-stimulated ATP production or
growth in PR-containing bacteria could be detected under carbon limiting conditions
(Gomez-Consarnau et al., 2007; Martinez et al., 2007; Gomez-Consarnau et al., 2010;
Steindler et al., 2011). In this study, we used marine flavobacterial strains to measure the
sensitivity to light of the PR light-transducing genes as DOC concentration was varied in a
culture media. To determine potential interference with very low levels of DOC caused by
endogenous carbon or carryover from the starter culture, the effect of the DOC
concentration was determined using a stationary phase culture grown on 1.1 mM glucose
in the dark. Then, the cells were washed twice using ASW. Cell density was directly
counted under the epifluorescence microscopy with SYBR Green (Applied Biosystems)
staining.
Before the beginning of the growth experiment with various carbon concentrations, we
confirmed the light-enhanced growth at low carbon concentration (0.14 mM C) on
Dokdonia sp. MED 134 and Persicivirgasp. S 1-08 (Fig. 6). In both strains, cell densities
were increased significantly, which were consistent with those seen in before. This shows
that further growth experiments with different carbon concentrations are needed to
investigate the PR-containing marine flavobacterial strains.
Flavobacteria
strains
(Dokdonia
sp.
MED134,
Winogradskyella sp.
PC-19,
Winogradskyella sp. PG-2, Gilvibacter sp. SZ-19, Persicivirgasp. S1-08, Tenacibaculum
sp. SZ-18; Yoshizawa et al, 2012) were grown under light and dark conditions while the
29
concentrations of DOC were varied. Our results showed that as the concentration of DOC
was increased there was a diminishing advantage of growth in the light over the dark (Fig.
7-11). This result is consistent with those of other studies analyzing the effect of different
carbon concentrations on light-induced increases in the growth yield in PR-containing
flavobacterium. Figure 7-11 shows characteristic growth curves for flavobacterial strains
grown on ASW media enriched with varied concentrations of carbon.
The measure of carbon concentrations showed that smaller amounts of carbon resulted
in a different growth yields between cultures grown in light and dark conditions.
Specifically, DOC concentrations below 1.1 mM showed a significant difference in the
maximum growth yield with increased yields in cultures grown in the light compared to
those in the dark (Fig. 7-11). In contrast, the difference between the light and dark cultures
was not significant at a DOC levels of 1.1 mM. The cell yield ratio between light and dark
showed a decreasing trend with increasing DOC concentration at DOC concentrations
greater than 1.1mM (Figure 7-11). Therefore, it can be concluded that proteorhodopsin has
a favorable impact on yields at low carbon concentrations. However, interestingly, the
inverse was true at high carbon concentrations (48.5mM C) in the dark, which resulted in
higher yields when grown in the dark than in the light. We could not fully explain the
reason at this time, but it has been suggested that PMF generated respiration could interact
with the PR.
In some microbial strains containing light transport-inducing rhodopsins, biomass
yields increased and rates of aerobic respiration decreased when the bacteria were grown
in light conditions (Gomez-Consarnau et al. 2007; Lami et al. 2009; Gomez-Consarnau et
al. 2010; Kimura et al. 2011). Several theories have been presented to explain the
mechanism, largely unknown, that could interfere with the ability of PRs to maintain
30
cellular proton mobility force even during respiratory distress. One theory states that
"backpressure" from the PMF directly interacts between the respiratory proteins and
proteorhodopsin, reducing its effect on proton pumping. As a result, PR proton pumping is
replaced by respiration. With the electrochemical potential decreased, the PRs are unable
to translocate protons and electrons across the cell membrane to a terminal electron
acceptor during cellular respiration.
Another possibility would be regulation of metabolic pathways, such as through a
decrease in available ATP, which results in fewer electron donors of the respiratory chain
being formed. These include NADH and the FADH 2 of the tricarboxylic acid (TCA) cycle.
Anabolic pathways competing for metabolites could result in a decrease in respiratory flux.
The high respiration rate may be caused by the excess nutrients which may, in turn,
interfere with the function of proteorhodopsin.
Studies on marine flavobacterial strains containing proteorhodopsin have similarly
found that the light-stimulated growth effect in PR-harboring flavobacteria only occurs at
low carbon substrate concentrations. However, the mechanism by which an increase in
carbon decreases the light effect was not elucidated in this study. To get a better
understanding, we performed transcriptomic analysis from cultures of Dokdonia sp.
MED134 at various carbon concentration and different incubation time.
31
so
(a)
60.2
an
0
C
0
40-
201
0
U
0Light
+
-4Id
2d
3d
4d
5d
6d
Dark
7d
Time (day)
40
(b)
30 -
an
8
0
20.
C
0
0
i
OW0I
U
-
Light
SDark
0.
Id
2d
3d
4d
Sd
6d
7d
Time (day)
Fig.6. Growth curves of Dokdonia sp. MED 134 (a) and Persicivirgasp. S 1-08 (b)
incubated in the light (o) or in the dark (o). Strains were grown in ASW enriched to 0. 14m
C. Error bars indicate standard deviation for triplicate cultures.
32
20-
(a)
(b)
6-
15
4.
10
50
0.
0*
Id
2d
3d
44
5d
68
70
Id
2d
3d
Time (Day)
70
(C)
4d
5d
6d
7d
Time (Day)
60
(d)
60
S40
40-
.- 30
20-
20
10
0
0-
+ LONh
--W- Dark
0
Id
2d
3d
4d
Time (Day)
5d
6d
7d
Id
2d
3d
4'd
Time
;d
6d
74
(Day)
Fig.7. Growth curves of Winogradskyella sp. PC-19 incubated in the light (o) or in dark (e). Winogradskyella sp. PC-19 was grown in ASW enriched to
0.14 mM C (a), in ASW enriched to 0.39 mM C (b), in ASW enriched to 1.1 mM C (c), and , in ASW enriched to 48.5 mM C (d).
20
(a)
(b)
15 -
I
10-
IC.)
