AN ABSTRACT OF THE THESIS OF
Terah Diana Wright for the degree of Master of Science in Microbiology
presented on March 28, 1997. Title: Bacterioplankton Diversity in the Lower
Ocean Mixed Layer.
Abstract approved:
Redacted for Privacy
Stephen J. Giovannoni
Microorganisms play an important role in the biogeochemistry of the ocean
surface layer, but the species composition of marine bacterial communities is
poorly understood, largely due to the limitations of classical cultivation
techniques. Furthermore, the spatial and temporal distributions of specific
bacterioplankton species are virtually unexplored. Recently, new
information about these distributions has come from DNA sequencing and
oligonucleotide probe hybridizations. In this study, a clone library of bacterial
6S rRNA genes collected from a depth of 250 m in the western Sargasso Sea
at the Bermuda Atlantic Time Series Station (BATS) was phylogenetically
analysed. The analysis indicates the presence of a novel microbial group, the
5AR324 lineage, which is most closely related to the
subdivision of the class
Proteobacteria. A specific oligonucleotide probe (SAR324R) was constructed
for the purpose of examining the distribution of this gene cluster in the water
column. The results from hybridization experiments showed that the
SAR324 gene cluster is a significant component of the bacterioplankton
community in the lower ocean surface layer of both the Atlantic and Pacific
Oceans. A second cluster of genes related to SAR2O2 a member of the
Oceans. A second cluster of genes related to SAR2O2 a member of the
Chioroflexus/Herpetosiphon phylum
was also observed. The SAR2O2-
related gene clones were shown by DNA sequence analysis to be highly
divergent, with significant variability in the predicted secondary structures of
the corresponding 16S rRNA molecules. The significance of this variability is
currently unknown, but could be important for the design of specific
oligonucleotide probes. Although 16S rRNA sequence data rarely provides
persuasive information regarding the physiological capabilities of
bacterioplankton, SAR196, also discovered by randomly sequencing the BATS
250 m clone library, was an exception. SAR196 was shown to be
phylogenetically related to Nitrospira marina, a nitrite-oxidizing bacterium.
The distribution and abundance of this species in the water column have yet
to be studied, but its presence in the lower ocean surface layer suggests that it
could play a role in nitrification. The diversity of genes observed in this
clone library and the stratification of microbial populations suggest that
bacterioplankton communities are composed of multiple bacterial species
that are functionally specialized and adapted for growth at certain positions in
the water column.
© Copyright by Terah Diana Wright
March 28, 1997
All Rights Reserved
BACTERIOPLANKTON DIVERSITY IN THE
LOWER OCEAN MIXED LAYER
by
Terah Diana Wright
A THESIS
submitted to
Oregon State University
in partial fulfillment of
the requirements for the
degree of
Master of Science
Completed March 28, 1997
Commencement June, 1997
Master of Science thesis of Terah Diana Wright presented on March 28, 1997.
APPROVED:
Redacted for Privacy
Major Professor, representing Microbiology
Redacted for Privacy
Chair of Department of Microbiology
Redacted for Privacy
Dean of Graduate School
I understand that my thesis will become part of the permanent collection of
Oregon State University libraries. My signature below authorizes release of
my thesis to any reader upon request.
Redacted for Privacy
Terah Diana Wright, Author
ACKNOWLEDGMENT
I would like to express my sincere gratitude to Dr. Stephen Giovannoni
for his enthusiasm and encouragement during this study. I am grateful to
the Bermuda Biological Station BATS group and Nanci Adair for collecting
and processing nucleic acid samples and to Frank Whitney, Institute of Ocean
Sciences, Sidney, B.C., for the Pacific Ocean physical data. I extend my
appreciation to Doug Gordon, Kevin Vergin, Brian Lanoil, Mike Rappé, Ena
Urbach, Marcelino Suzuki, and Kate Field for their useful comments
regarding the manuscript. This work was supported by National Science
Foundation grant OCE 9016373 for the study of microbial diversity at BATS,
Department of Energy grant FG0693ER61697, and by an N. L. Tartar Research
Fellowship.
CONTRIBUTION OF AUTHORS
Kevin Vergin was involved in the construction of the clone libraries
and the blots and assisted in data collection for this study. Philip Boyd
assisted in the collection of the water samples and physical data from Ocean
Station PAPA. The experimental research was performed in the laboratory of
Dr. Stephen Giovannoni who also assisted in the interpretation of the results
and in the writing and editing of the manuscript.
TABLE OF CONTENTS
Chapter
Pg
Thesis Introduction
2.
Molecular Ecology
4
Objective
7
A Novel Subdivision Proteobacterial Lineage From the
Lower Ocean Surface Layer
8
Abstract
9
Introduction
10
Materials and Methods
12
Sampling and nucleic acid extraction
Cloning
Gene sequencing and phylogenetic analysis
Hybridization
Accession numbers
3.
1
12
13
13
14
16
Results
16
Discussion
36
Acknowledgements
39
References
39
Secondary Structure Analysis of SAR202Related Clones
44
Introduction
44
Materials and Methods
48
Sampling and nucleic acid extraction
Cloning
Gene sequencing and phylogenetic analysis
Accession numbers
Results and Discussion
48
48
48
49
49
TABLE OF CONTENTS (Continued)
Chapter
4.
Page
Phylogenetic Analysis of Clone SAR196
Introduction
57
57
Materials and Methods
Sampling and nucleic acid extraction
Cloning
Gene sequencing and phylogenetic analysis
Accession numbers
59
59
59
59
Results and Discussion
5.
Thesis Summary
64
Bibliography
66
Appendices
71
LIST OF FIGURES
Figure
Page
Phylogenetic tree showing relationships of the SAR324 cluster
and representative bacterial 16S rRNA genes
18
2-2.
Proposed secondary structure for the SAR324 16S rRNA gene
25
2-3.
The thermal stability of the SAR324 probe
29
2-4.
Phylogenetic tree showing relationships among genes within
the SAR324 cluster
31
The distribution of the SAR324 gene in 16S rDNA amplicons
and high molecular weight (HMW) RNA prepared from
plankton samples
34
Phylogenetic tree showing relationships of SAR2O2 and
SAR3O7 to representative bacterial 16S rRNA genes
45
2-1.
2-5.
3-1.
3-2.
Secondary structural model of SAR2O2 16S rRNA
51
3-3.
16S rRNA structural variation among SAR2O2 gene clones
53
4-1.
Phylogenetic tree showing relationships between the SAR196
gene clone and representative bacterial 16S rRNA genes
61
LIST OF TABLES
Page
Table
2-1.
2-2.
Sequence similarities among the SAR324 cluster and 16S
rRNA gene sequences from representatives within the
proteobacteria.
21
Signature nucleotides relating SAR324 and SAR276 to the
subdivision of proteobacteria
23
LIST OF APPENDICES
Page
Appendix
SAR324 16S rDNA Sequences
SAR214 16S rDNA sequence
SAR218 165 rDNA sequence
SAR248 16S rDNA sequence
SAR237 16S rDNA sequence
SAR257 165 rDNA sequence
5AR276 16S rDNA sequence
SAR3O8 165 rDNA sequence
SAR324 16S rDNA sequence
5AR202 165 rDNA Sequences
5AR226 16S rDNA sequence
SAR242 16S rDNA sequence
SAR25O 16S rDNA sequence
SAR251 16S rDNA sequence
SAR256 16S rDNA sequence
SAR259 16S rDNA sequence
SAR267 16S rDNA sequence
SAR269 16S rDNA sequence
SAR272 16S rDNA sequence
SAR317 16S rDNA sequence
C.
SAR196 165 rDNA Sequence
72
72
72
73
74
74
75
76
77
78
78
79
80
81
81
82
82
83
84
85
86
BACTERIOPLANKTON DIVERSITY
IN THE LOWER OCEAN MIXED LAYER
CHAPTER 1
THESIS INTRODUCTION
Our knowledge of bacterioplankton diversity has expanded in the
past decade, due partly to an increased awareness of the role of
bacterioplankton in oceanic biogeochemical cycles. Numerous studies have
shown that heterotrophic bacteria are responsible for the bulk of organic
carbon utilization and respiration in the sea (Cole et al., 1988; Ducklow and
Carison, 1992; Pomeroy, 1974). Although many species of bacteria with a wide
variety of metabolic capabilities are known to live in marine habitats, little is
known about the species composition of microbial communities or its
variation. The exploration of microbial diversity in the ocean is driven not
only by interest in the oceanic carbon cycle, but also by the observation that
the most abundant and perhaps most significant members of microbial
communities are undescribed. Often, these novel microbial species cannot be
confidently placed within any of the bacterial phyla originally described by
Woese (Woese, 1987).
Early studies of bacterial diversity in the oceans were conducted
with surface samples. The results from these experiments indicate that
indeed, the dominant species of bacterioplankton do not correspond to any
cultured species, but instead form novel phylogenetic lineages. Experiments
conducted to examine the distribution of undescribed bacterioplankton
revealed that species recovered from the surface layer were often dominant
2
only in the surface layer and were rare in the deeper layers of the water
column (Giovannoni et al., 1996; Giovannoni and Cary, 1993; Gordon and
Giovannoni, 1996). Furthermore, genetic comparisons among
bacterioplankton 16S rRNA gene clones revealed unknown bacterial lineages
common to both the Atlantic and Pacific Oceans (Fuhrman et al., 1993;
Mullins et al., 1995; Schmidt et al., 1991). These findings suggest that
bacterioplankton communities are stratified and that species overlap exists
between sampling sites. These observations have driven the exploration of
the deeper layers of the water column. This study is part of an ongoing effort
to characterize the bacterioplankton of the lower ocean surface layer.
The bulk of heterotrophic processes, including carbon fixation and
respiration by bacterioplankton, occur at the ocean surface and have been
shown to affect atmospheric concentrations of carbon dioxide and oxygen
(Carison et al., 1994; Keeling et al., 1993). It is therefore not surprising that
most studies of bacterioplankton diversity have been conducted with samples
from the euphotic zone. More recently, the lower ocean mixed layer, also
known as the aphotic zone, has been implicated in important biogeochemical
processes related to nutrient regeneration and carbon storage. Studies of
bacterioplankton diversity in the aphotic zone of the water column have
yielded results similar to the euphotic zone. The localization of species to
this region of the water column suggests that they are adapted to the upper
mesopelagic (150 to 500 m) or abyssal zones, and are likely to participate in
processes occurring at that depth.
Although phylogenetic relationships between novel and described
species can be inferred from 16S rRNA sequence data, determining the
biogeochemical role of novel species is a challenging task. This is due in part
3
to the metabolic diversity already known to exist within related groups of
organisms. Also, the phylogenetic relationships between the novel species
and the described species are often distant and do not support any firm
conclusions. Future studies aimed at addressing the metabolic features of
uncultured bacterioplankton would therefore necessitate the collection of
physical, chemical, and ecological data. Assessing the roles of
bacterioplankton in biogeochemical cycling could be invaluable information
for solving environmental problems that may have microbial solutions.
Determining the types of bacterial species in natural habitats must
precede both the assessment of their phenotypic expression and the
regulation of metabolic pathways and hence, biogeochemical cycles (Azam et
al., 1993 and 1995). Molecular techniques that make bacterioplankton
identification possible without cultivation have been developed to facilitate
studies of species composition and variation in marine environments.
Advances in technology have provided the basis for an increased
understanding of the diversity and potential physiological activities of
microorganisms in nature and how they may interact with one another.
Several different microbial habitats have been studied by these techniques,
including soil, ice, animals, insects, and deep-sea hydrothermal vents. This
study focuses on the marine environment, specifically the lower ocean mixed
layer of the Atlantic and Pacific Oceans.
Molecular Ecology
The recent advancement of marine microbial ecology has to a great
extent relied upon DNA sequencing and nucleic acid probe hybridization.
Until recently, the communities of microorganisms mediating oceanic
biogeochemical processes were virtually undescribed and undefined
phylogenetically because of the limitations of classical cultivation techniques.
In 1949, Winogradsky expressed concern over the use of conventional culture
methods to investigate natural populations of microorganisms, stating that:
"it is only a minority which develop in the conventional media that
are offered.. .The microflora is not understood either qualitatively or
quantitatively." (Winogradsky, 1949)
In 1984, Atlas reaffirmed Winogradskys comment when he made this
remark:
"bacteriologists who rely on cultural methods to identify species, face
the problem of selectivity and thus the inevitable underestimation of
community diversity." (Atlas, 1984)
A recent study by Suzuki et al. (1996) showed experimentally that genes from
cultured marine bacteria did not correspond to the dominant genes cloned
directly from environmental DNA or to genes available in public databases.
This information supports the idea that the most abundant marine
bacterioplankton species are not readily culturable and that significant
members of oceanic microbial communities remain phylogeneticafly
undescribed.
5
DNA sequencing and the construction of oligonucleotide probes have
made it possible for microbial ecologists to take a phylogenetic census of any
microbial niche. These techniques have been especially useful in the study of
oceanic environments in which limitations of enrichment methods have
prevented microbial ecologists from assessing the diversity of the niche.
Numerous studies of the identitities and distributions of bacterioplankton in
the Atlantic and Pacific Oceans have been conducted (Fuhrman et al., 1993;
Giovannoni et al., 1996; Gordon and Giovannoni, 1996; Lee and Fuhrman,
1991; Mullins et al., 1995; Schmidt et al., 1991). In one such study, 60 bacterial
16S rDNA genes from a mixed population of DNA were cloned and analyzed
(Britschgi and Giovannoni, 1991; Giovannoni et al., 1990; Mullins et al., 1995).
The gene clones were identified as members of the c proteobacteria
subdivision, y proteobacteria subdivision, and cyanobacteria, but only four of
the 60 clones could be identified with cultured species. In a review by
Giovannoni et al. (1995), the 60 gene clones from the Sargasso Sea were also
compared phylogenetically with 16S rDNA gene clones recovered from the
North Pacific gyre (ALOHA station; Schmidt et al., 1991), sites near Bermuda
(Fuhrman et al., 1993), and the Western California Current (Fuhrman et al.,
1993). This analysis showed that closely-related lineages occur in clone
libraries even though the libraries have been prepared by different methods.
This suggests that previously unrecognized bacterioplankton groups are
common in the surface waters of subtropical oceans.
Chemically synthesized oligonucleotide probes, which take advantage
of sequence variation in different regions of the 16S rRNA molecule, have
been used extensively in hybridization studies to examine the temporal and
spatial variation of bacterioplankton community members. A recent study by
Giovannoni et al. (1996) reported the distribution of a clone (SAR2O2)
recovered from a depth of 250 m at the Bermuda Atlantic Time Series Station
(BATS) in the Atlantic Ocean. Clone SAR2O2, phylogenetically related to the
Chioroflexus/Herpetosiphon phylum, was shown by hybridization
experiments to be stratified in the lower region of the mixed layer, peaking at
200 m in the Atlantic Ocean and between 100 and 150 m in the Pacific Ocean.
