Prevalence of the Chloroflexi-Related SAR202 Bacterioplankton Cluster throughout the Mesopelagic Zone and Deep Ocean

Abstract

Since their initial discovery in samples from the north Atlantic Ocean, 16S rRNA genes related to the environmental gene clone
cluster known as SAR202 have been recovered from pelagic freshwater, marine sediment, soil, and deep subsurface terrestrial
environments. Together, these clones form a major, monophyletic subgroup of the phylum Chloroflexi. While members of this diverse group are consistently identified in the marine environment, there are currently no cultured
representatives, and very little is known about their distribution or abundance in the world's oceans. In this study, published
and newly identified SAR202-related 16S rRNA gene sequences were used to further resolve the phylogeny of this cluster and
to design taxon-specific oligonucleotide probes for fluorescence in situ hybridization. Direct cell counts from the Bermuda
Atlantic time series study site in the north Atlantic Ocean, the Hawaii ocean time series site in the central Pacific Ocean,
and along the Newport hydroline in eastern Pacific coastal waters showed that SAR202 cluster cells were most abundant below
the deep chlorophyll maximum and that they persisted to 3,600 m in the Atlantic Ocean and to 4,000 m in the Pacific Ocean,
the deepest samples used in this study. On average, members of the SAR202 group accounted for 10.2% (±5.7%) of all DNA-containing
bacterioplankton between 500 and 4,000 m.

Full text

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, May 2004, p. 2836–2842 Vol. 70, No. 5
0099-2240/04/$08.00⫹0 DOI: 10.1128/AEM.70.5.2836–2842.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.
Prevalence of the Chloroflexi-Related SAR202 Bacterioplankton
Cluster throughout the Mesopelagic Zone and Deep Ocean†
R. M. Morris,
1
M. S. Rappe´,
2
E. Urbach,
3
S. A. Connon,
4
and S. J. Giovannoni
1
*
Department of Microbiology, Oregon State University, Corvallis, Oregon 97331
1
; Hawaii Institute of Marine
Biology, School of Ocean and Environmental Science and Technology, University of Hawaii at
Manoa, Kaneohe, Hawaii 96744
2
; Department of Plant Pathology, University of
Wisconsin—Madison, Madison, Wisconsin 53706
3
; and Idaho State
University, Pocatello, Idaho 83209
4
Received 12 December 2003/Accepted 31 January 2004
Since their initial discovery in samples from the north Atlantic Ocean, 16S rRNA genes related to the
environmental gene clone cluster known as SAR202 have been recovered from pelagic freshwater, marine
sediment, soil, and deep subsurface terrestrial environments. Together, these clones form a major, monophy-
letic subgroup of the phylum Chloroflexi. While members of this diverse group are consistently identified in the
marine environment, there are currently no cultured representatives, and very little is known about their
distribution or abundance in the world’s oceans. In this study, published and newly identified SAR202-related
16S rRNA gene sequences were used to further resolve the phylogeny of this cluster and to design taxon-specific
oligonucleotide probes for fluorescence in situ hybridization. Direct cell counts from the Bermuda Atlantic time
series study site in the north Atlantic Ocean, the Hawaii ocean time series site in the central Pacific Ocean, and
along the Newport hydroline in eastern Pacific coastal waters showed that SAR202 cluster cells were most
abundant below the deep chlorophyll maximum and that they persisted to 3,600 m in the Atlantic Ocean and
to 4,000 m in the Pacific Ocean, the deepest samples used in this study. On average, members of the SAR202
group accounted for 10.2% (ⴞ5.7%) of all DNA-containing bacterioplankton between 500 and 4,000 m.