0-
01
2
1~~
03
04
-0-I
00
06
D7
DI
02
03
04
Do
D7
Time (Day)
Time (Day)
(c)
(d)
30
E 20
20
15
0
0*
Dl
02
03
D4
ime (Day)
05
06
D7
DI
02
03
04
06
06
i 1
07
Time (Day)
Fig.8. Growth curves of Winogradskyella sp. PG-2 incubated in the light (o) or in dark (9). Winogradskyella sp. PG-2 was grown in ASW enriched to 0.14
mM C (a), in ASW enriched to 0.39 mM C (b), in ASW enriched to 1.1 mM C (c), and), in ASW enriched to 48.5 mM C (d).
14-
6-
(b)
(a)
1210
4-
-
I
0
3-
24C
1-
20-
Id
2d
3d
4d
5d
id
6d
2d
3d
4d
5d
6d
a0
(C)
(d)
30LA
60-
E
2
-
20 -
40-
0
1520-
10 0
5-
r~i-~1
L~J
00-
-
Id
2d
3d
4d
Time (Day)
Sd
+Light
-Dak
Od
1d
2d
3d
4d
5d
Od
Time (Day)
Fig.9. Growth curves of Gilvibactersp. SZ-19 incubated in the light (o) or in dark (e). Gilvibactersp. SZ-19 was grown in ASW enriched to 0.14 mM C
(a), in ASW enriched to 0.39 mM C (b), in ASW enriched to 1.1 mM C (c), and ), in ASW enriched to 48.5 mM C (d).
a*
(a)
.
(b)
30.
25-
-5'
14
12.
-
20.
10
a.
6-
10-
4-
C)
5-
2.
0-
0Id
(c)
2d
3d
4d
5d
Gd
7d
so-
(d)
Id
2d
3d
18
2d
38
4d
5d
4d(d
Sd
Od
7d
35
30
25-
40-
2,0-
20-
5
1d
2d
3d
4d
Time (Day)
5d
Gd
7d
7y
Time (Day)
Fig.10. Growth curves of Tenacibaculum sp. SZ-18 incubated in the light (o) or in dark (9). Tenacibaculum sp. SZ-18 was grown in ASW
enriched to 0.14
mM C (a), in ASW enriched to 0.39 mM C (b), in ASW enriched to 1.1 mM C (c), and), in ASW enriched to 48.5 mM C (d).
(b)
10.
2.0-
II
A
4.
-0.5-
0.
DAY I
DAY 2
DAYS3
DAYS
DCA YS
DAYS6
DAY I
DAY 2
AY3
ime
DAY4
DAYS
DAYS
Time
125
M
i
S.
3-
4.
2-
= 2-
j
0DAY I
DAY 2
DAY 3
Mu.
DAY
4
DAY S
DAY
6
DAY I
DAY
j
2
DAY 3
F-0--- tE
DAY 4
DAYS
DAY I
TM.
Fig.11. Growth curves of Dokdonia sp. MED134 incubated in the light (o) or in dark(*). Dokdonia sp. MED134 was grown in unenriched ASW (0.05
mM C) (a), in ASW enriched to 0.14 mM C (b), in ASW enriched to 1.1 mM C (c), and ), in ASW enriched to 48.5 mM C (d). Error bars denote standard
deviation for the triplicates.
Table 3. Maximum cell density (106 cells ml-) of PR-containing marine flavobacterial strains
ASW +0.05mM C
ASW +0.14mM C
ASW + 0.39mM C
ASW + 1.1mM C
ASW + 48.5mM C
Strain
L
Dokdonia sp. MED134
wj
D
L
D
L
D
L
D
L
D
0.23
0.06
9.32
6.28
-
-
46.7
47.0
442
529
Winogradskyella sp. PC-19
-
-
7.30
4.72
17.5
18.3
47.2
59.6
349
659
Winogradskyella sp. PG-2
-
-
10.1
7.44
18.2
19.0
59.9
95.3
41.0
288
0.13
0.05
9.27
6.31
-
-
47.5
50.4
526
712
3.26
2.46
1.97
1.87
2.56
2.84
3.27
3.51
3.53
3.43
00
Gilvibactersp.
SZ-19
Persicivirgasp. S 1-08
Tenacibaculum sp. SZ-18
-
-
Table 4. Specific growth rate (day-1) of PR-containing marine flavobacterial strains
ASW + 0.05mM C
ASW + 0.14mM C
ASW + 0.39mM C
ASW + 1.1mM C
ASW + 48.5mM C
Strain
L
D
L
D
L
D
L
D
L
D
Dokdonia sp. MED134
1.53
0.62
3.20
2.96
-
-
3.94
4.04
4.99
5.14
Winogradskyella sp. PC-19
-
-
1.93
1.75
1.94
2.22
2.17
2.60
3.38
3.95
Winogradskyella sp. PG-2
-
-
0.74
0.73
0.81
0.82
1.16
1.29
1.04
1.29
Gilvibactersp. SZ-19
0.76
0.49
1.44
1.31
-
-
3.43
3.70
4.20
4.31
1.55
1.47
1.97
1.87
2.56
2.84
3.27
3.51
3.53
3.43
wj
Persicivirgasp.
S1-08
Tenacibaculum sp. SZ-18
-
-
(a)
ASW+
0.05 mM C
ASW+0.14mM C
(b)
20
20
15
-+-Ught
9 10 -
-U*-Dark
-.-
-
Ught: y = 3.2044x + 6.0143
Ught: y =31.5296x + 7.6954
R =0.9997
.......
S 10 Dark: y = 0.6242x +8.5632
R= 0.996
5
5
Dark: y = 2.9639x + 6.181
R2= 0.9986
-Light
-+Dark
0
0
1
2
3
1
TIme(Day)
(C)
2
3
TIme(Day)
ASW+1.1 mM C
ASW+ 48.5 mM C
(d)
20 -
25
Ught: y =4.9851x +4.6964
R = 0.9739
J~.