This was the first study showing that significant species from phylogenetic
groups other than oxygenic bacterioplankton have stratified distributions in
the water column. A similar study by Gordon and Giovannoni (1996)
demonstrated the distribution of clone SAR4O6, recovered from an 80 m
seawater sample at BATS. Clone SAR4O6, a distant relative of the genus
Fibrobcicter and the green sulfur bacterial phylum, was shown by
hybridization experiments to be stratified between 150 and 200 m in the
Atlantic Ocean and between 100 and 150 m in the Pacific Ocean.
Although little is known about the impact of stratified microbial
communities on the cycling of nutrients, a recent report by Carison et al.
(1994) suggests that stratification may be an important factor of carbon cycling
in the ocean surface layer. Other oceanic processes, such as nitrification, may
also be affected by the dynamics of significant community members;
however, data linking such processes to stratified bacterioplankton groups
has not been reported. Although the overall activity of bacterioplankton in
the aphotic zone is much reduced compared to activity in the euphotic zone,
localization of groups indicates participation by bacterioplankton in distinct
processes occurring in specific regions of the water column.
7
Objective
The purpose of this study is to further characterize the
bacterioplankton of the lower ocean mixed layer. The introduction provides
the background information on the work that has been done in the area of
microbial diversity of the marine environment. Chapter 2, "A Novel
Subdivision Proteobacterial Lineage from the Lower Ocean Mixed Layer,'
describes the SAR324 lineage, discovered by random sequencing of a clone
library that was constructed from a 250 m seawater sample. Chapter 2 has
been accepted for publication and has therefore been included in the form
accepted by the Applied and Environmental Microbiology journal. Chapter 3,
"Secondary Structure Analysis of SAR202-Related Clones," is a detailed
analysis of the secondary structure of the 16S rRNA molecules of several
SAR202-related clones. Chapter 4, "Phylogenetic Analysis of Clone 5AR196,"
describes the discovery of SAR196, a 16S rRNA gene clone related to the
nitrite-oxidizing bacterium Nitrospira marina. The final chapter is a brief
summary of the work presented here and contains any conclusions and
future directions of study.
A NOVEL 6 SUBDIVISION PROTEOBACTERIAL LINEAGE
FROM THE LOWER OCEAN SURFACE LAYER
Terah D. Wright', Kevin Vergin1, Philip W.
and Stephen I. Giovannoni1
Boyd2,
1Department of Microbiology
Oregon State University
Corvallis, Oregon 97331
2Department of Oceanography
University of British Columbia
Vancouver, British Columbia, Canada v6t 1z4
Submitted to Applied and Environmental Microbiology
Accepted for publication January 15, 1997
This paper represents a portion of the work described in a thesis to be
submitted to Oregon State University Department of Microbiology in partial
fulfillment of the requirements for an MS degree.
Abstract
A small subunit ribosomal RNA (16S rRNA) gene lineage (SAR324)
affiliated with the proteobacteria (DP) was discovered in a 16S rRNA gene
clone library prepared from a water sample collected from 250 m in the
western Sargasso Sea. This clone library of nearly full-length amplicons of
bacterial 16S rRNA genes has been the subject of previous studies aimed at
identifying bacteria that inhabit the lower ocean surface layer. The novel
lineage was identified by randomly sequencing clones that did not hybridize
to oligonucleotide probes specific for several abundant bacterioplankton
groups identified in previous studies. Phylogenetic analysis indicated that
SAR324 was most closely affiliated with the DP, although it showed no
specific relationship to any DP 16S rRNA genes in databases. Eight of the
clones in the library of 148 clones were identified as members of the SAR324
lineage by hybridization to an oligonucleotide probe specific for SAR324.
Subsequent hybridizations showed that the SAR324 group is stratified in the
lower surface layer of both the Atlantic and Pacific Oceans, with maxima
between 160 and 500 m. The repeated discovery of sequences belonging to
different gene clusters with similar distributions in this region of the water
column suggests that microbial communities in the lower surface layer may
be functionally specialized.
10
Introduction
The sequencing of 16S rRNA genes and the application of group-
specific oligonucleotide probes has provided much new information on
bacterioplankton diversity, and has revealed previously unseen structure in
the spatial and temporal distributions of microorganisms in marine systems
(17, 18, 34). An interesting and unforeseen conclusion of these studies is that
a majority of the genes recovered from seawater belong to a limited number
of phylogenetic groups. In a recent review, we reported that 86% of all
bacterial genes (n=440) recovered from seawater fall within eight phylogenetic
groups (16). Elsewhere it has been reported that all archaeal ribosomal RNA
genes recovered so far from seawater belong to two phylogenetic groups (6,
11). These conclusions are important to microbial ecologists seeking to
understand the relationship between microbial diversity and functional
specialization within microbial communities because they suggest that a
relatively limited array of molecular probes may be sufficient for monitoring
the population dynamics of the majority of bacterioplankton. Equally
important has been the observation of similar stratified patterns in the
distributions of some of the major bacterial groups in different oceans (17, 18,
26).
Collectively, these observations support the view that common
principles may underlie the organization of bacterioplankton communities in
temperate oceans.
Although common themes are emerging from investigations of
bacterioplankton diversity, novel genes of potential ecological significance
continue to be discovered (17, 18, 26). Bacterioplankton 16S rDNAs have been
recovered from surface, 100, 200, and 500 meters samples from subtropical and
11
temperate regions of the oceans, as well as Antarctica, and the eastern and
western continental shelves of the U.S.A. (2, 6, 7, 11, 12, 14, 28). The majority
of clones belong to the cyanobacteria (SAR6, SAR7) and proteobacteria
(SAR11, SAR86, SAR83, SAR116 clusters) divisions. Many genes from novel
lineages related to
Fibrobacter
(Marine Group A and SAR4O6), the green non-
sulfur bacteria (SAR2O2), and the gram-positive bacteria (NH16-9, BDA1-5)
have also been found, as well as numerous, unique clones that are rarely
encountered (12, 14, 17, 18).
The vertical stratification of photosynthetic bacterioplankton
populations is well-known, but only recently have investigations with
oligonucleotide probes shown that many of the most abundant
bacterioplankton lineages species of unknown physiology are also highly
stratified (4, 32). Lee and Fuhrman (23) showed that community DNAs from
the Pacific Ocean varied significantly among samples from 25, 100, 500, and
1,000 meters. Early hybridization studies with phylogenetic group-specific
oligonucleotide probes indicated that bacterial genes cloned from surface
samples were often dominant only in the upper surface layer (2, 14, 30). More
recently, we have found that four uncultured microbial groups (SAR2O2,
SAR4O6, SAR11G1 subcluster, and marine archaea group I) form stratified
populations at Atlantic and Pacific Ocean sites (17, 18, unpublished data).
These results suggest that stratification may be an important property of
community structure in marine systems, and that unique communities
might occur in the lower surface layer.
The work presented here is part of an ongoing effort to characterize the
bacterioplankton of the lower surface layer at the Bermuda Atlantic TimeSeries study site (BATS) in the western Sargasso Sea. Previous analyses of
12
this clone library from 250 m resulted in the discovery of a novel gene
lineage, SAR202, and a deep-water phylogenetic subgroup of the SAR11
cluster (14, 17). Here we describe another novel gene clone lineage (SAR324),
which is most closely affiliated with the
subdivision of the proteobacteria,
and show that it also forms stratified populations in the lower surface layer of
both the Atlantic and Pacific oceans.
Materials and Methods
Sampling and nucleic acid extraction.
Water samples were collected from BATS (31°50'N, 64°10'W) and from
ocean station PAPA in the subarctic north Pacific Ocean (approximately 50°N,
145°W) with Niskin bottles attached to a CTD (conductivity, temperature, and
depth) rosette. The samples from PAPA have not been studied previously;
however, the BATS samples used in this study have been described elsewhere
in previous studies of other bacterioplankton groups (16). Monthly time
series samples (30) were collected from BATS at two depths (0 and 200 m)
from August 1991 to February 1994. In addition to monthly samples, samples
were collected at BATS from 40, 80, 120, 160, and 250 meters ten times during
the same period. The samples from ocean station PAPA were collected in
September 1995 from depths ranging from 0 to 3300 m. 24 to 48 liters of
seawater was filtered from each depth. A Sea-Bird CTD was used to measure
continuous profiles of temperature.
13
Total cellular nucleic acids were extracted from the filters by procedures
optimized for small sample sizes, as described elsewhere (16).
Cloning.
Prokaryotic 16S rRNAs were amplified for cloning from the mixed
population genomic DNAs by PCR with Taq polymerase (Promega, Madison,
Wis.) and bacterial 16S primers (27F, AGA GTT CAT CMT CCC TCA C;
1522R, AAG GAG GTG ATC CAN CCR CA) as described previously (12, 16).
The clone library was constructed using the plasmid vector pCRII (Invitrogen,
San Diego, Calif.) as described in the manufacturer's instructions.
Transformants were screened for full-length insertions by EcoRl restriction
digestion. Clones were numbered discontinuously from 177 to 325 and stored
in LB (10 g/liter tryptone, 5 g/liter NaCl, 5 g/liter yeast extract, and 50 pg/m1
Kanamycin)/7.0% DMSO at -80°C.
Gene sequencing and phylogenetic analysis.
Plasmid DNAs were purified for sequencing from clones grown
overnight at 37°C in Luria Bertani broth using a Prep-A-Gene DNA
Purification Kit (Bio-Rad Laboratories, Hercules, CA) or a QlAprep Spin
Plasmid Miniprep Kit (Qiagen, Inc., Chatsworth, CA) according to the
manufacturer's instructions. Plasmid DNAs were sequenced bidirectionally
with universal and bacterial primers using an Applied Biosystems 373A
automated sequencer as described previously (2, 12, 14, 21). DNA sequence
14
data was manually aligned to bacterial sequences obtained from the
Ribosomal Database Project (RDP) using the program GDE, supplied by Steve
Smith (Millipore Corporation, Bedford, MA) (24). Sequences were evaluated
by the program CHECK_CHIMERA, also provided by the RDP, to aid in the
identification of chimeric gene artifacts. Phylogenetic relationships were
inferred by the neighbor-joining method and by parsimony using the
Phylogeny Inference Package (PHYLIP) version 3.4 (8, 28). Regions of
ambiguous alignment and hypervariability were excluded from the analysis.
Secondary structure analysis of the 16S rRNA gene was performed with the
program gRNAid, supplied by Shannon Whitmore (Mentor Graphics,
Wilsonville, OR).
Hybridization.
Vertical profiles of SAR324 rRNA and rDNA amplicons were
measured by hybridizations to dot blots as described previously (16, 17). For
the rDNA replicates used to generate error bars, bacterial rDNAs were
amplified in three separate reactions from seawater using bacterial 16S rDNA
primers (27F; 1492R, GGT TAC CTT GTT ACG ACT T) (12). For
hybridizations to environmental high molecular weight RNA, 100, 50, 20,
and 10 ng of each RNA sample was blotted, and the slopes of the lines were
determined by linear regressions. Nucleic acids were adsorbed onto Zetaprobe
membranes (Bio-Rad Laboratories, Inc., Carson City, Calif.), cross-linked by
TJV radiation and baking, and stored dessicated at -20°C before probing.
An oligonucleotide probe specific for the SAR324 lineage (SAR324R;
CGA AAG ACC CTC CGG) was designed to complement positions 625-639 of
15
the 16S rRNA
gene(Escherichia
coli numbering system). The probe was
prescreened for potential cross-reactivity with the program CHECK_PROBE,
provided by the RDP (24). T4 polynucleotide kinase was used to label the 5'
terminus of the oligonucleotide probe with [y-32P]ATP as described previously
(30). The empirical melting temperature (T1) of the probe was determined
by quantifying the amount of probe hybridized to dot blots of SAR324 rDNA
after 15-mm washes at temperatures from 30 to 55°C.
The rDNA and RNA blots were hybridized in Z-Hyb buffer (1 mM
EDTA, 0.25 M Na2HPO4, 7% SDS, pH 7.2) containing
Ca.
50 ng radiolabeled
oligonucleotide probe as described previously (16, 17). Follwing
hybridization, the blots were exposed to Phosphorlmager plates (Molecular
Dynamics, Sunnyvale, CA), followed by quantification with a Molecular
Dynamics Phosphorimager SI and IMAGEQUANT software. Data were analyzed
as described previously, with the hybridization of the bacterial probe 338R
used as a denominator so that variation in the hybridization of the specific
probe (SAR324R) is expressed in relative units that are proportional to
bacterial RNA; changes in the plotted values represent variation in the
proportion of bacterial RNA contributed by the SAR324 group. SAR324
rDNA hybridization values are expressed as percentages, since SAR324 genes
(amplicons) were available for use as standards for normalization, as
described previously (17, 18). Amplicons were not used to normalize the
rRNA hybridization data, since this would have assumed that free energy of
binding for the probe to RNA targets was the same as for DNA targets, an
assumption that is unlikely to be true.
16
Accession numbers.
Nucleotide sequences were filed in Genbank under the following
accession numbers: SAR324, U65908; SAR257, U65909; SAR237, U65910;
SAR214, U65911; SAR248, U65912; SAR3O8, U65913; SAR218, U65914;
SAR276, U65915.
Results
SAR324 and seven related genes were identified in a library of 148
bacterial 16S rRNA gene clones from a 250 m Sargasso Sea sample. Because so
many bacterioplankton genes had already been identified, and probes were
available, the library was screened by hybridization with radiolabeled
oligonucleotide probes that are specific for the bacterioplankton lineages
(SAR11, SAR83, SAR406 and SAR202) that had previously been shown to be
numerically significant in this and other rRNA clone libraries (2, 17, 18).
Clones that did not hybridize to these probes were selected at random for
phylogenetic analyses, and the 5' and/or 3' regions of the 16S rRNA genes
were sequenced. Of the 37 clones that were randomly sequenced, three
(SAR248, SAR276, and SAR324) appeared to be loosely affiliated with the DP
in preliminary phylogenetic analyses. Subsequently, complete bidirectional
sequences were determined for these clones. The SAR248, SAR276 and
SAR324 gene sequences were evaluated with the RDP programs SIMRANK
and CHECK_CHIMERA (25). CHECK_CHIMERA provided results which
supported the conclusion that the genes were not chimeric artifacts, but no
inferences regarding their phylogeny could be drawn from the low
SAB values
17
that were obtained (0.4-0.5) by the SIM_RANK analysis. SIM_RANK results
are expressed as
SAB
values, the number of shared oligomers of seven bases,
divided by either the number of unique oligomers in the submitted sequence
or the database sequence.