The discovery that previously unidentified bacterioplankton
16S rRNA gene sequences predominate in the ocean’s lower
surface layer was one of the first pieces of evidence to suggest
that marine bacterioplankton communities are stratified (8, 13,
47). The environmental gene clone SAR202 and close relatives
were among the groups recovered from seawater in early in-
vestigations of bacterioplankton diversity at the Bermuda At-
lantic time series study (BATS) site in the north Atlantic
Ocean (13). Shortly thereafter, close relatives were detected in
seawater samples from 1,000 m in the Atlantic Ocean and
3,000 m in the Pacific, rapidly extending the apparent range of
this group of microorganisms throughout the mesopelagic
zone and into the deep ocean (12).
Interestingly, SAR202 organisms and relatives are members
of the Chloroflexi phylum, one of the 11 original phyla de-
scribed by comparative 16S rRNA sequence analysis (45). The
Chloroflexi line of descent is thought by many to have diverged
early in the evolution of the domain Bacteria (29). Represen-
tatives of this phylum occupy a wide variety of habitats; Chlo-
roflexi-related sequences have been identified in geothermal,
soil, freshwater, marine, wastewater, and subsurface environ-
ments. In addition, the few cultivated representatives exhibit a
diverse range of phenotypes, including anoxygenic photosyn-
thesis (e.g., Oscillochloris and Chloroflexus) (23, 30), thermo-
philic organotrophy (Thermomicrobium) (21), and chlorinated
hydrocarbon reduction (Dehalococcoides ethenogenes) (27).
The phenotypic characteristics of the SAR202 clade of bacteria
cannot be inferred from their phylogeny because of the diverse
physiological traits exhibited by cultured representatives within
this phylum (19, 33, 36).
Since their initial identification in BATS 250-m seawater,
environmental gene clones related to the SAR202 cluster have
been found in deep subsurface, soil, marine sponge, and fresh-
water environments (4, 7, 18, 41) and further sequences have
been found in various seawater samples (2, 14, 46). While
cultivation-independent rRNA gene cloning and sequencing
results suggest that members of this diverse group are ubiqui-
tous and potentially abundant in the marine environment,
there are well-known sources of potential methodological bias
that prohibit absolute cellular quantification from these data.
Variable lysis efficiency between microbial cell types, variations
in rRNA gene copy number, and PCR-induced biases and
artifacts are just a few of the factors that confound and restrict
quantitative estimates of abundance from gene clone library
data (34, 37, 44). However, direct cell counts using fluores-
cence in situ hybridization (FISH) with rRNA-targeted oligo-
nucleotide probes have been used to accurately count cells in
natural samples (1, 6, 28).
In general, small, slow-growing microbial cells such as plank-
tonic marine bacteria have traditionally been difficult to detect
by FISH. Subsequently, various strategies have been used to
decrease background noise and increase signal intensity and
counting accuracy. Strategies have included the use of multiple
fluorescently labeled oligonucleotide probes (25), signal am-
plification methods such as tyramide signal amplification (35),
and unlabeled helper oligonucleotide probes (11). Our strategy
has been to use multiple oligonucleotide probes that target
different regions of the same 16S rRNA to produce an additive
effect on signal intensity (28) and, in this case, to ensure that all
* Corresponding author. Mailing address: Department of Microbi-
ology, Oregon State University, Corvallis, OR 97331. Phone: (541)
737-1835. Fax: (541) 737-0496. E-mail: steve.giovannoni@orst.edu.
† This is HIMB contribution 1179 and SOEST contribution 6337.
2836

available SAR202-related 16S ribosomal DNA (rDNA) se-
quences recovered from seawater were targeted by at least one
probe.
The available data suggest that members of the SAR202
cluster are ubiquitous and that they may play an important role
in lower-surface and deep-ocean biogeochemistry. However,
no data about their physiology or cellular abundance are avail-
able. There are currently no cultured representatives of the
SAR202 cluster or published quantitative abundance esti-
mates. In this study, we used newly identified SAR202-related
16S rDNA sequences from marine bacterioplankton with pub-
lished SAR202 cluster sequences recovered from a variety of
environments to further resolve SAR202 phylogeny and to
design oligonucleotide probes for quantitative FISH. We re-
port SAR202 cluster cell counts from the BATS site in the
Atlantic Ocean and the Hawaii ocean time series (HOT) site
(station ALOHA) in the Pacific Ocean. In addition, depth
profiles from coastal waters were obtained from five stations
along the Newport hydroline (NH35 to NH127), extending
from just off the Oregon coast to the edge of the north Pacific
gyre.