0
15
Ught: y
a 10-
=3.9392x +
5.1 49
20
-+-LUght
j5
-m-Dark
R2=0.9982
-- Ight
--- Dark
R2= 0.995
0
01
Dark: y = 5.1442x + 4.3696
R = 0.9897
10
Dark: y = 4.036x + 5.0083
2
Time (Day)
3
1
2
TIme(Day)
3
Fig. 12. Specific growth rate (p) determined by regression analysis based on the growth experiment of strain Dokdonia sp. MED134.
(a) ASW + 0.05 mM C, (b) ASW + 0.14mM C, (c) ASW + 1.1 mM C, (d) ASW + 48.5 mM C.
Specific growth rate of proteorhodopsin-containing marine flavobacterial strains
The maximum specific growth rates (g) are one good way of expressing the relative
ecological success of a species or strain in adapting to its natural environment or the
experimental environment imposed upon it.
The maximum specific growth rates (the slope of a plot of the natural log of cell numbers
and time yields the specific growth rate, g) at various carbon concentrations were determined
for PR-containing flavobacterial strains. The bacterium's growth was monitored during the
exponential phase to measure the specific growth rate at each carbon concentration, as shown
in Figure 12 and Table 4.
The maximum cell density and specific growth rate reached are
shown in Table 3 and 4, respectively.
The specific growth rates of the isolates under various growth conditions were summarized
in graphs and compared to each other (Fig. 13 and 14). The g values of the strains under light
in ASW plus 0.14 mM C and LNHM plus 48.5 mM C ranged from 0.74 to 3.20 day-1 and
1.04 to 4.99 day-1, respectively (Table 2), and these values were lower than p value of
cultures in the dark condition at each strain.
All five strains in the Flavobacteria showed maximum g values at low carbon
concentrations (0.05 mM C and 0.14 mM C) in light-incubated cultures, while they showed
maximum g values at high carbon concentrations (Fig. 13 and 14). Interestingly, growth
characteristics of the PR-containing flavobacterial strains were generally switched at media
containing high carbon concentration. From this result, DOC concentration could be one of
the criteria for differentiating light-dependent growth within the PR-harboring flavobacteria
strains.
41
(a)
Dokdonia sp. Strain MED134
6-
3
+Light
0
0.05mM C
0. 14 nM C
1.1 nM C
48.5 niM C
Carbon concentrations added to ASW
(b)
Winogradskyellasp. PG-2
6
5
4
3
0.05 mM C
0.14 nl
C
1.1 NIM C
48.5 niMI C
Carbon concentrations added to ASW
Fig. 13. Summary of specific growth rates (g) of Dokdonia sp. MED134 (a) and
Winogradskyella sp. PG-2 (b).
42
(a)
Winogradskyellasp. PC-19
4
-+-Light
-U-Dark
0
0.14 nM C
(b)
0.39 mM C
111mM C
48.5 nAM C
Carbon concentrations added to ASW
Gulvibactersp. SZ-19
4
o
_-__
-_--_
0.14 mM C
-_
0.39
IM C
1.1 IIM C
48.5 uM C
Carbon concentrations added to ASW
Tenacibaculunt sp. SZ-18
4
--- Light
-U-Dark
0
0.14 mM C
0.39
NM C
1.1mM C
48.5 mn
C
Carbon concentrations added to ASW
Fig. 14. Summary of specific growth rates (p) of Winogradskyella sp. PC-19 (a), Gilvibacter
sp. SZ-19(b) and Tenacibaculum sp. SZ-18 (c).
43
Culture experiments with an inhibitor of retinal biosynthetic pathway, MPTA
To determine the effect of retinal biosynthesis inhibitor on light-dependent growth, we
studied culture experiment with the MPTA in two different strains of PR-containing
flavobacteria: Dokdonia sp. MED134 which was broadly studied and Gilvibactersp.
SZ-19.
In previous studies assessing the role of PR in light-induced growth in strain
Dokdonia sp. MED134, it was observed that only light corresponding to the
wavelengths absorbed by PR supported growth (Gomez-Consarnau et al. 2007; Kimura
et al. 2011). Additionally, culture experiments on MPTA, an inhibitor of lycopene
cyclization in the retinal biosynthetic pathway, were conducted to provide further
evidence of the role of PR in stimulating growth in Dokdonia sp. MED 134 (Kimura et
aL 2011).
To assess the effectiveness of MPTA as an inhibitor of f-carotene synthesis (the direct
precursor of retinal), MED134 was grown on Marine agar plates with and without
MPTA. Bacterial cells accumulating
p-carotene
display yellow or orange colonies. In
the presence of MPTA, MED134 produced light pink colonies, indicating the
accumulation of lycopene (Fig. 15). In the absence of MPTA, yellow colonies were
observed on agar plates (Fig. 16). Our results indicate that MPTA effectively prevented
J-carotene generation, the precursor for retinal, in both Dokdonia sp. MED134 and
Gilvibactersp. SZ- 19.
44
(a)
(b)
(C)
Fig. 15. Colony images of Dokdonia sp. MED134 in culture experiment with a MPTA.
(a, b) Dokdonia sp. MED134 was grown on Marine Agar (Difco 2216) plates with
MPTA (a) and without MPTA (b). The strain was also grown on agar plate amended
with methanol as negative control (c).
45
(a)
(b)
Fig. 16. Colony images of Gilvibactersp. SZ-19 in culture experiment with a MPTA. (a,
b) Gilvibactersp. SZ-19 was grown on Marine Agar (Difco 2216) plates with MPTA (a)
and without MPTA (b). The strain was also grown on agar plate amended with methanol
as negative control (c).
46
Growth characteristics of representative flavobacterial strains on MPTA
containing media
To determine the role of MPTA as an inhibitor of p-carotene, culture media at both
low and high concentrations of DOC were used. Culture experiment with MPTA was
performed in both Dokdonia sp. MED134 and Gilvibactersp. SZ-19.