Phylogenetic analyses indicated that the novel genes formed a
monophyletic group that included no cultured representatives, thus
conforming to the definition of an environmental gene cluster (14, 26). In
separate phylogenetic comparisons of 5' and 3' domains, the genes behaved
similarly; neither domain alone showed a significant affiliation with any
phylogenetic group other than the DP (data not shown). A phylogenetic tree
inferred by the neighbor-joining method from full-length sequences of
SAR324, SAR248, SAR276 and other 16S rRNA sequences representing the
proteobacteria is shown in Fig. 2-1 (29). Bootstrap resampling (100 replicates)
of the data was used to provide statistical support for the phylogenetic
position of the SAR324 lineage (8, 10). SAR324 and related gene clones always
formed a monophyletic cluster within the DP; however, bootstrap values
supporting the DP as a monophyletic dade were low (64%). The branching
orders within the dade were not well-supported by bootstrap replicates;
hence, the deepest branches are shown here as a polytomy. Bootstrap support
for the DP group was improved considerably (from 64 to 88%) by omitting
Desulfovibrio desulfuricans and Bdellovibrio bacteriovorus from the analysis
(data not shown). The inclusion of the SAR324 lineage in this analysis caused
no rearrangement of relationships or significant changes in bootstrap values
of previously sequenced genes.
Figure 2-1. Phylogenetic tree showing relationships of the SAR324 cluster and
representative bacterial 16S rRNA genes. This tree was inferred by the
neighbor-joining method and included Ca. 1020 nucleotide positions in the
analysis. The number of bootstrap replicates out of 100 that supported each
branch is shown above (neighbor-joining) and below (parsimony) the nodes.
Values less than 50% are not shown. The DP are shown as a polytomy
because the branching order was not well-resolved.
SAR276
SAR32
-SAR248 1- Cluste
- SAR324J
Bdellovibrio bacteriovorus
metallireducens
Pelobacter acetylenicus
Myxococcus xanthus
Nannocystis exedens
Desulfovibrio desulfuricans
Chromo bacterium violaceum
Azoarcus denitrificans
Methylomonas methylovora
Oceanospirillum linum
Photobacterium phosphoreum
Escherichia coli
Roseobacter denitrificans
Rhizobium fredii
Bartonella bacilliformis
AR7
0.10
Figure 2-1.
20
Primary sequence similarities and signature sequence analyses
confirmed the loose association of the SAR324 gene lineage with the DP.
Sequence similarities among and within the four subdivisions of the
proteobacteria and SAR324, SAR248, and SAR276 were calculated from
Ca.
1020 nucleotide positions (Table 2-1). Only one member each of the a, j3, and y
subdivisions are shown; however, similarity matrices involving larger data
sets gave similar results (data not shown). Although the similarity between
SAR324 and members of the DP were low (0.856-0.907), they are not unusual
given the range of similarity values among characterized DP (0.867-0.987).
Among the DP, D.
desulfuricans
had the lowest similarity value (0.854) when
compared to SAR324, consistent with the observation that removal of this
sequence from the phylogenetic analysis significantly increased the bootstrap
values supporting the DP as a monophyletic dade.
The SAR324 sequences were compared to 16S rRNA signature
sequences for the bacterial phyla and their subdivisions, which were
previously published by Woese (35) and Haddad et al. (20). The highest
observed percentage of shared signature positions for SAR324 was to the DP
(87-90%) (Table 2-2). Among 51 characterized DP that were similarly analyzed,
the correspondence of nucleotide identities at signature sequence positions
for the DP was 86-100% (data not shown).
The proposed secondary structure for members of the SAR324 cluster is
structurally unique and conserved within the group (Fig. 2-2). Seven of the
nine signature sequence mismatches between SAR324 and the DP are
compensatory base changes (changes in variable nucleotides that preserve the
secondary structure of the 16S rRNA molecule) across regions of double-
stranded pairing. The inset illustrates variable region two in SAR276, which
21
Table 2-1. Sequence similarities (based on ca. 1020 nucleotide positions)
among the SAR324 cluster and 16S rRNA gene sequences from
representatives within the proteobacteria. The boxed numbers refer to the
similarities among the subdivision of proteobacteria and the SAR324 group.
1
2
I
SAR276 (
2
SAR248 ()
I 09531
3
SAR324 (ö)
(0.947 0.985
4 Bdellovibrio bacteriovorus ()
5
Ceo bacter metallireducens ()
6 Pelobacter acetylenicus (8)
3
4
5
6
7
8
9
10
11
-
-
0.873 0.873 0.873
-
J 0.906 0.888
0.890
0.896J
0.907 0.889
0.892
0.898 0.957f
-
-
7
Myxococcus xanthus (6)
(0.882 0.878 0.881
0.871 0.921 0.9251
8
Nannocystis exedens (6)
(0.879 0.867 0.868
0.873 0.895 0.895 0.987(
9
Desulfovibrio desulfuricans (6)
(0.856 0.851
0.854
0.867 0.895 0.898 0.876
0.881 (
10 Chromobacterium violaceum (3)
0.872 0.873
0.871
0.848 0.870 0.873 0.858
0.841 0.849
11 Oceanospirillum linurn(y)
0.863 0.865
0.864
0.868 0.890 0.883
0.883
0.872 0.869 0.893
12
0.855 0.857 0.857
0.860 0.890 0.885
0.868
0.845 0.864 0.852 0.882
Roseobacter denitrificcins (a)
Table 2-1.
12
-
-
-
-
-
-
23
Table 2-2. Signature nucleotides relating SAR324 and SAR276 to the
subdivision of proteobacteria.
24
Position a
Position
DP
SAR324/276
+1-
875
U:c
U
+
877
Y:a
C
U/C
+
+
C
+/+
878
Y:a
C
+
906
A:g
C
C/U
+
916
129
C
C
U
+/-
929
C
C
129:1
A
A
+
947
G:u
A
G
G
A
199
R
Y
C/A
+/-
948
Y
C
+
U
+
976
C
+
C
C
C
C
+
1015
A:g
237
+
1024
G:c
C
C
U
242
C:g
C
+
1026
C
C
284
C:c
C
+
1116
Y
U
+
370
C
C
+
1120
Y:G
C
+
371
C:a
+
1219
A
A
+
390
C:u
+
1233
R
C
+
391
C
A/C
U/C
C
+
1234
C:a
398
C
1246
G:u
+
438
G:u
1252
A
U
U
A
449
A
+
1260
G:Y
U/G
+
C
A
+
1291
C:g
1297
Y
C
U
+
+
+
1298
C:a
+
1325
C
C
U
+
A/C
+
107
US
124
233
236
485
C
496
G:a
502
A:g
513
C
U
U
A
543
U:c
554
U:a
A
A
U
U
U:c
640
689
690
C:a
+
+
+
+
+
+
+
+
1421
+
1426
C
Y
U:R
C
+
1431
Y:a
A
+
A
A/C
+
1437
C
C
+
R:u
A
+
1441
G:u
+
+
1443
C
+
1460
+
1464
A
G
+
1465
C:u
812
C
C
C
U
C
C
C
+
1467
Y
G
C
A
C
C/U
C
822
R:u
A/G
+
1469
A:u:c
A
+
823
R:u
1481
U:c
+
1520
G:c
C/U
C
+
A:g
G
A
+
825
871
U
U
+
564
698
722
760
C
C
/
A
A/U
50
b
SAR324/276
C
A
C
44
a
DP
Y:a
G:a
+
+
+
+
+
+
+
E. coli numbering system.
DP signature nucleotides.
Match (+) or mismatch (-) between the SAR324 lineage and the DP.
Table 2-2.
25
Figure 2-2. Proposed secondary structure for the SAR324 16S rRNA gene.
The DP signature sequences shared by SAR324 are marked with an asterisk.
The lowercase letters represent DP signature nucleotides, which are different
than the SAR324 nucleotide at that position. The target site for the SAR324
probe is shown between E. co/i positions 625-639. The dashed box indicates
variable region two of the molecule, the region of variation between SAR324
and SAR276. The inset shows variable region two of SAR276.
720
AAG
UAI3A
AA
A
G
U
U
A41GUGGUCCUUA
U0
AGAC-G
A GA
GU
G
U
A
GC
aA
AACCCcGG
111111
A
a-c
A-U
G
ACG
A
C
0
u
G
A
U
C0
*GJ??U(JU
A
AAU
5aA C0
A
A
a-c
c-a
A-U
U
A
UAAC_G
A GbfACacjc_a
ac
CA
UA
Cu
CA
A
A
AU
C
A50A034j
ua
a
u
320
GGCG
UC
5A
aj
A/>a
0GA UA.)
a-C
0.41
A
A.
UA
c:' USGU
C"GG
G0c0
U
0 AU
C
A
A
:
I
UA??A
GUUGQCaUC
a
t
1520
A
U
C
A
C
C
U
SAR248
SAR276
SAR324
U
0
A
*0. U
U
132U
C
A
A * u.
a-c
C
A
UAO
AG
a
A
U9AA
G.A
G.0
C
A
G
041
A
C
a
9,
G1)
AG
G_.
a
Gu 5
CAGG
C AC
A0
UG
C
a
a
A5
U A
C
c
2j
G'j
a
U-u
A-U
C-0
C-a
A
G
-
A
C
C
A A?GA (I1?16
AAa
/
CACAA\"/A
GaG
CA
ACAC
AAA
c;*
120
Ca
A)
tiC4
A
\
AAA
AU141 C-S
UA&*
ApU
U ??
U CCO3A
G'U
a. A
ACGI)
UAACC
A
c
a
a
ac
A
U
GC
C.
ac
c_a
A_tJA
3,
C-a
a-S U
A.
U
A
a-c
a
228
U
C0?G
UC
G-C
G
a .0
o .0
?Yuu??°
Ca
AOCA U& CGA0000A A
a_c
U-A
AC OC_GLJcuaaaAC
U- APc
A
A
'
A
UA
U-A
AU
a
a-c
UU
A
V2
Figure 2-2.
A
a-c
0-c
UA
U-A
A
u
0
U. G
a
CUCOJUG A
A0AU000
UUAA
A
U
A
U-
COS
C
U
A
c
;acd'JUAAU uUA
ACS
A
A
a
ACcuuAE1200
A
.0
a
CUCGUaAA
41A'C-a a
ac
c
A A d'5a A
?AGA',.F
a
5_41
A041
a-a
U-A
a
520-A
G
a.0a
ACA
c
C
o
A
o
G.
C
A
a-c
ca
c-a
a
CC
A
A
ic
CA
G
A-81 5
AGOCa
041
a-c
AUc
1UOç U-A
0
A
a
a
au
Uc
a-c
a-c
UA
a-c
AACC0U
LIC? C
cGuSSAa Sc/c,
c
a
u
1
Ua_cU
U_AA
A
A0
a
A
C&JULXA
A
Aa_cC
0C
A-U
GAG
C-G
U.S
620-C
1120
C
AAUGC.10
c
11?
Yi
A
A005 GOGGAUS ACGG GCC
GA
SC
aA
A
A-U AGO
C-G
U
AG
A-U
a-C
U
U
S
A
AA
C
U.G
a.u4
a_C
0_C
C5
C-S
0_C
SAR324 Probe
-AA
u.
CGAA
a
U
?GA
G
AAA
AA
U
C
SAR276 V2 Region
A
27
shows a deletion, relative to SAR324, that is the only topological secondary
structure variation found so far within the SAR324 lineage (19). Variable
region two contains two insertions or deletions (indels) relative to E. coli, the
first from positions 183-194, and the second from positions 203-218. The first
indel contains 25 and 11 nucleotides for SAR324 and SAR276, respectively. A
comparison of 51 other characterized DP revealed variations of 14-23
nucleotides in this same region. For the second indel, two of the 51 DP
analyzed had the same deletion (B. bacteriovorus and Nannocystis exedens).
The structural variation of the 16S rRNA gene within the SAR324 sequence
cluster is consistent with variation seen in other clades previously
encountered in this 250 m clone library (reference 17 and unpublished data).
We call attention to this variation because it underscores the substantial
variability within this group, which suggests the presence of multiple
bacterial species.
The discovery of multiple genes of a common type, like those
presented here, is evidence that members of the SAR324 gene lineage form a
novel, diverse cluster and are not chimeric artifacts. For example, SAR248
and SAR324 differ by only 22 nucleotides, which are distributed throughout
the gene. The nucleotide differences are confined mostly to hypervariable
loops and compensatory base changes in stem regions; hence, they introduce
no incongruities to secondary structural models. The possibility that two
genes with unique similarities in conserved and variable regions could result
from in vitro recombination in a complex gene mixture is unlikely.
An oligonucleotide probe (SAR324R), designed to specifically hybridize
to the SAR324 lineage (Fig. 2-2), was evaluated with the RDP program
CHECKPROBE (25). There were a minimum of three mismatches with any
I.1
known 16S rRNA gene sequence, and a minimum of five mismatches with
all other sequenced clones from the 250 m clone library. The specificity of the
probe was also shown empirically using blotted arrays of 16S rDNA genes
from several cultured and uncultured bacterioplankton under stringent
hybridization conditions (data not shown). The empirical Tm for this
oligonucleotide probe was determined to be between 40 and 45°C, which
supported the selection of 40°C as the stringent wash temperature (Fig. 2-3).
No cross-hybridization to unrelated genes was encountered. The radiolabeled
oligonucleotide was used to screen the 250 m Sargasso Sea library, and six
additional SAR324-related clones were detected by strong hybridization
signals.
The sequencing of the additional clones provided further evidence for
the highly diverse nature of the SAR324 lineage, and further supported the
specificity of the probe as a marker for a monophyletic microbial group. One
of the clones was a chimera (SAR2O6), which was detected after partial
sequencing of the 3' and 5' ends of the gene (data not shown).
The remaining five clones were phylogenetically related to 5AR324, although
the genes encompassed substantial variation in the form of nucleotide
substitutions similar to those described above (Fig. 2-4). A similarity matrix
based on ca. 300 nucleotide positions revealed that five (SAR324, 5AR257,
SAR237, SAR214, and SAR248) of the eight clones in the SAR324 lineage were
97-99% similar to each other in an analysis that excluded hypervariable
regions. Seven of the eight clones had secondary structures comparable to
SAR324 in variable region two (Fig. 2-2).
The results of the hybridization analyses indicate that SAR324 is
vertically stratified in the water columns of both the Atlantic and Pacific
29
Figure 2-3. The thermal stability of the SAR324 probe. The empirical Tm of
the probe was determined by quantifying the amount of probe hybridized to
dot blots of SAR324 rDNA after 15 mm washes at a range of increasing
temperatures (30 to 55°C).
30
2
C)
C
0
1.5
CD
CD
C)
N
V
L.
z .0>1
I
1
0.5
\.
30
35
40
45
50
Temperature (°C)
Figure 2-3.