MATERIALS AND METHODS
Sample collection. North Atlantic Ocean seawater was collected at the BATS
site (32°N, 64°W) from a total of 10 depths between 1 and 3,600 m. Surface
samples (1 to 250 m) were collected on 5 February 2001, while samples from
depths ⬎250 m were collected on 6 February 2001. Central north Pacific Ocean
samples were collected at station ALOHA (45°N, 158°W), the HOT study site,
from a total of seven depths on 15 December 2002. Water from the eastern
Pacific Ocean coastal transect was collected from various depths along the
Newport hydroline (44°N) at stations NH15 (25°W), NH35 (53°W), NH55
(22°W), NH65 (36°W), NH85 (126°W), and NH127 (127°W). Five samples (1, 10,
30, 100, and 500 m) were collected on 7 May 2002, and five samples (20, 110, 600,
1,000, and 2,700 m) were collected on 8 May 2002 at station NH127. All samples
were collected in Niskin bottles on conductivity, temperature, and density device
rosettes and transferred to primary collection bottles. Atlantic subsample vol-
umes of 500 ml were immediately fixed in filtered formalin at a final concentra-
tion of 10% and stored at ⫺80°C for up to 6 months. Pacific subsample volumes
of 10 to 250 ml were immediately fixed in filtered, buffered paraformaldehyde at
afinal concentration of 2% and stored at 4°C for 6 to 8 h. Fixed samples were
filtered onto white 0.2 ␮m-pore-size polycarbonate filters (GE Osmonics,
Minnetonka, Minn.), immediately placed in slide boxes containing silicon desic-
cant, and stored at ⫺20°C.
Cloning. Bacterial 16S rRNA gene clones from the original BATS 250-m clone
library were prepared as described previously (13). In short, DNA was amplified
from a mixed population of genomic DNA by PCR using primers specific for
bacterial 16S rRNA genes. A clone library was constructed with the plasmid
vector pCRII (Invitrogen, San Diego, Calif.) from the resulting PCR amplicon.
The clones were assigned the prefix SAR, numbered discontinuously from 177 to
325, and stored at ⫺20°C in Luria-Bertani (LB) broth containing 10% (wt/vol)
glycerol. Two new SAR202-related clone sequences were identified in a clone
library constructed from February 1992 BATS 200-m seawater (prefix D92). The
D92 bacterial 16S rDNA library was prepared essentially as described above, but
by a streamlined protocol for clone library analysis (42). rRNA genes were
amplified from environmental DNA for cloning by PCR with Taq polymerase
(Fermentas, Hanover, Md.) and variations of commonly used bacterial primers
8F (AGRGTTYGATYMTGGCTCAG) and 1492R (GGYTACCTTGTTACG
ACTT) (24). Amplifications were performed in a PTC-0200 thermocycler (MJ
Research, Cambridge, Mass.) under the following conditions: 35 cycles of an-
nealing at 55°C for 1 min, elongation at 72°C for 2 min, and denaturation at 94°C
for 30 s. A single band of the predicted length was observed by agarose gel
electrophoresis. The clone library was constructed with the pGEM-TEasy (Pro-
mega, Madison, Wis.) vector by following the manufacturer’s instructions. Indi-
vidual clones were numbered sequentially from D92-01 to D92-96.