First, strains were grown in ASW enriched to 0.14 mM C and amended with MPTA
for low carbon concentration media. When Dokdonia sp. MED134 and Gilvibactersp.
SZ-19 were incubated in ASW without MPTA, bacteria grew well to 1.14 x 106cells/ml
and 1.17 x 106 cells/ml in the presence of light, respectively (Fig. 17a and Fig. 18a).
However, cultures incubated in ASW with MPTA in the light showed significantly
lower yields than those in ASW without MPTA. In culture experiments in the dark with
or without MPTA, cell density of both strains similar to the results with MPTA-treated
condition in the light (Fig. 17a and Fig. 18a).
Second, Dokdonia sp. MED134 and Gilvibactersp. SZ-19 were grown on the media
of ASW enriched to 48.5 mM C and amended with MPTA for high carbon concentration
(Fig. 17b and 18b). Under culture experiment with MPTA at high carbon concentration
conditions, whether present of light of not, Dokdonia sp. MED 134 were reached up to
5.15 x 107 cells/ml and 5.77 x 10 7 cells/ml in the light or in the darkness, respectively,
which was at least two times higher than the maximum cell density in the MPTA-treated
cultures. Also, the similar results were shown in the Gilvibactersp. SZ-19.
Although the results of MPTA growth experiment at high carbon concentration are
hard to explain clearly at this point, we can still suggest that PR bound to retinal has a
47
important role in the light-stimulated growth of the marine flavobacterial strain
containing proteorhodopsin.
48
14
(a)
12
-
JE 10
-
u6
Z
x
0
---
LightControl
-0-
LIghtMPTA
Dark Control
-y-a-
id
2d
3d
4d
DarkMPTA
6d
Sd
Time (Day)
70
(b)
60
50
U
40
0
7'
30
07
20
0
U
10
+
-0-v--
0
LightControl
Light -MPTA
DwlControl
_a
-10
id
2d
3d
4d
5d
6d
Time (Day)
Fig. 17. Growth curves of Dokdonia sp. MED 134 in culture experiments with a MPTA inhibitor.
(a, b) Dokdonia sp. MED 134 was grown in ASW enriched to 0.14 mM C with and without
MPTA (a), in ASW enriched to 48.5 mM C with and without MPTA (b). The cultures in
enriched ASW amended with MPTA exposed to light (o) and in the dark (A), and in the
enriched ASW without MPTA in culture exposed to light (e) and in the dark (V). The
epifluorescence microscopy was used to observe and count bacterial cells.
49
(a)
14*
12 -
10 -
Le
6v 4
0
2-0---a-
0
1d
2d
3d
4d
Sd
UghtControl
Lght MPTA
DarkContrl
Dark MPTA
6d
Time (Day)
70
(b)
60 50 040
.0
30 -
20 10 -e- LightControl
-0- LightMPTA
-r- DarkControl
A Dark.MPTA
0
Id
2d
3d
4d
Sd
6d
Tine (Day)
Fig. 18. Growth curves of Gilvibactersp. SZ-19 in culture experiments with a MPTA inhibitor.
(a, b) Gilvibacter sp. SZ-19 was grown in ASW enriched to 0.14 mM C with and without MPTA
(a), in ASW enriched to 48.5 mM C with and without MPTA (b). The cultures in enriched ASW
amended with MPTA exposed to light (o) and in the dark (A), and in the enriched ASW without
MPTA in culture exposed to light (e) and in the dark (V). The epifluorescence microscopy was
used to observe and count bacterial cells.
50
CHAPTER 3. TRANSCRIPTOMIC ANALYSIS OF LOW AND HIGH CARBON
CONCENTRATIONS ON LIGHT-DEPENDENT GROWTH OF Dokdonia sp.
MED134
3.1 Introduction
A branch of biology 'genomics' was coined by Thomas Roderick as a name for the
new journal Genomics in 1986, and refers to a new scientific discipline of mapping,
sequencing, and analyzing genomes (McKusick 1997). And, at the beginning of this
century, genomics greatly changed the face of biology with the completion of largescale genome projects. Not only has the scale of molecular biology changed, but also its
scope. Genomics is now undergoinga transition or expansion from the mapping and
sequencing of genomes to an emphasis on genome function (Debouck and Metcalf
2000; DeLong et al. 2006; Rusch et al. 2007; Rodrigue et al. 2009). To reflect this shift,
genome analysis may now be divided into "structural genomics" and "functional
genomics". Structural genomics represents an initial phase of genome analysis such as
linkage analysis, molecular cytogenetics, physical mapping, expressed sequence tag
(EST) sequencing, genome sequencing and genome organization (McKusick 1997).
And functional genomics refers to the development and application of global (genomewide or system-wide) experimental approaches to assess gene function by making use
of the information and reagents provide by structural genomics (Hieter and Boguski
1997; Tringe and Rubin 2005). It is characterized by high throughput or large-scale
experimental methodologies combined with statistical and computational analysis
(bioinformatics) of the results (Fig. 19)
51
Functional genomics studies for the definition of the functions of large sets of genes
that are involved in or regulate processes in plant growth and development are called
plant functional genomics.
In functional genomics, 'transcriptomics", the study of gene expressions at
transcription level, is one of the flourishing areas that will have a great impact in
understanding the functional role of genes (Eymann et al. 2002; Ernst et al. 2005;
DeLong et aL. 2006; Toledo-Arana et al. 2009). Since mRNA is the initial and
intermediate product of gene expression, most cases studies at the transcriptional level
may reflect the function of the respective genes. To analysis transcriptomes in largescale, several genomic technologies were developed. They include: PCR-based assays,
serial analysis of gene expression (SAGE) (Velculescu et al. 1995), rapid analysis of
gene expression (RAGE), expressed sequence tags (ESTs) (Poretsky et al. 2009),
macroarrays (Steward et al. 2004) and microarray (biochip) technology (Lindell et al.
2007; Zinser et al. 2009).