55
31
Figure 2-4. Phylogenetic tree showing relationships among genes within the
SAR324 cluster. This tree was inferred by the neighbor-joining method from
Ca. 300 nucleotides.
SAR324
Cluster
[IIMIJ
Figure 2-4.
NJ
33
Oceans (Fig. 2-5). In hybridizations to 30 consecutive time-series samples
from two depths (0 and 200 m) in the Sargasso Sea, SAR324 was always found
to be more abundant at 200 m than at 0 m (data not shown). A one-tailed ttest assuming unequal variances indicated that the SAR324 lineage was
proportionately three times more abundant at 200 m than at 0 m (P = 1.0 X 10
4).
The time-series data are consistent with the data obtained from the rDNA
and rRNA vertical profiles and support the hypothesis that the SAR324
lineage is located in the lower surface layer and mesopelagic.
The SAR324 probe was hybridized to amplified rDNA prepared from
vertical profiles of seawater samples, and to high molecular weight RNA
from the Atlantic Ocean and Pacific Ocean to more accurately determine the
position of SAR324 in the water column. In both cases, the SAR324 cluster
was found to be most abundant in the aphotic zone, peaking at 200 m at
BATS, and at 500 m in the profiles from ocean station PAPA (Fig. 2-5),
although this difference between sites may have been due to the different
depth ranges sampled. Similar to other uncultured bacterioplankton, the
absolute abundance of SAR324 rRNA could not be accurately estimated from
hybridization of oligonucleotide probes to rRNA because no pure SAR324
RNA is available for standardization. However, rDNA amplicons from the
target organism (SAR324) are available, therefore, the rDNA hybridization
values in Fig. 2-5A are expressed in percent. At the position of the maximum
in its distribution in the Atlantic samples, SAR324 comprised 18% of bacterial
rDNA amplicons, indicating that it is a very abundant group.
34
Figure 2-5. The distribution of the SAR324 gene among 16S rDNA amplicons
and high molecular weight (HMW) RNA prepared from plankton samples.
(A) The distribution in percent of SAR324 rDNA, as a proportion of bacterial
rDNA, in the upper 250 m at BATS. The means and standard deviations are
shown for triplicate PCR reactions from a single nucleic acid sample. (B)
Hybridization of the 5AR324 probe to HMW RNA from the the upper 250 m
at BATS, expressed in relative units. (C) Hybridization of the SAR324 probe
to high-molecular-weight RNA from the upper 3300 m at ocean station
PAPA, expressed in relative units.
ATLANTIC
A
PACIFIC
Temperature (°C)
B
C
ATLANTIC
CTemperature (°C)
0
50
0
250
\
500
4
50-
0
100
100750
a.
C)
c
150
0NN
150-
1000
C
1500
200
200-
2501
0
I
I
5
10
15
20
25
rDNA Specific Hybridization (%)
/
3300
0
250-
I
0
0.05
I
0.10
0.15
0.20
0.01
0.02
0.03
0.04
rRNA Relative Hybridization
0
0.25
rRNA Relative Hybridization
Figure 2-5.
c)
c.rI
36
Discussion
The data presented here reveal the existence of a previously unknown
bacterioplankton group, show that they have a wide biogeographical
distribution, and provide insight into their ecological role by demonstrating
that these organisms are most abundant in the lower ocean surface layer.
Furthermore, the evidence shows that this phylotype is in fact a diverse but
monophyletic gene cluster, and therefore might be regarded as a collection of
species.
The particular emphasis of this investigation was dictated by a longterm research strategy that will utilize fluorescent probes to identify single
cells in future studies of environmental samples. From the beginning, it has
been clear that strategies involving ribosomal RNA probes for uncultured
bacteriplankton groups would only be sound if thorough sequence databases
that explored the genetic diversity within gene clusters were available for
probe design (14, 27). Recently, Amann and colleagues (1) obtained perplexing
results when hybridizing fluorescent probes to natural populations of beta-i
proteobacteria in activated sludge. The data verified that the diversity of
genes in environmental gene clusters indeed represented real diversity at the
cellular level, but also showed that the specificity of probes could not easily be
extrapolated from the analysis of a limited dataset of environmental
sequences.
The physiology of the SAR324 gene cluster is unknown, and cannot be
deduced from its observed phylogenetic associations; however, the
physiological variability of the DP provides a background for the construction
of hypotheses regarding the activity of the SAR324 group. Metabolically, the
37
subdivision is mainly divided into two groups: the aerobes (bdellovibrios and
the myxobacteria) and the anaerobes, which use sulfate or other inorganic
compounds as electron acceptors. Although unclear in this analysis, it is
possible that the sulfide producers form the deepest branch within the DP,
and that the bdellovibrios and myxobacteria represent aerobic adaptations
(35). Furthermore, previous studies characterized the bdellovibrios as a
phylogentically heterogenous group composed of some 'fast-clock" species,
which has further complicated the resolution of the phylogenetic positions of
the organisms within the DP (35).
The SAR324 lineage represents a unique cluster within the DP. Teske
and colleagues (33) have recently obtained 16S rRNA sequences related to
genera within the DP, but phylogenetic analyses similar to those described
here failed to indicate any specific association between the genes reported in
that study and the SAR324 lineage. The relationships reported here are the
most significant relationships uncovered following a thorough search of
public sequence databases.
The hybridization data are presented as SAR324 rDNA abundance
among PCR amplicons, in percent, and relative rRNA abundances. As we
have shown previously, these measures often lead to qualitatively similar
conclusions where general trends in the ecological distributions of
bacterioplankton are the subjects of interest (17, 18). Although relative gene
frequencies are sensitive to the distribution of rDNA copy number and
genome size, as well as cell numbers along environmental transects, they
nonetheless represent a type of information that is very informative, though
it may not correspond directly to biomass or microbial activity (21). Likewise,
relative rRNA abundance has its pitfalls, most notably in the fact that it
measures protein synthesizing activity and so may underestimate the
abundance of populations that are temporarily inactive. Notwithstanding the
novelty of these measures, they are emerging as useful indicators of microbial
distributions that complement other types of measurements, such as biomass,
that often have their own limitations (24).
Although the physiology of the SAR324 cluster is unknown, the
diversity and proportionally high abundance of this group in the aphotic
zone suggests that this is a group of related species that are functionally
specialized for life in abyssal regions of the ocean. Evidence of other
microorganisms (SAR4O6, SAR2O2 and deep water variants of SAR11) that
specifically inhabit the aphotic zone has been described (17, 18). Collectively,
these data suggests that the aphotic zone bacterioplankton community may be
a more specialized microbial community than was generally thought
previously. Organic carbon is exported to the aphotic zone by sinking
particles, and by injection of dissolved organic carbon during winter mixing
(3). As a major constituent of the aphotic bacterioplankton community, it
seems likely that the SAR324 cluster participates somehow in these processes.
Although the overall activity of microbes in abyssal regions of the oceans is
much reduced relative to microbial activity in the euphotic zone, the ocean
depths nonetheless sustain a significant biomass of microorganisms that are
likely to be important in the ecology of the oceans (5). Further studies based
on the data presented here will be aimed at elucidating the population
genetics and ecological role(s) of these species.
39
Acknowledgements
We are grateful to the Bermuda Biological Station for Research BATS
group and Nanci Adair for collecting and processing nucleic acid samples
from BATS, to Nelson Sherry and Michael Lipsen for sampling from ocean
station PAPA, and to Frank Whitney, Institute of Ocean Sciences, Sidney,
B.C., for the Pacific Ocean physical data. We also thank Douglas Gordon,
Brian Lanoil, Michael Rappé, Ena Urbach, Marcelino Suzuki, and Kate Field
for their many helpful suggestions.
This work was supported by NSF grant OCE 9016373 and DOE grant
FG0693ER61697 to S.J.G, and by an N. L. Tartar Fellowship to T.D.W.
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12. Fuhrman, J. A., K. McCallum, and A. A. Davis. 1993. Phylogenetic
diversity of subsurface marine microbial communities from the Atlantic
and Pacific oceans. Appi. Environ. Microbiol. 59:1294-1302.
13. Giovannoni, S. J. 1991. The polymerase chain reaction, p. 177-203. In E.
Stackenbrandt and M. Goodfellow (ed.), Sequencing and hybridization
techniques in bacterial systematics. John Wiley & Sons, Inc., New York.
14. Giovannoni, S. J., T. B. Britschgi, C. L. Moyer, and K. G. Field. 1990.
Genetic diversity in Sargasso Sea bacterioplankton. Nature 345:60-63.
15. Giovannoni, S. J., E. F. DeLong, G. J. Olsen, and N. R. Pace. 1988.
Phylogenetic group-specific oligodeoxynucleotide probes for identification
of single microbial cells. J. Bacteriol. 170:720-726.
41
16. Giovannoni, S. J., M. S. Rappé D. Gordon, E. Urbach, M. Suzuki, and K. G.
Field. 1996. Ribosomal RNA and the evolution of bacterial diversity, p.
63-85. in D. McL. Roberts, P. Sharp, G. Alderson, and M. Collins (ed.),
Evolution of microbial life. Cambridge University Press, Great Britain.
17. Giovannoni, S. J., M. S. Rappé, K. L. Vergin, and N. L. Adair. 1996. 16S
rRNA genes reveal stratified open ocean bacterioplankton populations
related to the green non-sulfur bacteria. Proc. Nati. Acad. Sci. 93:79797984.
18.
Gordon, D. A., and S. J. Giovannoni. 1996. Detection of stratified
microbial populations related to Chiorobiuni and Fibrobacter species in
the Atlantic and Pacific oceans. Appl. Environ. Microbiol. 62:1171-1177.
19. Gray, M. W., D. Sankoff, and R. J. Cedergren. 1984. On the evolutionary
descent of organisms and organelles: A global phylogeny based on a
highly conserved structural core in small subunit ribosomal RNA.
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20. Haddad, H., F. Camacho, P. Durand, and S. C. Cary. 1995. Phylogenetic
characterization of the epibiotic bacteria associated with the hydrothermal
vent polychaete Alvinella pompejana. Appi. Environ. Microbiol. 61:16791687.
21. Kemp, P. F., S. Lee, and J. LaRoche. 1993. Estimating the growth rate of
slowly growing marine bacteria from RNA content. Appl. Environ.
Microbiol. 59:2594-2601.
22. Lane, D. J., K. G. Field, G. J. Olsen, and N. R. Pace. 1988. Reverse
transcriptase sequencing of ribosomal RNA for phylogenetic analysis.
Meth. Enzymol. 167:138-144.
23. Lee, S. and J. A. Fuhrman. 1991. Spatial and temporal variation of
natural bacterioplankton assemblages studied by total genomic DNA
cross-hybridization. Limnol. Oceanogr. 36:1277-1287.
24. Lee, S., C. Malone, and P. F. Kemp. 1993. Use of multiple 16S rRNAtargeted fluorescent probes to increase signal strength and measure
42
cellular RNA from natural planktonic bacteria. Mar. Ecol. Prog. Ser.
101:193-201.
25. Maidak, B. L., N. Larsen, M. McCaughey, R. Overbeek, G. Olsen, K. Fogel,
J. Blandy, and C. Woese. 1994. The Ribosomal Database Project. Nucleic
Acids Res. 22:3485-3487.
26. Mullins, T. D., T. B. Britschgi, R. L. Krest, and S. J. Giovannoni. 1995.
Genetic comparisons reveal the same unknown bacterial lineages in
Atlantic and Pacific bacteioplankton communities. Limnol. Oceanogr.
40:148-158.
27. Olsen, G. J., D. J. Lane, S. J. Giovannoni, and N. R. Pace. 1986. Microbial
ecology and evolution: a ribosomal RNA approach. Ann. Rev. Microbiol.
40:337-365.
28. Rappé, M. S., P. F. Kemp, and S. J. Giovannoni. 1995. Chromophyte
plastid 16S ribosomal RNA genes found in a clone library from Atlantic
Ocean seawater. J. Phycol. 31:979-988.
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method for reconstructing phylogenetic trees. Mo!. Biol. Evol. 4:406-425.
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chemically synthesized polynucleotides to form the DNA duplex
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the prochiorophyte Prochlorococcus in the central Pacific Ocean as
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33. Teske, A., C. Wawer, G. Muyzer, and N. B. Ramsing. 1996. Distribution of
sulfate-reducing bacteria in stratified fjord (Manager Fjord, Denmark) as
43
evaluated by most-probable-number counts and denaturing gradient gel
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Environ. Microbiol. 62:1405-1415.
34. Ward, D. M., R. Weller, and M. M. Bateson. 1990. 16S rRNA sequences
reveal numerous uncultured microorganisms in a natura' community.
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35. Woese, C. R. 1987. Bacterial evolution. Microbiol. Rev. 51:221-271.
44
CHAPTER 3
SECONDARY STRUCTURE ANALYSIS OF SAR2O2-RELATED CLONES
Introduction
The SAR2O2 lineage, first reported by Giovannoni et al. (1996), was
discovered by randomly sequencing clones from the same BATS 250 m library
described previously. The construction of the 250 m library was motivated by
hybridization experiments that indicated that gene lineages which were
abundant in the surface layer of subtropical oceans were rare in the deeper
layers. Phylogenetic analyses of complete 16S rRNA gene sequences showed
that the 5AR202 genes constitute a new deep-branching lineage of the
Chioroflexus/Herpetosiphon phylum, one of the 11 bacterial phyla originally
described by Woese (1987). The Chiorofiexus/Herpetosiphon phylum seems
to have diverged before the main radiation of bacteria and is associated with
four genera, all of which contain some thermophilic species: Chloroflexus
and Heliothrix, both phototrophs, and Herpetosiphon and
Therrnomicrobium, both chemoorganotrophs (Olsen and Woese, 1993; Fig. 31). In the study by Giovannoni et al (1996), a specific oligonucleotide probe
(SAR2O2AR) was constructed for the purposes of screening the clone library
and for studying the distribution of the 5AR202 genes in the water column.
From the hybridization experiments, ten additional clones were obtained and
the SAR2O2 gene cluster was shown to be stratified in the lower region of the
water column in the Atlantic and Pacific Oceans.
45
Figure 3-1. Phylogenetic tree showing relationships of SAR2O2 and SAR3O7
to representative bacterial 16S rRNA genes. The tree was inferred from
nearly complete sequences by the neighbor-joining method. The numbers of
bootstrap replicates that supported the branching order, from a total of 100
replicates, are shown above (neighbor-joining) and below (Wagner
parsimony) the internal segments. Values below 50% are not shown. Gene
sequences from Haloferax volcani and Methanococcuc voltae were used to
determine the root of the tree. From Giovannoni et al. (1996).