Gene sequencing and phylogenetic analysis. Complete 16S rRNA gene clone
sequences were obtained and added to an aligned database of ⬎12,000 homol-
ogous 16S rDNAs maintained with the ARB software package (26). Evolutionary
distance, parsimony, and maximum-likelihood phylogenetic analysis methods
were used in concert to identify robust phylogenetic relationships within the
SAR202 cluster data set and were performed with the program PAUP*, version
4.0 beta 10 (39). The tree topology was inferred by maximum likelihood employ-
ing a heuristic search with a tree bisection-reconnection branch-swapping algo-
rithm, a proportion of invariable sites of 0.2339, equal base frequencies, and a
gamma distribution of rate heterogeneity at variable sites with a shape parameter
of 0.6889 and four rate categories. Bootstrap proportions from 1,000 replicate
resampled data sets were used to estimate the relative confidence in monophy-
letic groups and were determined by evolutionary-distance and parsimony meth-
ods. Likelihood ratio tests were used to select a substitution model for evolu-
tionary distance calculations by employing the program Modeltest, version 3.06
(30a). The model selected was SYM⫹I⫹G (48), with the estimated proportion
of invariable sites set to 0.2339, equal base frequencies, and a gamma distribution
of rate heterogeneity at variable sites with a shape parameter of 0.6889 and four
rate categories. Distance matrices from bootstrapped data sets were calculated
with this model, and neighbor joining was used to generate trees for the boot-
strap analysis. Parsimony analyses employed a heuristic search, tree bisection-
reconnection, and a starting tree obtained by stepwise addition with random
sequence addition. All sequences used in this analysis were ⬎1,200 nucleotides
in length; 914 nucleotide positions remained after masking out hypervariable and
other ambiguously aligned regions from the alignment. In preliminary analyses,
a range of bacterial phyla were employed as outgroups. The choice of outgroup
did not influence the significant relationships shown in Fig. 1.
FISH. Hybridization reactions were performed essentially as described by
Glo¨ckner et al. (15) with the following modifications. Reactions were performed
on one-quarter membrane sections at 37°C for 12 to 16 h in hybridization buffer
(900 mM NaCl, 20 mM Tris [pH 7.4], 0.01% [wt/vol] sodium dodecyl sulfate
[SDS], 35% formamide) and two Cy3-labeled oligonucleotide probes (SAR202-
104R [GTTACTCAGCCGTCTGCC] and SAR202-312R [TGTCTCAGTCCC
CCTCTG]) specific for members of the SAR202 cluster and designed with the
ARB software package (26). Additionally, a control hybridization reaction was
performed with a low-stringency buffer containing 15% formamide and a Cy3-
labeled nonsense oligonucleotide (338F). All probes had a final concentration of
2ng␮l
⫺1
. Optimal hybridization stringency was achieved by washing the mem-
branes in hybridization wash (70 [SAR202] or 150 [338F] mM NaCl, 20 mM Tris
[pH 7.4], 6 mM EDTA, 0.01% SDS) for two 10-min intervals. An experimentally
determined temperature of dissociation (T
d
) specific for the SAR202 probe suite
(58.0°C) was used for all SAR202 hybridization reactions (see Fig. 2), and a
low-stringency T
d
(50.0°C) was used for all 338F control hybridization reactions.
Nucleic acid staining was achieved by transferring the membrane to a chilled
(4°C) hybridization wash containing DAPI (4⬘,6⬘-diamidino-2-phenylindole) at a
final concentration of 5 ␮gml
⫺1
for 10 min. The DAPI was rinsed for 2 min in
afinal hybridization wash chilled to 4°C. All reagents were filtered through a
0.2-␮m-pore-size filter.
Fluorescence microscopy. After the filters were mounted in Citifluor (Ted
Pella, Redding, Calif.), Cy3-positive and DAPI-positive cells were counted for
each field of view with a Leica DMRB epifluorescence microscope equipped with
a Hamamatsu ORCA-ER charge-coupled device digital camera, filter sets ap-
propriate for Cy3 and DAPI, and Scanalytics IPLab, version 3.5.6, scientific
imaging software. Consistent exposure times of 1 and 5 s were used for DAPI and
Cy3 images, respectively. Cy3 images were manually segmented in IPLab and
automatically made to overlie corresponding DAPI image segmentations in
order to identify positive probe signals coincident with DAPI signals. Consistent
size, morphology, and signal intensity criteria were used for all cell counts.