Among these technologies microarray technology was
considered as the powerful tool for transcriptomes analysis. The DNA microarray is
invented for studying gene expression patterns simultaneously on a global scale. Large
numbers of different gene fragments are immobilized in an ordered array on a solid
support; it is available for the interrogation of transcriptomes. To do this, an entire
population of mRNAs from a tissue can be simultaneously hybridized, in a single
experiment, with tens of thousands of known DNA probes that are immobilized at
precisely known positions on the array. After the hybridization, the expression levels of
each gene can be determined with a high-resolution laser scanner, and computer
programs can group genes that are up- or down- regulated in response to the
52
interrogated stress. Therefore, it is possible to detect the differences in relative
abundance of several mRNAs between two sets of conditions run in parallel.
Despite these advantages (Zhou and Thompson 2002), a non-targeted, sequence
approach is increasingly used to profile community transcripts due to the technological
limitations of microarrays. While microarrays employ complex quantification
algorithms, increasingly more complex gene expression solutions are required. Factors
that can affect probe specificity include outputs of signal intensity instead of nucleotide
sequence identities. Furthermore, microarrays depend on massively parallel nucleic acid
hybridization and therefore have a risk of cross-hybridization of highly related
sequences. The scope and size of studies enabled by non-targeted, sequence is evident in
a 2005 study that generated and sequenced a cDNA clone library of -400 clones by
random priming of microbial community RNA collected from a hypersaline lake
(Poretsky et al. 2005).
Sequence-based profiling of community transcriptomes on a large scale has benefited
from technological advances. Next-generation sequencing techniques can perform
millions of sequence reads in a run. These include pyrosequencing (Margulies et al.
2005), Illumina technology (formerly Solexa sequencing), and Ion Torrent technology
(Ion Torrent Systems, Inc., Guilford, CT). Pyrosequencing, in particular, has been
increasingly used in microbial ecology and its use has been peer reviewed many times
since 2007. First used in soil analysis (Leininger et al. 2006), it is conducive to a
number of open ocean microbial assemblage experiment procedures, including
measuring size-fractionation and collecting bacterioplankton biomass.
A major advantage of next-generation sequencing applying metatranscriptomic
analysis is the lack of a need for cloning in the process of processing the extracted RNA
53
(rRNA subtraction, amplication and so forth) and converting it into cDNA (Fig. 20). In
terms of analysis, metatranscriptomic technology provides insight into the regulation
and expression patterns of the biological processes of genes at the molecular level (Frias
Lopez et al., 2008). Accordingly, it is being more widely applied to understand the
unknown and increasingly complex biology of microbes under different and untested
environmental conditions. Methodological and technological approaches and protocols
continue to evolve.
54
Genomics
Gene
(Genome)
MTranscriptomnics
mRNA
niRNAI
(Transcriptomie)
Protein
-
Metabolite ---
Phenotpe
Proteomics
(Proteome)
+
BioinformaticsV
--
1
ru
Microorganis
bolorli
Phenonics
(Phenome)
Fig. 19 Scheme showing how the integration of results from different technological
levels of functional genomics leads to construction of a virtual microorganism.
55
sample
DNA and RNA
extraction
DNA
PCR amplification wI
T7-appended primers
16S ar d 23S rDNA
in vitro transcription,
T7 polymerase
biotin-la beled antisense
16 5 and 23S RNA
total RNA
total RNA
hybridizatio
biotin-labeled ds 16S and 23S rRNA
unlabeled ss RNA
bead binding,
magnetic separation
16S and 23S rRNA
rRNA-subtracted RNA
RNA amplification,
Illumina sequencing
Fig. 20. Scheme of sample processing pipeline for the metatranscriptomic analysis;
adapted from Stewart et al (2010).
56
3.2. Materials and methods
Cultivation for transcriptomic analyses
Our experiment was designed to determine the effect of DOC concentration on gene
expression of PR-containing Dokdonia sp. MED134 under light and dark growth
conditions. For transcriptomic analyses, MED134 was grown in ASW amended with
FSM to yield DOC concentrations of low (0.14mM C) and high (48.5mM C),
respectively.
To examine the gene expression of Dokdonia sp. MED134 at low carbon
concentration under both light and darkness, MED134 was grown in 4L of ASW
enriched to 0.14 mM C at 22 C in the darkness for 2 days. At this time, 500 mL of
culture was filtered on to a pore-size 0.22 mm Durapore membrane filter (25mm
diameter, Millipore), yielding the TO sample. The remaining culture was split in two 1 L
flasks that were incubated again at 22
C under the continuous white light
(approximately 150 mmol of photons m-2 s-1) or in darkness. After 2 more days, the
cultures were taken from two time points, mid to late exponential phase and stationary
phase and filtered onto Durapore membrane filters (Millipore), yielding samples Li
(exponential phase and light conditions) and L2 (stationary phase and light conditions)
and Dl (exponential phase and dark conditions) and D2 (stationary phase and dark
conditions), respectively. Sampling time points were determined by pilot experiment.
The same procedures were performed in cultures of high carbon concentration (48.5
mM C) condition. All filtered samples were immediately placed into screw-cap tubes
containing 1 mL of RNAlater (Ambion, Austin, TX, USA) and stored at -80 C until
RNA extraction. All treatments were performed in triplicate.
57
Total RNA extraction
Total RNA was extracted from the filter samples using a modification of the mirVana
miRNA isolation kit (Ambion) as described previously (Shi et al., 2009; McCarren et al.,
2010). Briefly, Sterivex filter cartridges containing samples were taken from the -80 C
freezer, thawed on ice and the RNAlater was carefully removed by pushing through
with a syringe. Then, the filters were immersed with Lysis/Binding (Ambion) and mixed
to lyse attached cells. The yellowish color on the filter due to cells disappeared during
vortexing suggesting cells were lysed. Total RNA was extracted from the lysate
according to the manufacturer's protocol. Remaining genomic DNA was removed using
a TURBO DNA-free kit (Ambion). Finally, extracted total RNAs (pellets) from the
filters were purified, concentrated, and resuspended in 50 pl DEPC treated RNase-free
water by using the MinElute PCR Purification Kit (Qiagen) and stored at -80 C.
ribosomal RNA (rRNA) subtraction
rRNA frequently represents 53% - over 90% of the total RNA (Frias-Lopez et al.,
2008; Stewart FJ et al., 2010). Thus, rRNA needs to be removed so that the majority of
sequencing effort goes to mRNA.