46
Leptonema illini
63
Spirochaeta bajacaliforniensis
Bacteroides fragilis
100
Flavo bacterium aqua tile
Chiorobiurn vibrioforme
Oceanospirillum linum
Desulfuromonas ace toxidans
Chlamydia trachomatis
Demo coccus radiodurans
98
SAR6
100
Synechococcus PCC 6301
Megasphaera elsdenii
Heliobacterium chiorum
Bacillus subtilis
SAR2O2
SAR3O7
The rmomicrobium roseum
Chloroflexus aurantiacus
Herpetosiphon aurantiacus
Pirellula staleyi
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Thermotoga maritima
Fervido bacterium ice landicum
Geo toga subterranea
ifex pyrophilus
0.10
Figure 3-1.
IGREEN
NON-SULFUR
BACTERIA
47
The widespread distribution of the SAR2O2 gene lineage suggests that bacteria
asoociated with this gene cluster may be significant members of the
bacterioplankton community in the lower ocean mixed layer. This was one
of the first demonstrations of microbial stratification based on the application
of novel methods for surveying populations of uncultured species; many
similar studies soon followed.
The diversity of the SAR2O2 gene cluster was first described from
sequencing analyses of clones that hybridized to the SAR2O2AR
oligonucleotide probe. The genes discussed in this chapter are among the
clones that did not hybridize to the SAR2O2AR probe, but were instead
discovered by random sequencing. These unique genes also appear to be
members of the SAR2O2 lineage; however, the sequences are divergent from
the members of the SAR2O2 cluster first described. The most interesting
observation to report from these analyses is the substantial variability in the
secondary structure at variable regions one and two of the 16S rRNA
molecule. The continued investigation of this novel lineage is driven by its
possible significance in the ecology of the lower ocean mixed layer.
Materials and Methods
Sampling and nucleic acid extraction.
This procedure was performed as described in Chapter 2 with the
following exception: no samples from ocean station PAPA were collected (all
samples analyzed were collected from BATS).
Cloning.
The procedure for cloning was performed as described in Chapter 2.
Gene sequencing and phylogenetic analysis.
The procedure for gene sequencing and phylogenetic analysis was
performed as described in Chapter 2 with the following exception: not all
genes reported here are complete, bidirectionally sequenced genes (some
partial sequences are reported).
49
Accession numbers.
The sequences described in this chapter have not been submitted to
Genbank, but will be submitted upon publication of the results obtained in
this study.
Results and Discussion
The clones described in this chapter were discovered by randomly
sequencing the BATS 250 m clone library that has been mentioned
previously. Phylogenetic analyses showed that these genes (SAR226, SAR242,
SAR25O, SAR251, SAR256, SAR259, SAR267, SAR269, SAR272, and SAR317)
represent highly divergent lineages that are most closely related to the
SAR202 gene cluster first described by Giovannoni et al. (1996); however,
these genes did not hybridize to the SAR2O2AR probe. These recently
discovered genes, when compared to the 16S rRNA genes of the described
SAR2O2 lineage, present not only the lowest similarities seen within gene
clusters, but also extreme variability within the secondary structures of the
16S rRNA molecule at variable regions one and two (Gray et al., 1984). The
significance of this variation is unknown but may indicate the presence of
multiple bacterial species.
The sequencing of the ten clones recovered in the original study and of
those discovered in this study provides evidence that the SAR2O2 gene
cluster is highly diverse. In the study by Giovannoni et al. (1996), the
secondary structural model for the SAR2O2 gene product was used to examine
the sequence for any unusual base pairing in conserved helices or loop
structures (Fig. 3-2). The majority of the structure within the 16S rRNA
molecule was conserved; however, two new structural phenotypes in
variable regions one and two were observed among the newly discovered
clones (Fig. 3-3). Although variable regions one and two within the molecule
were significantly different among clones, diagnostic base substitutions and
secondary structure signatures associated with the Chloroflexus/
Herpetosiphon phyluth and the SAR2O2 lineage were conserved. In five of
the ten new clones examined, all five contained the unusual 15 base deletion
between positions 1123 and 1147 (data not shown). Furthermore, the loop
structure between positions 607 and 630 was present in eight of the ten clones.
Diagnostic base substitutions defined for the Chloroflexus/Herpetosiphon
phylum were also common among several of the clone types. These
observations suggest that diversity or similarity within novel gene clusters
cannot be predicted and is unknown until further sequences are explored.
This information is potentially significant when considering oligonucleotide
probe design, which capitalizes on sequence variation in different regions of
the 16S rRNA molecules.
51
Figure 3-2. Secondary structural model of 5AR202 16S rRNA. Arrows
indicate signature nucleotides of the Chioroflexus/Herpetosiphon phylum.
The shaded areas indicate unusual secondary structural features that are
unique to members of the Chloroflexus/Herpetosiphon phylum. (A) Loop
structure at E. coli positions 607-630. (B) The 15 base pair deletion in the
region of positions 1123-1147. From Giovannoni et al. (1996).
52
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Figure 3-3. 16S rRNA structural variation among SAR2O2 gene clones. (A)
Secondary structure of variable regions one and two of the two clones
(SAR2O2 and SAR3O7) originally described by Giovannoni et al. (1996). (B)
Secondary structure variation at variable regions one and two of newly
described clones related to SAR2O2 showing the insertion at variable region
one (X) relative to SAR2O2 and SAR3O7. (C) Secondary structure variation at
variable regions one and two of newly described clones related to 5AR202
showing the insertion at variable regions one (X) and two (Y) relative to
SAR2O2, SAR3O7, and other newly discovered clones.
54
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57
CHAPTER 4
PHYLOGENETIC ANALYSIS OF CLONE SAR196
Introduction
Phylogenetic analyses of rRNA genes rarely provide persuasive
information on the pFiysiological potential of microbes. SAR196 was an
unusual exception. SAR196 is another gene clone from the BATS 250 m
clone library that was discovered by random sequencing. Phylogenetic
analyses indicate that this gene clone is most closely related to the cultured
species Nitrospira marina, a nitrite-oxidizing bacterium. This species was
originally isolated in pure culture from a sample collected at a depth of
approximately 200 m from the Gulf of Maine in the Atlantic Ocean (Watson
et al., 1986). N. marina differs from the three recognized terrestrial
chemolithotrophic nitrite-oxidizing genera in that it possesses comma- to
helical-shaped cells, divides by transverse fission in a single plane and has
been shown to be intrinsically marine, growing as a strict aerobe in 70-100%
seawater mineral medium with nitrite as the sole energy source. Members of
the genus appear to be ubiquitous in oceanic environments, including both
the water column and sediments. Furthermore, members of this genus are
widespread, having been isolated from New York harbor sediments, Black
Sea water samples, Woods Hole Harbor, Cape Cod beaches and salt marshes,
and from Atlantic water off the coast of Africa (Watson et al., 1986). It's
possible that N. marina may be one of the most prevalent chemolithotrophic
nitrite-oxidizing species in marine environments.
Phylogenetically, there has been some debate regarding the position of
N. marina.
Based on 16S rRNA sequence data, it has recently been shown
that species of the genus Nitrobacter are members of the a subdivision of the
Proteobacteria, while Nitrococcus belongs in the y subdivision. Nitrospina
and Nitrospira were similarly placed within the class Proteobacteria, but in
the
subdivision; however, no specific relationship to each other or to other
members of the subdivision were shared (Teske, et al., 1994). A more recent
study describes a novel obligate chemolithoautotrophic, nitrite-oxidizing
bacterium, Nitrospira moscoviensis, isolated from a partially corroded iron
pipe (Ehrich et al., 1995). This novel species is similar to N. marina in
morphology and substrate range, and has been shown to be phylogenetically
related to N. marina. A phylogenetic analysis that included the bacterial
phyla as well as archaeal sequences used as outgroups revealed that the novel
species and N. marina represent a phylogenetically coherent group together
with the leptospirilla.
Clone SAR196 is phylogenetically associated with N. marina, as shown
by analysis of complete 16S rRNA gene sequence and the use of phylogentic
programs. The analysis presented here confirms the association of N. marina
and SAR196 to the leptospirillum group and shows that organisms belonging
to the Nitrospira genus are, therefore, not members of the S subdivision of
the class Proteobacteria.
59
Materials and Methods
Sampling and nucleic acid extraction.
This procedure was performed as described in Chapter 1 with the
following exception: no samples from ocean station PAPA were collected (all
samples analyzed were collected from BATS).
Cloning.
The procedure for cloning was performed as described in Chapter 1.
Gene sequencing and phylogenetic analysis.
The procedure for gene sequencing and phylogenetic analysis was
performed as described in Chapter 1.
Accession number.
The SAR196 gene has not been submitted to Genbank, but will be
submitted upon publication of the results obtained in this study.
Results and Discussion
Clone SAR196 was among the unidentified clones chosen at random
for phylogenetic analysis by complete bidirectional sequencing of the 16S
rRNA gene. As mentioned in previous chapters, the complete SAR196 gene
sequence was evaluated with the RDP programs SIM_RANK and
CHECK_CHIMERA (Olsen et al., 1991). CHECK_CHIMERA provided results
supporting the conclusion that the gene was not chimeric; however, no firm
conclusions regarding the phylogeny of SAR196 could be drawn based on the
low SIM_RANK values obtained
(SAB
0.530). SIM_RANK results, although
inconclusive, are sometimes useful in determining which phyla or domain
the gene of interest could be distantly related to. In this study, SIM_RANK
results were used as a starting point, so to speak. The low value of 0.530 was
the highest SAB value obtained and corresponded to an unidentified
proteobacterium; therefore, a thorough phylogenetic analysis was performed.
A variety of methods were employed to determine the evolutionary
position of the SAR196 gene clone. Two phylogenetic trees, inferred by the
neighbor-joining method from the full length SAR196 gene sequence and
other representative 16S rRNA sequences, were constructed in the
preliminary analysis. One of the phylogenetic trees represented the
Proteobacteria exclusively, while the other tree included other Bacterial phyla
in addition to the Proteobacteria (data not shown). Both data sets included ca.
975 positions in the mask to exclude regions of ambiguous alignment and
Aqufex
pyrophilus was used as the outgroup. The SAR196 sequence did not
show a significant affiliation with any of the subdivisions of the
Proteobacteria, but instead was related to N. marina (Fig. 4-1).
Figure 4-1. Phylogenetic tree showing relationships of the SAR196 gene clone
and representative bacterial 16S rRNA genes. This tree was inferred by the
neighbor-joining method and included Ca. 975 nucleotide positions in the
analysis. The number of bootstrap replicates out of 100 that supported each
branch is shown above (neighbor-joining) and below (parsimony) the nodes.
Myxococcus xanthus
1
ö
Pelobacter acetylenicus
Agrobacterium tumefaciens
I
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92
100
100
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71
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100
100
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93
T7Photobacterium phosphoreum
}
Escherichia coli
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Nitrospira marina
Pz
1eI]
SAR196
100
Nitrospira moscoviensis
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0.10
Figure 4-1.
NJ
63
Although the phylogenetic position of N. marina is somewhat controversial,
the results shown here indicate that N. marina and relatives are closely
associated with Leptospirillum sp. This is in agreement with the result
obtained by Ehrich et al. (1995), but in opposition with the conclusions of
Teske et al. (1994), whose results suggest that N. marina is phylogenetically
affiliated with the
subdivision of the class Proteobacteria.
The presence of the SAR196 16S rRNA gene clone in the BATS 250 m
library is not surprising since its closest relative, N. marina, has been cultured
from a variety of marine samples, including one retrieved from 200 m in the
Gulf of Maine in the Atlantic Ocean. The distribution and abundance of this
gene clone in the water column are unknown, but the physiological
characteristics of its closest relative N. marina suggest that it may play a role
in nitrification in the western Sargasso Sea.
64
CHAPTER 5
THESIS SUMMARY
A 16S rDNA clone library constructed from a mixed population of
environmental nucleic acid was analysed using DNA sequencing programs
and oligonucleotide probe hybridization experiments. The SAR324 lineage
and SAR2O2 lineage are two novel lineages which have been discovered by
random sequencing of the 148 clones contained in the library. The abundance
of genes corresponding to the SAR2O2 and SAR324 clusters suggests that
bacteria harboring these genes may be significant members of
bacterioplankton communities in the lower ocean mixed layer. SAR196, a
gene clone related to a nitrite-oxidizing bacterium (Nitrospira marina), was
also among the randomly sequenced clones. Although the abundance of this
gene in the lower ocean mixed layer is currently unknown, bacterioplankton
with this gene may participate in nitrification in the ocean.
The SAR324 lineage forms a monophyletic cluster within the
subdivision of class Proteobacteria. Sequence and structural analyses of the
16S rRNA gene within the SAR324 gene cluster showed significant variability
among clones, which suggests the presence of multiple bacterial species. An
oligonucleotide probe designed to specifically hybridize to members of the
SAR324 cluster was used to scan the library of 148 clones and to determine the
position of the genes in the water column. From the hybridization analyses,
five additional clones were detected and SAR324 was shown to be vertically
stratified, peaking at 200 m at BATS, and at 500 m at ocean station PAPA in
the Pacific. At the position of its maximum in the Atlantic, SAR324
comprised 18% of bacterial rDNA amplicons, suggesting that it is a very
65
abundant group. Although the physiology of SAR324 is currently unknown,
its presence in the aphotic zone suggests that members of the the SAR324
cluster may participate in biogeochemical processes that are unique to the
abyssal regions of the ocean.
Several clones related to 5AR202 were also discovered by randomly
sequencing the BATS 250 m clone library. The clones discussed in this report
were among clones that did not hybridize to the specific oligonucleotide
probe constructed by Giovannoni et al. (1996). 16S rRNA secondary structure
analyses of several of the clones showed significant variation in variable
regions one and two of the molecule, suggesting that the SAR2O2 cluster is a
highly divergent group that is composed of many species. To date, 22 gene
clones related to SAR2O2 have been recovered from the BATS 250 m clone
library. This suggests that species containing the SAR2O2 gene cluster are
potentially significant members of the bacterioplankton community in the
lower ocean mixed layer.
Although most bacterioplankton species recovered to date are not
represented by cultured species, clone SAR196 is an exception. SAR196 was
shown by complete 16S rRNA gene sequencing and phylogenetic analysis to
be related to Nitrospira marina, a nitrite-oxidizing species that has been
isolated from a variety of marine habitats. Although the phylogeny of N.
marina is controversial, the data reported here further supports the
relationship between N. marina and Leptospirillum sp.
The phylogenetic
relatedness between SAR196 and N. marina suggests that bacterioplankton
species harboring the SAR196 gene may participate in nitrification in the
ocean. Further studies should be aimed at determining the vertical
organization of this gene in the water column.