Negative control counts were determined from the 338F hybridization using the
same technique and subtracted from positive probe counts to account for objects
detected with the Cy3 and DAPI filter sets in the absence of the positive probe
set, such as autofluorescent cells.
Nucleotide sequence accession numbers. Gene sequences were deposited in
GenBank and given accession numbers AY534087 through AY534100.
RESULTS
A combination of methods were used to determine phylo-
genetic relationships among 16S rRNA gene sequences from
members of the original SAR202 cluster (13); published rela-
tives were identified by searching public nucleotide sequence
databases (GenBank and the RDP-II), published reference
sequences from other major subgroups of the Chloroflexi (33),
and newly sequenced environmental gene clones recovered
VOL. 70, 2004 OCEANIC ABUNDANCE OF THE SAR202 CLUSTER 2837

FIG. 1. Phylogenetic tree of the SAR202 cluster and representatives of the phylum Chloroflexi. rRNA gene sequences from cultivated
microorganisms are shown in boldface, while sequences derived from cultivation-independent studies are labeled with the environment from which
they were derived and clone name. GenBank accession numbers are shown in parentheses. Nodes supported by bootstrap replicates ⬎70% in
evolutionary-distance (above) or parsimony (below) analyses are indicated. The scale bar corresponds to 0.05 substitutions per nucleotide position.
Dashed brackets, subclusters (numbered 1 to 4). “Original”indicates the phylogenetic depth of the original SAR202 cluster (13).
2838 MORRIS ET AL. APPL.ENVIRON.MICROBIOL.

from the BATS study site in the north Atlantic Ocean. All of
the analyses showed that the rRNA gene clones from pelagic
marine bacterioplankton within the phylum Chloroflexi fell in-
side a single monophyletic cluster (Fig. 1), but the addition of
newly identified clones greatly expanded the genetic diversity
of this cluster relative to that based on the original observa-
tions (13). The first two full-length gene clones published in
1996, SAR202 and SAR307, are 94.9% similar. Currently, the
most dissimilar Chloroflexi marine bacterioplankton gene clone
sequences are 78.7% similar (D92-36 and SAR259 in Fig. 1).
Within the Chloroflexi phylum, the closest relatives to the
SAR202 cluster could not be identified with the 16S rRNA
gene sequence data and analysis methods currently available.
Unlike clusters from other predominant groups of marine
bacterioplankton, such as the SAR86 (31, 38) and Pelagibacter
(SAR11) clusters (32, 33, 38), marine bacterioplankton envi-
ronmental gene clones of the SAR202 cluster are not mono-
phyletic; sequences retrieved from nonmarine and/or non-
planktonic communities are interspersed throughout the
marine bacterioplankton clones. For example, environmental
gene clones from freshwater bacterioplankton of Crater Lake,
Oreg. (41), sponge symbionts from shallow marine environ-
ments (18), deep-sea sediments (unpublished data), and ter-
restrial soils (references 4 and 7 and unpublished data) are
dispersed throughout the SAR202 cluster. Four subclusters
within the SAR202 cluster were supported by high bootstrap
proportions (Table 1; Fig. 1). While all four contained gene
clones from marine bacterioplankton, only one was exclu-
sively so (subcluster 2). In addition to marine bacterioplank-
ton, subcluster 1 contained clones recovered from marine
sponge and freshwater bacterioplankton communities, sub-
cluster 3 contained clones from marine sponge, deep-sea
sediment, and forest soil communities, and subcluster 4 con-
tained a clone from a deep-sea sediment community. Sev-
eral clones did not fall within the four monophyletic sub-
groups but instead formed independent lines of descent
within the SAR202 cluster (e.g., clones SAR242, SAR269,
and FTL256 in Fig. 1).