To remove bacterial 16S and 23S rRNA molecules from total RNA samples, we carried
out the subtractive hybridization using sample specific biotinylated rRNA probes as
previously described by Stewart et al (Stewart et al., 2010). Ribonucleotide probes were
generated from the total DNA extracted from the Dokdonia sp. MED 134.
Templates for probe were first prepared by PCR using strain-specific primers flanking
58
nearly the full length of the bacterial 16S gene and 23S rRNA gene and Herculase II
Fusion DNA Polymerase (Stratagene, La Jolla, CA, USA), with reverse primers
modified to contain the T7 RNA polymerase promoter sequence. Biotin-labeled
antisense rRNA probes were generated by in vitro transcription (IVT) using T7
promoter-containing 16S and 23S amplicons as templates based on MEGAscript High
Yield Transcription Kit (Ambion, Austin, TX, USA). Biotinylated rRNA probes were
hybridized to rRNA in the total RNA sample. The labeled double-strand rRNA was then
removed via hybridization to streptavidin-coated magnetic beads (New England Biolabs,
Ipswich, MA, USA),
followed by magnetic separation.
After subtracted
RNA
purification, purified RNA was confirmed the removal of bacterial rRNAs using a 2100
Bioanalyzer (Agilent, Santa Clara, CA, USA).
RNA
amplification,
complementary
DNA
(cDNA)
synthesis
and
Illumina
sequencing
The rRNA-subtracted RNA was amplified using the ScriptSeq" v2 RNA-Seq Library
Preparation kit (Epicentre, Madison, WI). cDNA was synthesized according to the
manufacturer's protocol.
In brief, the rRNA-depleted sample is fragmented using the RNA Fragmentation
Solution. The fragmented RNA was converted to double-stranded cDNA via randomsequence primers containing a tagging sequence at their 5' ends. The 5'-tagged cDNA is
then tagged at its 3' end by the terminal-tagging reaction to make di-tagged at both its
5'- and 3'-end, single-stranded cDNA. Following purification, the di-tagged cDNA is
amplified by PCR, which completes the addition of the Illumina adaptor sequences,
amplifies the library for subsequent cluster generation and adds an optional Illumina
59
Index. The amplified RNA-seq library is purified using AMPure kit (Beckman Coulter
Genomics, Danvers, MA, USA) and is ready for cluster generation and sequencing. The
resulting cDNA libraries were then pyrosequenced on the MiSeq platform (Illumina).
60
3.3 Results and Discussions
Cultivation for transcriptomic analyses
Before
the
beginning
of the
transcriptomic
analysis
with
various
carbon
concentrations, we confirmed the light-enhanced growth on Dokdonia sp. MED134 at
both low (0.14 mM C) and high (48.5 mM C) carbon concentration. Sampling time
points were also determined by this pilot experiment.
Cell densities
were increased
significantly in light condition at low DOC
concentration whereas MED134 grew well slightly in darkness at high DOC
concentration, which were consistent with those seen in before. This shows that further
transcriptomic analysis with different carbon concentrations are needed to investigate
the PR-containing marine flavobacterial strains. Twelve samples were collected at TO,
TI and T2 of low DOC condition and TO, TI and T2 of high DOC condition under both
light and darkness, respectively (Fig. 21).
61
14
(a)
T2
TI
121lo8-
S6 TO
.a
C
4
2-1
0-
-0-- Ught
Dark
Od
2d
3d
4d
5d
6d
Time (Day)
(b)
70
60-
T2
TI
S0.
30-
TO
2010 -
0
-
-0- Ught
*e Dark
Od
2d
3d
4d
5d
6d
Mime (Day)
Fig. 21 Determining optimal sampling time points for transcriptomic experiments, based on
growth curves of Dokdonia sp. MED 134. Growth curves of Dokdonia sp. MED 134 incubated in
the light (o) or in the dark (*). MED 134 was grown in ASW enriched to 0. 14 mM C (a), and in
ASW enriched to 48.5 mM C. Error bars indicate standard deviation for triplicate cultures. Red
arrows represent the sampling time points for each condition.
62
Transcriptomic analyses
Illumina sequencing of Dokdonia sp. MED134 transcriptomes (samples TO, TI
triplicates in light, TI triplicates in dark at low carbon concentrations) yielded 3.16E4 to
6.72E5 reads per sample (Table 5). Poor quality reads were removed by demultiplexing,
dereplication, removal of short reads and reads with low fastq quality scores. Quality
control was performed using pipelines for CLC Genomics workbench. Table 5 reflects
that the number of reads that survive quality-control step in read processing via our
scripts across samples.
Transcriptomic analyses revealed that several genes showed significant upregulation
in the presence of light at Ti and T2 low carbon condition (Table 6). In particular, two
predicted
Fe
assimilation
proteins
(MED134_06839,
MED134_06834,
MED134_22457) were significantly upregulated in the light at TI low carbon condition
(Table 6). In T2 low carbon cultures, genes of a proteorhodopsin (MED134_07119),
retinal
biosynthetic
enzymes
(MED134_13081),
putative
adhesion
protein
(MED134_03969) are exhibited significant upregulation in the light (Table 7). Further,
bacterial cryptochrome gene (MED134_10201) also exhibited significant upregulation
in the light at T2 low carbon cultures. These results suggested the importance of the
proteorhodopsin for the light-stimulated growth in the light.