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71
APPENDICES
72
Appendix A - SAR324 Cluster 16S rDNA Sequences
SAR214 16S rDNA Sequence
SR2i.4
Accession
U65911
DNA
322 bp
17 -NAY- 1996
ORIGIN
1 GAACGCUGGC GGCAUGCUUA ACACAUGCAA GUCGAACGAG AAAGCTJCJCUtJ CGGAAGUGAU
61 UAAAGUGGCG CACGGGUGAG UAACGCGUAG ACAAUCUGCC CUUCAGUCCG GGACAACUUTJ
121 UCGAAAGGGG AGCUAAUACC GGAUAACAAU GUUUAACAUA AGtJTJGAAUAU UtJGAAAGCUC
181 CGGCGCUGAU GGAUGAGUCU GCGUCCCAUtJ AGCtJtJGUUGG UGCGGUAGAG GCGCACCAAG
241 GCUACGAUGG GUAGCGGGUtJ UGAGAGGACG AUCCGCCACA CUGGAACUGA GACACGGUCC
301 AGACtJCCUAC GGGAGGCAGC AG
SAR218 16S rDNA Sequence
SAR2 18
Accession
U65914
DNA
322 bp
20-MAY-1996
ORIGIN
1 GAACGCUGGC GGCAUGCUUA ACACAUGCAA GUCGAACGAG AAAGCUTJCUU CGGAAGUGAU
61 UAAAGUGGCG CACGGGUGAG UAACGCGUGG AUCAUCUACC CtJCCAGUEJCG GGACAACUCU
121 UCGAAAGGGG AGCUAAIJACC GGAUAAUACC UUUGGUUUTJA UGGCCtJGAGG UTJGAAAGCUC
181 CGGCGCtJGAG GGAUGAGUCU GCGUACCAUU AGCUUGUTJGG UGGGGUAAAG GCCUACCAAG
241 GCGACGAUGG UIJAGCGGGUC UGAGAGGACG AUCCGUCACA CUGGAACUGA GUCACGGUCC
301 ANACUCCUAC GGGAGGCAGC AG
73
SAR248 16S rDNA Sequence
SAR24 8
Accession U65912
DNA
1509 bp
25 -MAR- 1996
ORIGIN
1 AGAGUULJGAU CAUGGCtJCAG GACGAACGCU GGCGGCAUGC CUIJAUACAUG CAAGUCGAAC
61 GAGAAAGCUU CUUCGGAAGU GAUUAAAGUG GCGCACGGGU GAGUAACGCG UAGACAAUCU
121 GCCCUUCAGU CCGGGACAAC UtJFJ(JCGAAAG GGGAGCUAAU ACCGGAUAAC AAUGUUUAAC
181 ACAAGUTJGAA UATJUUGAAAG CUCCGGUGCtJ GAUGGAUGAG UCUGCGUCCC AUUAGCUtJGU
241 UGGUGCGGUA GAAGCGCACC AAGGCUACGA UGGGUAGCGG GUtJtJGAGAGG ACGAUCCGCC
301 ACACUGGAAC UGAGACACGG UCCAGACUCC UACGGGAGGC AGCAGUGGGG AAUATJUGCAC
361 AAUGGCGGCA ACUCUGAUGC AGCAAUGCCG CGUGAGUGAA GAAGGCCCUtJ GGGUCGUAAA
421 GCUCUUUUAU GGGGGAAGAU GAUGACGGUA CCCCAGGAAU AAGCACCGGC UAACUACGUG
481 CCAGCAGCCG CGGUAAUACG UAGGGUGCGA GCGUtJGtJUCG GAAUUACUGG GCGUAAAGGG
541 CGCGCAGGCG GAGGUGUAAG UCGGAGGUGA AAGCCCGGGG CtJCAACCCCG GAGGGUCUtJtJ
601 CGAAACUGCA UCUCUAGAGA GGGUUAGGGG CCGGCAGAAU UCCUGGUGUA GAGGUGAAAU
661 UCGUAGAUAU CAGGAGGAAU ACCGGUGGCG AAAGCGGCCG GCUGGGGCCA CUCUGACGCU
721 GAGGCGCGAA AGCGUGGGGA GCAAACAGGA UUAGAUACCC UGGUAGUCCA CGCCGUAAAC
781 GAUGAGCACU GGACGUUCGG AGGGUUCGAC CCUUCUGGGU GUtJUCAGCUA ACGCAUtJAAG
841 UGCUCCGCCU GGGGAGUACG GUCGCAAGAC UAAAACtJCAA AGGAAUUGAC G000GCCCGC
901 ACAAGCGGUG GAACAUGUGG UUIJAAUUCGA UGCAACGCGA AGAACCUUAC CUGGUCtJUGA
961 CAUCCUCGGA CAUCUCCAGA GAUGGAGTJIJU CCUCUUCGGG GGCCGAGUGA CAGGUGCUGC
1021 AUGGCUGUCG UCAGCUCGUG UCGUGAGAUG UUGGGUUAAG UCCCGCAACG AGCGCAACCC
1081 CUGCCCUUAA IJUGCCAUCGG GUCAAGCCGG GCACtJTJUAGG GGGACtJGCCG GUGACAAGCC
1141 GGAGGAAGGU G000AUGACG UCAAGUCCUC AUGGCCUTJUA UGACCAGGOC UACACACGUG
1201 UtJACAAUGGG AGUCACAGAG GGAUGCUAAG CCGCGAGGCC AUGCCAAUCC CAGAAAAGCU
1261 CUCUCAGtJUC GGAUCGCAGU CUGCAACUCU ACCGCGUGAA GCtJGGAAUCG CUAGUAAUCG
1321 CGGUUCAGGA CGCCGCGGUG AAUACGUUCC CGGGCCLJtJGC ACACACCGCC CGUCACACCA
1381 UGGGAGUtJGG ACAGGGCAGA AGUCGCCGAG CUAACCUUCG GGAGGCAGGC GCCCAAGCUC
1441 UGGUUGACGA CUGGGGUGAA GUCGUPACAA GGUAGCCGUA GGAGAACCtJG CGGGUGGAUC
1501 ACCUCCUtJA
74
SAR237 16S rDNA Sequence
SAR2 37
Accession U65910
DNA
322 bp
20-MAY-1996
ORIGIN
1 GAACGCUGGC GGCAUGCTJUA ACACAUGCAA GUCGAACGAG AAAGCUUCtJ(.J CGGAAGUGAU
61 UAAAGUGGCG CACGGGUGAG UAACGCGUAG ACAAUCUGCC CtJtJCAGUCCG GGACAACUUU
121 CCGAAAG000 AGCUAAUACC GGAUCACUAU GtJUtJGACAUA AGUUGAAUAU UUGAAAGCUC
181 CGGCGCtJGAU GGAUGAGUCU GCGUCCCAW AGCUTJGUUGG UGCGGUAGAG GCGCACCAAG
241 GCtJACGAUGG GUAGCGGGtJtJ UGAGAGGACG AUCCGCCACA CtJGGAACUGA GACACGGUCC
301 AGACTJCCtJAC GGGAGGCAGC AG
SAR257 16S rDNA Sequence
SR2 57
Accession U65909
DNA
322 bp
20-MAY-1996
ORIGIN
1 GAACGCUGGC GGCAUGCUtJA ACACAUGCAA GUCGAACGAG AAAGCUUCUU CGGAAGUGAU
61 UAAAGUGGCG CACGGGUGAG UAACGCGUN ACAAUCTJGCC CUUCAGUCCG GGACAACUtJU
121 CCGAAAGGGG AGCtJAAUACC GGAUAACAAU GUtJUAACAUA AGUUGAAUAU TJtJGWGCUC
181 CGGCACUGAU GGAUGAGUCU GCGUCCCAtJtJ AGCUUGUUGG UGCGGUAGCG GCGCACCAAG
241 GCCACGAUGG GUAGCGGGUU UGTGAGGACG AUCCGCCACA CTJGGAACUGA GACACGGUCC
301 AGACUCCUAC GGGAGGCAGC AG
75
SAR276 16S rDNA Sequence
SAR27 6
Accession
U65915
DNA
1494 bp
27 -OCT- 1995
ORIGIN
1 AGAGUUUGAU CCUGGCUCAG AACGAACGCU GGCGGCAUGC CtJtJAUACAUG CAAGUCGAAC
61 GAGAAAGUCA CUUCGGUGGC GAGUAGAGUG GCGCACGGGU GAGUAACGCG UAGAUAAUCU
121 ACCCUUCAGU CUGGGACAAC UCUUGGAAAC GGGAGCIJAAU ACCGGUAAA ACCUtJCGGGU
181 UGAAAGAUUU ACCGCUGGGG GAUGAGUCTJG CGUACCAUtJA GCUUGUUGGU 000GUAAUAG
241 CCUACCAAGG CGACGAUGGU UAGCGGGUCTJ GAGAGGACGA UCCGCCACAC UGGAACUGAG
301 ACACGGUCCA GACUCCUACG GGAGGCAGCA GUAGGGAAUA UtJGCGCAAUG GGGGCAACCC
361 UGACGCAGCA AUGCCGCGUG AGUGAAGAAG 000tJtJCGGGU CGUAAAGCUC UUUUAU0000
421 GAAGAUGAUG ACGGUACCCC AUGAAUAAGC ACCGGCUAAC UACGUGCCAG CAGCCGCGGU
481 AAUACGUAGG GUGCGAGCGU UGUIJCGGAAU UACUGGGCGU AAA0000GUG CAGGCGGAtJTJ
541 GGCAAGCCGG AGGUGAAAGC CCGGGGCUCA ACCCCGGAGG GUCUEJFJCGGA ACTJGCCAGUC
601 UtJGAGAGGGU CAGGGGCCAG CGGAAUtJCCU GGUGUAGAGG UGAAPLJUCGU AGAGAUCAGG
661 AGGAACACCG GCGGCGAAAG CGGCUGGCtJG GGGCCACIJC[J GACGCUGAGG CGCGAAAGCG
721 UGGGGAGCAA ACAGGAUtJAG AUACCCUGGU AGUCCACGCC GUAAACGAUG GGCACUAAGC
781 GUCCGAC000 UUCGACCCCG UFJGGGUGCtJG CAGUUAACGC GUUAAGUGCC CCGCCUGGGG
841 AGUACGGUCG CAAGACUAAA ACUCAAAGGA AUtJGACGGGG GCCCGCACAA GCGGUGGAAC
901 AUGUGGTJTJtJA AtJUCGACGCA ACGCGAAGAA CCUtJACCUGG GTJUUGACAUC CCCGGCCGAC
961 ACCAGAGAUG GUGUtJtJtJCJC UtJCGGAGACC GGGUGACAGG UGCUGCAUGG CUGtJCGtJCAG
1021 CtJCGUGUCGU GAGAUGUtJGG GUUAAGUCCC GCAACGAGCG CAACCCUUGC CCUCAGUtJGC
1081 CAGCAGUUCG GCUGGGCACU CUG0000GAC UGCCGGUGAC AAGCCGGAGG AAGGUGGGGA
1141
1201
1261
1321
1381
1441
UGACGUCAAG UCCUCAUGGC CUtJUAUAUCC A000CUACAC ACGUGUtJACA AU0000GUCA
CAAAGGGCAG CGAAACGGUG ACGUGGAGCG AAUCCCAAAA AAGCCCUCUC AGTJIJCGGAUC
GCAGUCUGCA ACUCGACUGC GUGAAGCUGG AAUCGCUGGU AAUCGCGGAU CAGCACGCCG
CGGUGAAUAC GUDCCCGGGC CUUGCACACA CCGCCCGUCA CACCAUGGGA GU(JGACAGGG
GCAGAAGCCG CCGAGCCAAC CUUCGGGAGG CAGGCGUCCA AGCUCCGGtJU GAUGACUGGG
GUGAAGUCGU AACAAGGUAG CCGUAGGAGA ACCUGCGGAU GGAUCAACUC CUUA
76
SAR3O8 16S rDNA Sequence
F+.. ;1cI,J:]
Accession U65913
DNA
323 bp
29-MAY-1996
ORIGIN
1 GAACGCUGGC GGCAUGCCUA ACACAUGCAA GUCGAACGAG AAAGtJtJCCtJtJ CGGGAGCGAU
61 UWGUGGCG CACGGUGAG UAACGCGUAG ACAAIJCUGCC CTJtJCAGUCUG GGACAACtJtJtJ
121 UCGAAAGGAG AGCUAUACC GGAUAACAAU GUU(JAACCtJA AGUUAAGUAU UUGAAAGCUtJ
181 UAUGUGCtJGA AGGAGOGGUC UGCGUCCCAU UAGCUAGUUG GIJAAGGUAAA GGCUUACCAA
241 GGCGACGAUG GGUAGCGGGU UUGAGAGGAC GAUCCGCCAC ACUGGAACAG AGACACGGUC
301 CAGACtJCCUA CGGGAGGCAG CAG
77
SAR324 16S rDNA Sequence
SAR3 24
Accession U65908
DNA
1509 bp
25-MAR-1996
ORIGIN
1 AGAGUUUGAU CCUGGCUCAG AACGAACGCTJ GGCGGCAUGC CtJAACACAUG CAAGUCGAAC
61 GAGAAGGCtJtJ CUTJCGGAAGTJ GAUUAAAGUG GCGCACGGGU GAGUAACGCG UAGACAAUCtJ
121 GCCCIJUCAGU CCGGGACAAC UUUCCGAAAG GGGAGCUAAU ACCGGAUAAC AAUGUUUAAC
181 AUAAGUUAAA UAUUUGAAAG CTJCCGGCGCU GAUGGAUGAG UCUGCGUCCC AUUAGCUUGU
241 UGGUGCGGUA GUGGCGCACC AAGGCCGCGA UGGGUAGCGG GUUUGAGAGG ACGAUCCGCC
301 ACACUGGAAC UGAGACACGG UCCAGACUCC UACGGGAGGC AGCAGUGGGG AAUAUrJGCAC
361 AAUGGAGGAA ACUCUGAUGC AGCAAUGCCG CGUGAGUGAA GAAGGCCCtJtJ GGGUCGUAAA
421 GCUCUUUtJAU G000GAAGAU GAUGACGGUA CCCCAAGAAU AAGCACCGGC UAACtJACGUG
481 CCAGCAGCCG CGGUAAUACG UAGGGUGCGA GCGUtJGUUCG GAAUtJACUGG GCGUAAAGGG
541 CGCGCAGGCG GAGGUGCAAG UCGGAGGUGA AAGCCCGGGG CUCAACCCCG GAGGGUCUUtJ
601 CGAAACUGCA UCCCUAGAGA GGGUCAGGGG CCGGCAGAAU UCCUGGUGUA GAGGUGAAAU
661 UCGUAGAUAU CAGGAGGAAU ACCGGUGGCG AAAGCGGCCG GCUGGGGCCA CtJCUGACGCU