Two oligonucleotide probes were designed to target mem-
bers of the SAR202 cluster. The probe SAR202-104R was
designed to target a region corresponding to positions 104 to
121 of the Escherichia coli 16S rRNA. This probe matched
perfectly 20 of 30 members of the SAR202 cluster possessing
complete or nearly complete 16S rDNA sequences and 15 of
TABLE 1. Probe specificity for members of the SAR202 cluster
Clone Source
No. of: Reference or
source
SAR202-104R
mismatches
SAR202-312R
mismatches
Subgroup 1
SAR272 Sargasso Sea seawater, 250 m 0 0 This study
SAR256 Sargasso Sea seawater, 250 m 0 0 This study
SAR188 Sargasso Sea seawater, 250 m 1 0 This study
D92-36 Sargasso Sea seawater, 200 m 0 0 This study
AEGEAN_116 North Aegean Sea seawater, 200 m 0 0 Unpublished
Arctic95A-18 Arctic Ocean seawater, 500 m 2 0 2
PAWS52f Sponge symbiont, 20–30 m, Pacific Ocean 0 0 18
CL500-9 Freshwater Crater Lake, 500 m 3 3 41
CL500-10 Freshwater Crater Lake, 500 m 0 1 41
Subgroup 2
SAR261 Sargasso Sea seawater, 250 m 1 0 This study
SAR292 Sargasso Sea seawater, 250 m 0 0 This study
SAR319 Sargasso Sea seawater, 250 m 0 0 This study
SAR317 Sargasso Sea seawater, 250 m 0 0 This study
SAR250 Sargasso Sea seawater, 250 m 0 0 This study
SAR194 Sargasso Sea seawater, 250 m 0 1 This study
Subgroup 3
SAR307 Sargasso Sea seawater, 250 m 0 0 13
SAR202 Sargasso Sea seawater, 250 m 0 0 13
D92-22 Sargasso Sea seawater, 200 m 1 0 This study
Arctic96BD-6 Arctic Ocean seawater, 500 m 0 0 2
TK04 Sponge symbiont, 7–15 m, Mediterranean Sea 0 0 18
MBAE74 Deep-sea sediment, Pacific Ocean 3 0 Unpublished
MBMPE46 Deep-sea sediment, Pacific Ocean 0 0 Unpublished
C083 Forest soil, Arizona 0 0 7
Subgroup 4
SAR259 Sargasso Sea seawater, 250 m 0 0 This study
MBMPE42 Deep-sea sediment, Pacific Ocean 4 0 Unpublished
SAR269 Sargasso Sea seawater, 250 m 0 0 This study
SAR242 Sargasso Sea seawater, 250 m 0 1 This study
H1.2.f Deep subsurface paleosol 2 0 4
MBAE68 Deep sea sediment, Pacific Ocean 2 0 Unpublished
FTL276 Trichloroethene-contaminated soil 1 1 Unpublished
VOL. 70, 2004 OCEANIC ABUNDANCE OF THE SAR202 CLUSTER 2839

19 marine bacterioplankton environmental gene clones in this
cluster (Table 1). Outside of the SAR202 cluster, probe
SAR202-104R matched perfectly the 16S rRNA gene se-
quence from the archaeaon Sulfolobus solfataricus (GenBank
accession no. X90483) and closely related environmental gene
clones and contained a single base mismatch with a wide va-
riety of published 16S rRNA gene sequences, including those
of several members of the SAR11 marine bacterioplankton
cluster of the alpha Proteobacteria. The probe SAR202-312R
was designed to target a region corresponding to positions 312
to 329 of the E. coli 16S rRNA. It matched perfectly 25 of 30
full-length members of the SAR202 cluster and 17 of 19 ma-
rine clones (Table 1). In addition, this probe matched perfectly
16S rRNA gene sequences from several members of candidate
division OP11 (20) and had a minimum of two mismatches with
all other known 16S rRNA gene sequences outside of the
SAR202 cluster. Of 30 full-length, or nearly full-length, gene
sequences within this cluster, only two (freshwater bacterio-
plankton clone CL500-9 and contaminated-soil clone FTL276
in Fig. 1) did not possess a target site that perfectly matched
that of one of the two SAR202 cluster probes (Table 1). All 19
full-length marine bacterioplankton gene clone sequences
within this cluster perfectly match at least one of the two
SAR202 cluster probes.