Also, MED134 has a gene associated with light sensing contains the BLUF (blue
light sensing using FAD) domain, which specifically responds to blue light (Gomelsky
and Klug, 2002). The BLUF domain is also found in the genome of Polaribacter sp.
MED152 (Gonzalez et al., 2008). In addition to light sensors, ABC transporter
(MED134_03404) and molecular chaperone DnaK (MED134_02409), which might
have important roles for response regulation, were significantly upregulated in the light.
63
Transcriptomic survey for High carbon conditions
In transcriptomic experiments of high carbon concentration condition, upregulated
genes were similar to the results with low carbon condition at T2 in the light T1 (Table
8). At TI in high carbon condition, genes of a proteorhodopsin (MED134_07119), ABC
transporter (MED134_03404), bacterial cryptochrome gene (MED134_10201), retinal
biosynthetic enzymes (MED134_13081), putative adhesion protein (MED134_03969)
are exhibited significant upregulation in the light (Table 8). However, the pattern of
upregulated genes in T2 high carbon condition was changed compared to the results of
low carbon condition. The lesser upregulation of a preoterhodopsin (MED134_07119)
was observed at T2 higher carbon concentration. Also, P-oxidation pathways involved
in ATP/energy production are up-regulated in the high carbon concentration at T2
culture with the former being statistically significant (Table 9). In particular, genes
encoding electron transfer flavoprotein alpha-subunit (MED134_12996),
transfer
flavoprotein
beta-subunit
(MED134_00130)
and Enoyl-CoA
electron
hydratase
(MED134_06229) exhibited significant upregulation in the dark.
Fatty acid
P-oxidation is a multi step process by which fatty acids are broken down by
various tissues to produce energy. The long chain acyl-CoA enters the fatty acid
p-
oxidation pathway, which results in the production of one acetyl-CoA from each cycle
of fatty acid
P-oxidation. This acetyl-CoA then enters the mitochondrial tricarboxylic
acid (TCA) cycle. The NADH and FADH 2 produced by both fatty acid P-oxidation and
the TCA cycle are used by the electron transport chain to produce ATP. The proton
gradient built up in electron transport chain then allows for ATP synthesis. These results
suggested the potential importance of the
P-oxidation for growth of PR-containing
MED 134 in the high carbon concentration condition.
64
Table 5. Number of reads remaining after quality control step in the pipeline for quality control.
Low
Carbon
67255
High
38174
0
Carbon
1
T2-Light
T1-Dark
Ti-Light
TO
39672
43456
45458
2
7
38888
3
40066
6
24880
5
36447
7
T2-Dark
31609
39134
3
40861
6
81880
15280
24691
9
10279
4
24187
8
363742
35520
8
2
68029
43189
9
25478
8
39483
2
35637
2
43794
7
Table 6. Preliminary analysis of functional enrichment of differential gene expression in low carbon condition at T1.
ON~
M~
Read number
Locus tag
ligh
light
dmr
dark
foldChange
log2FoldChange
pval
pval.
FDRadj
Genbank annotation
MED134_06839
107
27
0.3
-2.0
2.37E-05
0.017
Putative uncharacterized protein
MED134_06834
1798
463
0.3
-2.0
1.95E-09
5.55E-06
TonB-dependent outer membrane
receptor
MED134_22457
11827
3598
0.3
-1.7
1.16E-07
1.65E-04
#N/A
MED134_01225
109
360
3.3
1.7
5.00E-06
0.005
Secretion protein HlyD
Red character indicates significant upregulation in the light. Black character indicates significant upregulation in the darkness.
Table 7. Read number and significant upregulated genes in the light in low carbon condition at T2.
Read
Locus tag
number
foldChange log2FoldChange
pval
Genbank annotation
FDRadj
light
dark
MED134_07119
156
11
0.1
-3.8
7.39E-08
3.49E-05
Bacteriorhodopsin
MED134_07089
25
2
0.1
-3.7
4.94E-09
2.80E-06
Putative uncharacterized protein
MED134_10201
41
4
0.1
-3.4
1.29E-10
1.82E-07
Probable bacterial cryptochrome
Transcriptional regulator, MerR family
MED134_13081
69
11
0.2
-2.7
4.29E-09
2.80E-06
MED134_14276
34
6
0.2
-2.6
1.43E-05
0.005
Putative uncharacterized protein
MED134_04999
97
18
0.2
-2.4
1.95E-10
1.84E-07
Putative uncharacterized protein
MED134_01275
456
102
0.2
-2.2
3.15E-13
8.92E-10
Putative uncharacterized protein
MED134_13076
33
9
0.3
-1.9
2.36E-04
0.061
Phytoene dehydrogenase
MED134_05424
66
27
0.4
-1.3
0.001
0.140
ThiJ/PfpI family protein
MED134_03409
161
66
0.4
-1.3
9.89E-06
0.004
(dnaK) Molecular chaperone DnaK
MED134_03969
590
268
0.5
-1.1
4.27E-04
0.101
Putative adhesion lipoprotein
MED134_03404
206
99
0.5
-1.1
6.54E-05
0.021
ABC-1
MED134_11080
642
331
0.5
-1.0
8.57E-05
0.024
Putative uncharacterized protein
protein
Table 8. Read number and significant upregulated genes in the light in high carbon condition at T1.
Locustag
00
Read number
foldChange
log2FoldChange
pval
pval FDRadj
84
0.1
3.76E-13
1.07E-09
Bacteriorhodopsin
1368
73
2537
139
9
317
0.1
2.80E-10
3.98E-07
3.18E-04
0.023
Putative adhesion lipoprotein
1194
151
0.1
-3.8
-3.3
-3.0
-3.0
-3.0
MED134_04999
836
107
0.1
-3.0
MED134_10201
MED134_14271
MED134_14276
337
149
198
234
45
25
40
0.1
0.2
0.2
-2.9
-2.6
-2.3
59
0.3
-2.0
light
dark
MED134_07119
1197
MED134_03969
MED134_07089
MED134_01275
MED134_03404
MED134_13076
0.1
0.1
4.46E-07
4.95E-05
6.38E-05
Genbank annotation
Putative uncharacterized protein
Putative uncharacterized protein
0.024
ABC-1
6.79E-05
0.024
Putative uncharacterized protein
6.08E-08
1.46E-06
5.77E-05
0.001
0.029
Probable bacterial cryptochrome
9.18E-05
1.12E-04
0.032
Short chain dehydrogenase
Putative uncharacterized protein
Phytoene dehydrogenase
Table 9. Preliminary analysis of functional enrichment of differential gene expression in high carbon condition at T1.