721 GAGGCGCGAA AGCGU000GA GCAAACAGGA UUAGAUACCC UGGUAGUCcA CGCCGUAAAC
781 GAUGAGCAC[J AGACGUtJCGG AGGGIJUCGAC CCUtJCUGGGU GUEJGCAGCtJA ACGCAUtJAAG
841 UGCUCCUCCU GGGGAGUACA GUCGCAUGAC UAAAACUCAA CGGAAUUGAC G000GCCCGC
901 ACAAGCGGUG GAACAUGUGG UUUAAUUCGA UGCAACGCGA AGAACCt.JUAC CUGGUCUUGA
961 CAUCCUCGGA CAGCUCCAGA GAUGGAGUUU CCUCUtJCGGA GGCCGAGUGA CAGGUGCUGC
1021 AUGGCUGUCG UCAGCUCGUG UCGUGAGAUG UTJGGGUUAAG UCCCGCAACG AGCGCAACCC
1081 CUGCCCtJTJAA UtJGCCAUCGG GUCAAGCCGG GCACUUUAGG GGGACUGCCG GUGACAAGCC
1141 GGAGGAAGGU GGGGAUGACG UCAAGUCCtJC AUGGCCUUJA UGACCAGGGC UACACACGUG
1201 UUACAAUGGG AGUUACAGAG GGAUGCUAAG CCGCGAGGCC AUGCCAAUCC CAGAAAAGCU
1261 CtJCUCAGUUC GGAUCGCAGU CUGCAACUCK ACUGCGUGAA GCUGGAAUCG CUAGUAAUCG
1321 CGGUtJCAGGA CGCCGCGGUG AAUACGUIJCC CGGGCCUUGU ACACACCGCC CGUCACACCA
1381 UGGGAGUtJGG ACAGGGCAGA AGUCGCCGAG CUAACCTJtJCG GGAGGCAGGC GCCCAAGCUC
1441 UGGUtJGACGA CUGGGGUGAA GUCGUAACAA GGUAGCCGUA GGAGAACCtJG CGGUUGGAUC
1501 ACCtJCCUUA
Appendix B - SAR2O2-Related 16S rDNA Sequences
SAR226 16S rDNA Sequence
SAR2 26
DNA
748 bp
14 -JIlL- 1995
ORIGIN
1 TAACATGC AAGTCGAACG AGCGACCCGG GCTTGACCGG TTAGCTAG3 GCAGACGGCT
61 GAGTAACACG TAAGTAATT GCCCCGAAGA GGGGGATAAT CCAGAGAAAT CTGGCCTAAT
121 ACCCCGTACC TcccITrCCA GCCTGCTGGA TI'GGAAGAAA GGITI'CGGCC G'ITI'GGGAGA
181
241
301
361
421
481
541
AGCTTGCGGC TTATCAGGTA GTTGGTGGGG TAATGGCCTA CCAAGCCGAA GACGAGTAAC
CGGTGTGGA GCACGATCGG TCAGAGGGGG ACTGAGACAC GGCCCCCACT CCTACGGGAG
GCAGCAGCAG GGAATCTI'GC GCAATGGGCG AAAGCCAC GCAGCGACAC CGCGTGGAGG
ATGAAGG'rrK TGRRNATTGT AANCTCCTrI' TATCAGGGAA GAGAAAGGAC GGTACCTnAT
GAATAAGGTT CGGnTAnCTA CGNCAGCA GCCGCGGCAA TACGTAGGAA CCGAGCGTI'G
TCCGAATTA CTGGGCGTAA AGAGCGCGTA GGTGG'WTGG TAAGTCTCGT GTGTNNTCTC
CCGGCTCANC TGGGAGGGGT CACGGTATAC TGnCAGACTI' GAGGGTAGCA GAGGAAAGCG
601 GNATI'CCCGG AGTAGTGG
AAATGCGTAG ATNCCGGGAG GnACACCAGA GGCGAAGGCG
ACACTGAGGC GCGAAGCG
661 GCWTCTGGT CTATCC
GGGAGCrIACC CGNATTAGAT
721 NCCCGGGTAG CCCACGCCCT AAACGATG
79
SAR242 16S rDNA Sequence
S.AR24 2
DNA
1481 bp
14-SEP-1995
ORIGIN
1 AGAGTTGAT CATGGCTCAG GACGAACGCT GGCGGCGCGC CTAATGCA
CAAGTCGAAC
61 GGTCTG GGCTTCGGCC CGCAGCGATA GTGGCAGACG GCTGAGTAAC ACGTAAGAAA
121 CCTGACCTCG GGAG000GAT AACCCATCGA AAGGTGTGCT ATACCCCGT ATCC3ATCC
181 CCCGCATGAG GGATCAGAA ATGTCTTCGG GCGCCGGG AGGGTCTTGC GTCCTATCAG
241 GTAGTI'GGGT GTGGTAACGG CTCACCAAGC CTAAGACGGG TAACCGGTGT GAGAGCATGA
301 TCGGTCAGAG GGGGACTGAG AAACGCCCCC ACTCCTCGGG GCAGCACGAA NCCTGACGC
361 AGCGACGCCG CGGGGGAA GANGCCTTA GGGTTGTAAA CCCCITrrCT GGGGGAAGAG
421 AGAGGACT ACCCCAGGAA TAAGCCCCGG CTAACTACGT GCCAGCAGCC GCGGTAATAC
481 GTAGGGGGCG AGCGTIGTCC GGAATI'ACTG GGCGTAAAGG GCGTGTAGGC GGCACACGAA
AAATCTCCCG GCTCAACTGG GAGGGGTCCG GGGAAACTCG TGAGCTTGAG
541 GTCTTCGG
CGTAGATA TCGGGAGGAA
601 GACAGCAGAG GAGGGTGGAA TCCCGGTGT AGTGGTGAAA
661 CACCAGGC GAAGGCGGCC CTCTGGGCTG TACCACGC TGAGGCGCGA AAGCGTGGGG
721 AGCAAACCGG ArTAGATACC CGGGTAGTCC ACGCTPAAA CTAGATGC TAGGTATGGG
781 GGGTATCGAA CCCCTCCGTG CCGAAGCTAA CGCGTI'AAGC ATCCCGCCTG GGGAGTACGG
841 CCGCAAGGCT AAAACTCAAA GGAATTGACG GGGGCCCGCA CAAGCAGCGG AGCGTGTGGT
901 TTAATTCGAT SCAAAGCGAA GAACCTI'ACC AAGGCTGAC ATCGGTAG TAACCCGT
961 GAAAGTC000 GG3ACCCT CGGGGAGCCG TCACAGGTGC TGCAGCTG TCGTCAGCTC
1021 GTGCCGTGAG GTG'rrGGG'rT AAGTCCCGCA ACGAGCGCAA CCCTCGTCGC TAGTI'GATrT
1081 CTCTAGCGAG ACAGCCCCTT AAAGA000GG AGGAAGGTGG GGATGACSTC APNTCAGCAT
1141
1201
1261
1321
1381
1441
GGCCCTACG CCTTGGSCTA CACACACSCT ACAATGATCG GGACAACGGG TTGCCAAACC
GCGAGGTGGA GCCAATCCCA TCAAACCCGA TCCCAGGG GATICAGGC TGAAACCCGC
CCATGAAC GCGGAGTGC TAGTAACCGC AGGTCAGCAT TACTGCGGTG AATACGTTCC
CGGGCCTI'GT ACACACCGCC CGTCACGTCA TGGAAGCTGG CAATGCCTGA AGTCCGTAGG
CTAACCCTI'C G000AGGCAG CGGMCGAGGG T000GCTAGT GACTGGGACG AAGTCGTAAC
AAGGTAGCTG TACCGGAAGG TGCGGGTGGA TCACCTCCTT A
E11]
SAR25O 16S rDNA Sequence
SAR2 50
DNA
940 bp
6-SEP-1995
ORIGIN
1 CCTAATGCAT GCAAGTCGAA CGAGCGACCC GGGCTTGCCC GGCTAGCTAG TGGCAGACGG
61 CTGAGTAACA CGTAAGTAAT TTGCCCCGAA GAGGGGGATA ATCCAGAGAA ATCI\GCCTA
121 ATACCCCGTA CCTrrCTrrc CGGCCTGCCG GATGGAAGA AAGGCTCCGG CGCITTGGGA
181
241
301
361
421
481
541
601
GAAGCfl3CG GCTTATCAGG TAGTGGG GGTAATGGCC TACCAAGCCG AAGACGAGTA
ACCGGTGTGA GAGCACGATC GGTCAGAGGG GGACAGAC ACGGCCCCCA CTCCTACGGG
AGGCAGCAGC AGGGAATCTT GCGCAATGGG CGAAAGCCTG ACGCAGCGAC ACCGCGTGGA
GGATGTAGGT CCTAGGATI TAAACTCCTT TTATCAGGGA AGAGAAAGGA CGGTACCTGA
AATAAGGT TCGGCThACT ACGTGCCAGC AGCCGCTA ATACGTAGGA ACCGAGCG'rT
GTCCGGATT ACTGGGCGTA AAGAGCGCGT AGGTGGTrI'G TTAAGTCTCA TGTGTAATCT
CCCGGCTCAA CTGGGA000G TCAGGATA CGCAGACT CGAGGGTAGC AGAGGAAAGC
GGAATCCCG GAGTAGTGGT GAAATGCGrT CGGGACGGAT TCACAGATGT TGCAGCTG
661 TCGTCAGCTC GTGTCGTGAG A'I\3'I'rGGGTT AAGTCCCGCA ACGAGCGCAA CCCCTGTCGT
721
781
841
901
TAGTACTAT GTCTAGCGAG
GTCGTCATGG CTCTTACGTC
TGCCACAGCG CGAGCTGGAG
AACTCGCCTh CATGAACG
ACTGCCCTCA
TGGGGCTACA
CGAATCCTCA
GAGTTGCTAG
CTGAGGAGAG GAAGGTGGGG ACGACGTCAA
CACACGCTAC AATGGCCGGA AACAATGGGC
AAGCCGGTCT CAGTTGGAAT TGCAGGCTGC
TAACCGTAGG
SAR251 16S rDNA Sequence
S.AR2 51
DNA
701 bp
3-AUG-1995
ORIGIN
1 GCGGCGTCCT TAACAAATGC AAGTCGTGCG AGCAATCGG GCTTCGGACT GACGCGAG
61 CGGCAGACGG CTGAGTAACG CGTAGGTAAT CTGCCCCTAG GTGAGGGAOA ACCAGCCGAA
121 AGGTI'GGCTA ATACCTCATA AGTI'CTCTAA GrI'GCGACTT AGAGAGGAAA GCTTCGAGC
181 GCCAGGAT GAGCCTGCGT CCCATCAGGT AGTIX3GCGGA GTAATAGCCC ACCAAGCCGA
241 AGACGGGTAG CTGGTCAG AGGACGATCA GCCAGAGGGG GACTGAGACA CGGCCCCCAC
301 TCCTACGGGA GGCAGCAGCA GGGAATArITG GGCAATGGGC GAAAGCCTGA CCCAGCGACG
361 CCGCGTGGAG GAAAGGAC CTPGGGTCGT AAAcTTcTT TCCGAGGG1A GAGAACGGAC
421 TGTACCGG CAATAAGCCC CGGCTAGCTA CGTGCCAGCA GCCGCAGTAA TACGTAGGGG
481 GCGAGCGTA CCCGGA'ITrA CTGGGCGTAA AGGGCGTGTA GGTGGCTCGG TTAGTCGITT
541 GTGAAAGACC TCAGCTTAAC TGAGGAAGTG CTGACGAAAC CACCGGGCTA GAGGACAGTA
601 GAGGCGAGTG GAATTCCCGG CGGAGCGGTG AAATGCGTAG ATCTCGGGAG GAACACCAGT
661 GGCGAAGGCG GCTCGCCG CTGTCCCTGA CACTAAGGCG C
SAR256 16S rDNA Sequence
SAR2 56
644 bp
3 -AUG- 1995
ORIGIN
1
61
121
181
241
301
CAAGGA ACGAGATACC GCCGCTCGCG GTGGTCTA GTGGCAGACG GCTGAGTAAC
ACGAGTGA TCTGCCTrI'G AGAGGGGGAT AACACAGGGG AAACCTGTGC TAATACCGCG
TACGCTCT TTCATAAAAG GGACAGAGGA AACGAGAACA GGTAACTGTI' CTCGCTCAAA
GATGAGCTCG CGTCCTATCA GGTAGTI'GGT GGGGTAATI'G CCTACCAAGC CTAAGACGGG
TAGCTGGTAT GAGAGTACGA TCAGCCAGAG GGGGACAG ACACGTCCCC CACTCCTACG
GGAGGCAGCA GCAGGGAATT TTCCACAATG GGCGAAAGCC AGATGGAGCG ATGCCGCGTG
361 AAGGATGAAG GCTrI'AGGGT CGTAAACrI'C TITI'CTCAGG GAAGAGTAAG GACTGTACCT
421 GAGGAATAAG TCACGGCTAA CTACGTGCCA GCAGCCGCGG TAATACGTAG GTGGCGAACG
481 TIGTCCGGAT TrA'rrGGGCG TAAAGGGTCC GTAGGGT TGGTAAGTCT TC3TGAAAG
541 GTACAGGCTI' AACCTGTAGA AGTCAGAAGA TACCTAAC CTCGAGACTG TCAGAGGAAC
SAR259 16S rDNA Sequence
SAR2 59
DNA
753 bp
14 -JUL- 1995
ORIGIN
1 AGAGTII'GAT CCTGGCTCAG GGTGAACGCT TGCGGCGTGC ttaAtgcA
CAAGTCGTGC
61 GAGCAATCG GGCTTCGGCC TGATGCGCGA GCGGCAGACG GCTGAGTAAC GCGTAGGTAA
121 TCTGCCCCTA GGTOAGGGAC AACCAGCCGA AAGGTTGGCT AATACCTCAT AAGrTCTCTA
181 AGTGCGACT TAGAGAGGAA AGCTTCGAG CGCCATGGGA 'R3AGCCTGCG TCCCATCAGG
241 TAGTGGCGG AGTAATAGCC CACCAAGCCG AAGACGGGTA GC3GTCTGA GAGGACGATC
301 AGCCAGAGGG GGACTGAGAC ACGGCCCCAC TCCTACGGGA GGCAGCAGCA GGGATTGG
361 CAATGGGCGA AAGCCACC CAGCGACGCC GCGTGGAGGA TGAAGGCCCT TGGCTCGTAA
421 TCTCTTC CGAGGGAAGA GAACGGACTG TACCTCGGGA ATAAGCCCCG GATAGCTACG
481
ACAGCAGC CGCAGTAATA CGTAGGGGGC GAGCGTCACC CGGATTACT G000GTAAAG
541 GGTGTCTAGG TGGCTCGGTT AGTCGTGTGT GAAAGACCTC AGCTTATC
AGGAAGTGCT