Direct cell counts from the Atlantic and Pacific Oceans were
obtained by hybridizing paraformaldehyde-fixed, filtered sea-
water samples with the two SAR202 cluster probes labeled
with Cy-3. The T
d
of 58°C for cells hybridizing to the SAR202
probe pair was empirically determined from 100-m Oregon
coast seawater (NH35). An axenic SAR11 cluster isolate (32),
fortuitously exhibiting a single base mismatch to probe
SAR202-104R, was used to evaluate the specificity of hybrid-
ization of this probe. SAR11 cells hybridized to the SAR202
probe pair showed a complete loss of probe-conferred fluores-
cence signal intensity between 49 and 55°C (Fig. 2). While it is
known that base composition and rRNA secondary structure
can affect in situ hybridization kinetics (9, 10), these results
indicate that SAR11 cells containing the target sequence with
a single base mismatch were excluded from counts reported in
this study.
Additional confidence in the cell count measurements came
from observations of the average morphology, size, and rela-
tive signal intensity of cells hybridizing to the SAR202 probe
suite. Probe-positive cells had a coccoid morphology and were
greater than 1 ␮m in diameter (Fig. 3) and unusually bright
(1,067 ⫾480 relative intensity units) compared to other pelagic
bacterioplankton hybridizations (Fig. 2). Because of the dis-
tinctive size, morphology, and signal intensity of cells hybrid-
izing to the SAR202 probe suite, there was very little ambiguity
in the scoring of cells from below the upper ocean surface
layer, where autofluorescent-cell counts are low.
The overall abundance of SAR202 cells remained surpris-
ingly constant below 500 m and accounted for an average of
(3.0 ⫾1.9) ⫻10
6
cells liter
⫺1
in Atlantic (BATS) and Pacific
(HOT) Ocean depth profiles (Fig. 4). On average, the SAR202
FIG. 2. SAR202 probe pair dissociation curve. SAR202 cells from
Oregon coast seawater (250 m; ■) and an axenic SAR11 culture in
exponential growth phase (F) hybridized to the SAR202 probe suite.
Temperatures indicate various stringency conditions associated with
the hybridization wash buffer.
FIG. 3. FISH image of SAR202 cells from the Pacific Ocean (2,700
m). Identical fields of view show DNA-containing cells stained with
DAPI and relatively large (cocci ⬎1␮m in diameter) target cells
stained with the SAR202 cluster probe pair labeled with Cy3. Images
were obtained with a Hamamatsu ORCA-ER charge-coupled device
digital camera.
FIG. 4. Group-specific FISH and prokaryotic-cell counts (DAPI-
stained particles) in Atlantic and Pacific Ocean gyres. Shown are
SAR202 (■) and DAPI (F) cell counts for the north Atlantic Ocean at
BATS sites (A) and in the central north Pacific Ocean at HOT sites
(B). The Atlantic Ocean profile is a composite consisting of surface
samples (1 to 250 m) and deep samples (1,000 to 3,600 m) taken from
two different casts in February 2001.
2840 MORRIS ET AL. APPL.ENVIRON.MICROBIOL.

group accounted for 10.2% (⫾5.7%) of DAPI-stained cells
present below the ocean surface layer. In surface waters,
SAR202 cell counts were ⱕ1.0 ⫻10
6
cells liter
⫺1
, at or below
the threshold of detection for surface waters. The threshold for
accurate counting of the less-abundant bacterioplankton
groups was higher in surface waters than in deep waters, due to
the high autofluorescent-cell and particle counts associated
with negative control probe hybridizations. Bulk nucleic acid
hybridization data suggest that DNA from the SAR202 group
decreases in surface waters (13), and surface cells (0 to 300 m)
positive for both probe hybridization and DAPI always lacked
green fluorescence (fluorescein isothiocyanate channel), indic-
ative of chlorophyll autofluorescence. These data reinforce the
ⱖ66% decrease in ocean surface layer SAR202 cells relative to
the numbers in deeper waters suggested by the in situ hybrid-
ization data.