Read number
Locus tag
M.
foldChange
log2FoldChange
pval
pval
Genbank annotation
FDRadj
light
dark
MED134_07119
27
8
0.3
-1.7
1.60E-04
0.051
Bacteriorhodopsin
MED134_00750
85
46
0.5
-0.9
2.90E-04
0.075
Purine nucleoside phosphorylase (EC 2.4.2.1)
MED134_22457
6752
4431
0.7
-0.6
1.27E-04
0.049
#N/A
MED134_12996
333
545
1.6
0.7
4.78E-05
0.023
Putative recognition particle-docking protein
MED134_00125
407
694
1.7
0.8
8.84E-06
0.005
Electron transfer flavoprotein alpha-subunit
MED134_05174
2604
4669
1.8
0.8
1.11E-07
1.11E-04
Putative heat shock ClpB protein
MED134_13401
737
1344
1.8
0.9
1.17E-07
1.1 1E-04
GrpE protein (Hsp-70 cofactor)
MED134_00130
311
574
1.8
0.9
4.66E-07
3.32E-04
Electron transfer flavoprotein (Beta subunit)
MED134_06234
136
257
1.9
0.9
3.79E-04
0.090
Thiolase family protein
MED134_06229
409
779
1.9
0.9
1.38E-04
0.049
Enoyl-CoA hydratase
MED134_03499
189
364
1.9
0.9
2.23E-04
0.063
Putative uncharacterized protein
MED134_04044
1158
2381
2.1
1.0
1.99E-10
0.000
Putative heat-shock related protein
Red character indicates significant upregulation in the light. Black character indicates significant upregulation in the darkness.
(a)
C-
-si
a
I
I
1
10
I
100
1000
10000
mean of normalized counts
(b)
C4
I
0
W.~'
~
CYI
I?.
II
10-01
10+00
I
10+01
10+02
1
1e+03
mean of normalized counts
Fig. 22. Plot of normalized mean versus log2 fold change for low carbon TI light
sample versus low carbon TI dark sample (a) and for low carbon T2 light sample versus
low carbon T2 dark sample (b).
70
(a)
CM -
a
t~J
10-01
10+00
10+01
16+02
le+03
mean of nomalized counts
en
(b)
q
C
0
-L
9
0
l-01
1+00
1o+01
1o+02
10+03
mean of normelized counts
Fig. 23. Plot of normalized mean versus log2 fold change for high carbon TI light
sample versus high carbon TI dark sample (a) and for high carbon T2 light sample
versus high carbon T2 dark sample (b).
71
(a)
N
0
(~1
'9
10
100
1000
10000
mean of normalized counts
(b)
0
"I.Av
10-01
10+01
le+03
mean of normalized counts
Fig. 24. Plot of normalized mean versus log2 fold change for TI light low carbon versus
TI light high carbon (a) and for low carbon TI dark sample versus high carbon TI dark
sample (b). Red dots indicate differentially expressed genes.
72
(a)
--
-
-
H
;
10+00
10-01
le+02
10+01
-
10+03
mean of normalized counts
(b)
-
--
I
10-01
V
1o+01
10+03
mean of nonnalized counts
Fig. 25. Plot of normalized mean versus log2 fold change for T2 light low carbon versus
T2 light high carbon (a) and for low carbon T2 dark sample versus high carbon T2 dark
sample (b). Red dots indicate differentially expressed genes.
73
Conclusions
The light-enhanced growth rates and cell yields in marine flavobacterial strains
demonstrate the proteorhodopsin-associated light-dependent growth effect at various
carbon concentrations for the first time in a native cell in a proteorhodopsin
photosystem (PRPS). In this study, we found that increasing concentrations of DOC in
the culture media decreased the effect of the PR-stimulated growth. Also, interestingly,
we found that cell densities of dark-incubated cultures with PR-containing
flavobacterial strains were higher than light-incubated cultures at high carbon
concentration. Together, the results show strong evidence that PR is beneficial in carbon
and respiration limited conditions. The results also show that this effect is seen in a
variety of different PR-containing flavobacteria, suggesting that this phenomena is
widespread and not just limited to a few strains.
To verify that proteorhodopsins are responsible for the observed effect, we conducted
a growth experiment in which we inhibited the synthesis of proteorhodopsin with an
inhibitor of MPTA. Cultures incubated in ASW with MPTA in the light showed
significantly lower yields than those in ASW without MPTA. In culture experiments in
the dark with or without MPTA, cell density of both strains similar to the results with
MPTA-treated condition in the light. This result demonstrated the critical role of PR in
light-enhanced growth.
Additionally, to better characterize the photophysiology of PR-containing marine
flavobacteria, metatranscriptomics studies were performed to see the gene expression
patterns in strain MED134 in this study. Metatranscriptomic analysis will lead to a
better understanding of functional capacity under different carbon conditions different
staged of growth. We still need to work more on metatranscriptomics analyses, but this
study has addressed several questions including:
74
"What are the active genes at
different DOC conditions in light?", "When are they active?", and "How do
flavobacteria to different DOC conditions in the dark compared to light" which provides
new insight into the photophysiological characteristics of PR-containing marine
flavobacteria. In the future, it will be useful to examine the gene expression patterns
associated with the higher growth rates and cell yields in flavobacterium strain
MED134 in both low-carbon and high-carbon media in even greater detail.
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