601 GACGAACCCA CCGTGTCTAG AGGACAGGG AGGCGAGTI'G TCCCCGGC GGAGCGGTGA
661 AATGCGTAGA TCTCGGGAGG AACAACAGTG GCGAAGGCGG CTCGCTIGT CTCCCA
721 CACTAAGGCG GCGAAAGCAT GGAGAGCAAC CCG
5AR267 16S rDNA Sequence
SAR2 67
DNA
712 bp
16-JUN-19 95
ORIGIN
1 GCGGCGCGCC
61 GCAGACGGCT
121 CTGGCCTAAT
181 CTrIGGGAGA
241 GACGAGTAAC
TAAAACATGC AAGTCGAACG AGCGACCCGG GCTTGCCCGG
GCCCCGAAGA GGGGGATAAT
GAGTAACACG TAAGTAAT
ACCCCGTACC 'rrrC'rrrCCG GCCTGCCGGA TTGAAAGAAA
ATCAGGTA GTTGGTGGGG TAATGGCCTA
AGCTTGCGGC
CGGTGTGAGA GCACGATCGG TCAGAGGGGG ACTGAGACAC
TTAGCTAGTG
CCAGAGAAAT
GGCTCCCGCG
CCAAGCCTAA
GGCCCCCACT
301 CCTACGGGAG GCAGCAGCAG GGAATCGC GCAATGGGCG AAAGCCAC GCAGCGACAC
361 CGCGGAGG ATGAAGGTCC TAGGATI'GTA AACTCC'rrrT ATCAGGGAAG AGAAAGGACG
421
481
541
601
661
GTACCATG AATAAGG'rTC GGCTAACTAC GACAGCAG CCGCGGTAAT
CGAGCGTTGT CCGGAAC TGGGCGTAAA GAGCGCGTAG GTGGTGTGrr
TGTcTcTCc CGGCTCAnCT GGGAGGGGTC AGnTATAC TGACAGTCTI'
GAGGAAAGCG GAATTCCCGG AGTAGTGGTG AAATGCGTAG ATACCGGGAG
GGCGAAGGCG GCTnTCTGGT CTATTCCA CACAGGCG CGAAAGCGTG
ACGTAGGAAC
ATATCTCATG
GAGGGTAGCA
GAACAACAGA
GO
SAR269 16S rDNA Sequence
S.AR2 69
DNA
1498 bp
14-SEP-1995
ORIGIN
1
61
121
181
241
301
361
421
481
541
601
661
721
781
841
901
961
1021
1081
1141
1201
1261
1321
1381
1441
NAAGTCGAAC
AGAGTIT3AT CATGGCTCAG GACGAACGCT AGCGGCGCGC CTAACA
GAGCGATCCC TCTCGGAGG GCTAGCGAGT GGCAGACGGC TGAGTAACGC GTAAGCAACT
TACCCAC'IXG CGGGGACAA CCCGGAGAAA TCCGAGCTAA TACCGCATGT GATGCCCGT
TGAACGGG TAACGAAAGG CTTCGGCCAC CAGTGGATGG GCTI'GCGTCC CATCAGGTAG
TIGTGAGGT AACGGCCCAC CAAGCCATCG ACGGGTAGCC GG[AGAG CACGACCGGC
CAGAGGGGGA CTGAGACACG GCCCCCACTC CTACGGGAGG CAGCAGCAGG GAATCIGCA
CAATGGGCGA AACCCTGACG CAGCGACGCA GCGTGGGGGA AGACGGCCTG CGGGTTGTAA
GGCTAACTAC
ACCCCTITI'C TCGGGGAAGA AGATCACG GTACCCGAGG AATAAGCC
G3CCAGCAG CCGCGGTAAT ACGTAGGASS CAARNGI'I'GT CCGGITITAC WGGGCGTAAA
GGGAGAGCAG GTGGCAGT TAGTCCGG TGCAAGCTCC AGGCTCAACT TGGAGAGGTC
TACGGATACT GCTCGGCTI'G AGGGCGGTAG AGGAGCACGG AATTCCTGGT GTAGTGGTGA
AATGCGTAGA TATCAGGAGG AACACCGGTG GCGAAAGCAG TGCTCTGGGC CGTCCAC
ACTCAGGCTC GAAAGCGTGG GGAGCGAACC GGATAGATA CCCGGGTAGT CCACGCCCTA
AACGATGAGT GTTGGGTATG GGGGGTATCG ACCCCCTCCG TGCCGAAGCT CACGCGTAA
GCACTCCGCC TGGGAACTAC GGCCGCAAGG CTAAAACTCA AAGGAATI'GA CG0000CCCG
GITrAATTCG ATGCAAAGCG AAGAACCTTA CCTGGGCTIG
CACAPNCAGG GGAGCGTG
ACNLTCGGT GGTACCCTGC CCGAAAGGAT G000GACCCT TCGGGGAGCC GTCACAGACG
CTGCATGGCT GTCGTCAGCT CGTGCCGTGA GGTGTI'GGGT TAAGTCCCGC AACAAGCGCA
ACCCTCGTGA CTAGTI'GCAC TCTCTAGA GACTGCCTCG TAATTCGAGG AGGAAGGTGG
GGATGACGTC AAGTCATCAT GGCTCTI'ACG CCCAGGGCGA CACACACGCT ACAATGGGTA
GGACAACGGG CAGCCACGTC GCGAGACGGA GCAAATCCCT TAAACCTACC CCCAGTIGGG
ATTGCAGGCT GCAACTCGCC TGCATGAACG TGGAGTI'GCT AGTAACCGCA GATCAGCCAT
CCTGCGGTGA ATACGrrCCC GGGCCrrGTA CACACCGCCC GTCACGTCAT GGAAGCCGGT
AACGCTGAA GTCGCACAGC CAACCGTAAG GGGGCC3CG CCGAGGGCGG GACTGGTGAC
TGGGACGAAG TCGTAACAAG GTAGCTGTAC CGGAAGG3T GGCTGGATCA CCTCCTTA
SAR272 16S rDNA Sequence
SAR27 2
DNA
1290 bp
26-SEP-1995
ORIGIN
1 CATAATGCAT GCAAGTCGAA CGAAGTCCCA CCCTTCGGGG TGATGACTTA GTGGCAGACG
61 GCTGAGTAAC ACGTGAGA TCTGCCITIG GGTGAGGGAC AACAGAGGGA AACTTCTGCT
121 AATACCTCAT ACGATCCAAG ATCTAAGGTC TIGGATGAAA GGCCGGGT AACTGGTTGC
181 CGCCCTTAGA TGAGCTCGCG GCCTATCAGG TTGTTGGTGG GGTAATGGCC TACCAAGCCT
241 AAGACGGGTA GCTGGTGTGA GAGCACGACC AGTCAGAGGG GGACTGAGAC ACGGCCCCCA
301 CTCCTACGGG AGGCAGCAGC AGGGGAATCT TCCACAAG GCGAAAGCCT GATGGAGCGA
361 CGCCGCGA GGGATGAAGG CTCTAGGGTC GTAAACCTCT prrC'rrAGGG AAGAGTAAGG
421 ACGGTACCTA AGGAATAAGT GACGGCTAAC TACGTGCCAG CAGCCGCGGT AATACGTAGG
TGTAAGTCTC
481 TCACAAACGT TGTCCGGAAT TATI'GGGCGT AAAGGGTCCG TAGGCGGC
541 CTGTGAAATC TTCAGGCTCA ACTTGAAGAG GTCGGGGGAT ACTGCTGAAC TI'GAGACIA
601 CAGAGGCAAG TGGAATTCCC GGTGTGGTGG TGAAATGCGT AGTATCGGG AGGAACACCA
661 ATGgCgaaag cggcttgctg ggtcagttct gacgctgagg gacgaaagcg tgggtagcaa
721 accggattag atacccgggt agtccacgcc gtaaacgatg aatgctaggt tttcggggta
781 tcgaccccct gggagccgta gttaacgcga taagcattcc gcctggggAc TACGGTcgca
841 agactaaaac tcaaaggaat tgacgggggc ccgcacaagc aGCGGAGCGT GTGGtTTAAT
901 TCGA3CAAA GCGAAGAACC TI'ACCTAGGC PTGACMX3CA AATI'ACCGAT CCGAAAGG
GTGCCG3AG
961 AggcACCCKT AAGGGAGITT GCACAGGI3T TGCATGGCTG TCGTCAGC
1021 GTGTGGGTT AAGTCCCGCA ACGAGCGCA aCCCCTATCG CTAGTI'AATr TCTCTAGTGA
1081 GACTGCCCCT AAAAGGGAGG AAGGTGGGGA TGACGTCAAG TCATCATGAC TCTTACGTCT
1141 AGGGCTACAC ACACGCTACA ATGGCCAGTA CAGACGGTCG CTAAGCCGCA AGGTGGAGCC
1201 AATCCGATAA AGCTGGTCTC AGTTGGGAT GCAGGCTGCA ACTCGCCC ATGAACGCGG
1261 AGGCTAGT AAACGCAGGT CAGCACACTG
SAR317 16S rDNA Sequence
SAR3 17
DNA
753 bp
14-SEP-1995
ORIGIN
1 AGAGrrlGAT
61 GAGCGACCCG
CCCCGAAG
121
181 GGCCTGCCGG
CATGGCTCAG
GGCTTGCCCG
AGG GGATAA
ATTGAAAGAA
GACGAACGCT GGCGGTGCGC CTAATGCATG CAAGTCGAAC
GTI'AGCTAGT GGCAGACGGC TGAGTAACAC GTAAGTAATT
TCCAGAGAAA TC3GCCTAA TACCCCGTAC CrrTCITTCC
AGGCTCCGGC GCrrrGGGAG AAGCTTGCGG CTTATCAGGT
241 AGTPGGGG GTAATGGCCT ACCAAGCCTA AGACGAGTAA CCGGTGAG AGCACGATCG
301
361
421
481
541
601
GTCAGAGGGG
GCAATGGGCG
ATCTCCTITT
CGTGACAGCA
AGAGCGCGTA
CATGGATATA
GACTGAGACA CGGCCCCCAC TCCTACGGGA GGCAGCAGCA GGGATCTI'GC
AAAGCCTGAC GCAGCGACAC CGCGTGGAGG ATGAAGGTCC TAGGATITA
AATAAGGTTC GGTATATCTA
ATCAGGGAAG AGAAAGGACG GTACCTGA
GGCGCGGTAA TACGTAGGAA CCGAGCGTIG CCCGGAITTA CTGGGCGTAA
GGTGGTGTGT TATGTCTCAT GTGTAATCTC CCGGCTCATC TGGGAGGGGT
CTGACAGGCT TGAGGGTAGC AGAGGAAAGC GGCCTCCCG GAGTAGTGGT
661 GAAATGCGTA GATACCGGGA GGAACAACAG AGGCGAAGGC GGCTTCG TCTATCCCTG
721 ACACTGAGGC GCGAAAGCGT GGGGAGCGAC CCG
[ø1sJ
Appendix C - SAR196 16 S rDNA Sequence
SAR19 6
DNA
1530 bp
14-SEP-1995
ORIGIN
1 AGAGUUUGAU CAUGGCUCAG AACGAACGCU GGCGGCGCGC UtJAACACAUG CAAGUCGCAC
61 GAGAGGCUCU UCGGAGUAGU AAAGTJGGCGC ACGGGUGAGU AAUACAUGGG UAAUCUGCCU
121 UUGAGAGGGG AAUAACCAGC CGAAAGGCUA GCUAAUACCC CAUACGCUUC CGGUCCUUCG
181 GGUAAGGAAG GAAAGCUGCA UCGUGGAUGU GGCGCtJCGAA GAUGGOCUCA UGGCCUAUCA
241 GCUTJGUTJGGU AGGGUAACGG CCUACCAAGG CAACGACGGG UAGCUGGUCU GAGAGGAUGA
301 UCAGCCACAC UGGCACUGAG AUACGGGCCA GACUCCUACG GGAGGCAGCA GUGGGGAAUA
361 UUGCGCAAUG GGCGAAGCC UGACGCAGCG ACGCCGCGUG GGGGAAGAAG GIJTJCCCGGAU
421 UGUAAACCCC UUUUAGGAGG AAAGAUGGGG UGGGUAACCA CCUUGACGGtJ ACCUCCAGAA
481 AAAGCCACGG CUAACUUCGU GCCAGCAGCC GCGGUAAUAC GAAGGUGGCG AGCGUTJGUUC
541 GGAtJUIJACUG GGCAUAAAGA GCACGUAGGC GGUUtJAGUAA GCCCCCUGUG AAAGCUCCGG
601 GCUUAACCCG GAAAGGUCGG GGGGUACUGC UAAGCUAGAG GGCGGGAGAG GAGCGCGGAA
661 U[JCCCGGtJGU AGCGGUGAPJI UGCGUAGAUA UCGGGAAGAA GGCCGGUGGC GAAGGCGGCG
721 CUCUGGAACG CAUJtJGACGC UGAGGUGCGA AAGCGUG000 AGCAAACAGG AUtJAGAUACC
781 CUGGUAGUCC ACGCUGUAAA CGAUGGGCAC UAAGUGUCGG CAGAUtJACUG UCGGUGCCGC
841 AGCUAACGCA GUAAGUGCCC CGCCUGGGGA GUACGGCCGC AAGGUUGAAA CUCAAAGGAA
901 UUGACGGGGG CCCGCACAAG CGGUGGAGCA UGUGGUUUAA UUCGACSCAA CGCGAAGAAC
961 CTJtJACCCAGG UUAGACACGC UtJGUAUUAGG AACCCGAAAG GGUGACGAGU CCTJtJCGGGAC
1021 AGCUtJGCGCA GGUGCOGCAU GSCUGUCGtJC AGCUCGUGCC GUGAGGUGUU GGGWAAGUC
1081 CCGCAACGAG CGUAACCCCtJ GUCUUCAGtJU GCCAUCGGGU GAUGCCGAGC ACUCtJGGAGA
1141 GACUGCCCAG GAUAACGGGG AGGAAGGUGG GGAUGACGUC AGUCAGCAU GGCCUUUAUG
1201 CCUGGGGCUA CACACGUGCU ACAAUGGCCG GCACAAAGGG UtJGCAAUCCC GCGAGGGGGA
1261 GCCAAUCCCA AAAAACCGGC CUCAGIJUCAG AUUGUGGUCU GCAACtJCGWC CACAUGAAGG
1321 UGGAAUCGCU AGUAAUCGCG GRUCAGCACG CCGCGGUGAA AACGUUCCCG GGCCUUGUAC
1381 ACACCGCCCG UCACACCACG AAAGTJCAGCU GUACCTJGACG UCACUGGAGC UAACCCGCAA
1441 GGGAGGCAGG UGCCCACGGU AUGGUUGGUG AUtJGOGGUGA AGUCGUAACA AGGUAGCCGU
1501 AGGGGAACCU GUGGUUGGAU CACCUCCTJUA