Depth profiles from stations along the Newport hydroline,
which extended from the Oregon coast to the edge of the north
Pacific gyre, showed a similar trend in the depth-specific dis-
tribution of the SAR202 group (Fig. 5). SAR202 cell counts
were highest just below the deep chlorophyll maximum
(DCM), reaching 27 ⫻10
6
cells liter
⫺1
in the 100-m sample
from station NH35 and accounting for an average of (12 ⫾8.3)
⫻10
6
cells liter
⫺1
just below the DCM. Average abundance
values below SAR202 surface maximums declined to (2.5 ⫾
1.5) ⫻10
6
cells liter
⫺1
but persisted throughout the water
column to a maximum depth of 2,700 m at station NH127.
These results confirm previous findings, showing a peak in
relative SAR202 high-molecular-weight rRNA and 16S rRNA
amplicon abundance just below the DCM (13) and extend their
known range to depths throughout the mesopelagic zone and
deep ocean.
DISCUSSION
SAR202 is intriguing because of the apparently lengthy evo-
lutionary history and extraordinary metabolic diversity of the
phylum Chloroflexi and also because organisms with this di-
verse and complexly structured cluster resides in the deep
pelagic zone of oceans and some lakes (12, 41). In this study we
have added to the sparse information about the SAR202 clus-
ter by identifying the associated cell morphology, providing
accurate numbers of cells in the water column, and providing
a detailed phylogeny for the group.
The data show that SAR202 cluster organisms occur
throughout the mesopelagic zone, constituting about 10% of
the microbial population there. They probably account for a
somewhat larger proportion of deep-ocean microbial biomass,
because they are larger than the average bacterioplankton cell
(43). Their considerable abundance suggests an important
role, but as yet no information about their metabolic activity
has come to light. One aspect of the mesopelagic environment
is the relatively constant availability of macronutrients (N and
P), which are deficient in surface waters, where they likely
drive competition among species (40). Energy for microbial
metabolism is scarce in the deep ocean most of the time and
mainly comes from the oxidation of recalcitrant organic com-
pounds (semilabile dissolved organic carbon [DOC]), ammo-
nium, and nitrite and from the metabolism of more-labile
substrates originating from the indigenous fauna and sinking
organic material (5, 22). The introduction of surface DOC to
the upper mesopelagic zone by convective events associated
with winter storms constitutes a large periodic input of DOC to
the upper mesopelagic zone (3, 16, 17) and may sustain some
elements of the microbial community that reside there.
The SAR202 cluster has been eclipsed because of interest in
some of the more abundant bacterioplankton groups, but they
occupy an important position in the bacterioplankton pantheon
and will undoubtedly be a subject of keen interest as environmen-
tal genome sequences, environmental monitoring, and possibly
cultures provide more information about this group.
ACKNOWLEDGMENTS
We acknowledge Terah Wright, who identified several SAR202
clones from the BATS 250 m clone library, and Rachel Parsons, who
prepared the BATS samples used for FISH.
FIG. 5. Group-specific FISH of samples taken from off the Oregon coast. Squares, SAR202 depth profiles showing direct cell counts from
NH35, NH55, NH65, NH85, and NH127; lines without squares, chlorphyll concentrations.
VOL. 70, 2004 OCEANIC ABUNDANCE OF THE SAR202 CLUSTER 2841

This study was supported by the following grants from the National
Science Foundation: An Oceanic Microbial Observatory (MCB-
9977918) and Bacterial Activity in the NE Pacific (OCE-0002236).
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