APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Feb. 2006, p. 1680–1683
Copyright © 2006, American Society for Microbiology. All Rights Reserved.
Vol. 72, No. 2
Characterization of Bacterial Communities Associated with Deep-Sea
Corals on Gulf of Alaska Seamounts†
Kevin Penn,1Dongying Wu,1Jonathan A. Eisen,1,2and Naomi Ward1,3*
The Institute for Genomic Research, 9712 Medical Center Drive, Rockville, Maryland 208501; Johns Hopkins University,
Charles and 34th Streets, Baltimore, Maryland 212182; and Center of Marine Biotechnology,
701 East Pratt Street, Baltimore, Maryland 202123
Received 22 June 2005/Accepted 8 November 2005
Although microbes associated with shallow-water corals have been reported, deepwater coral microbes are
poorly characterized. A cultivation-independent analysis of Alaskan seamount octocoral microflora showed
that Proteobacteria (classes Alphaproteobacteria and Gammaproteobacteria), Firmicutes, Bacteroidetes, and Ac-
idobacteria dominate and vary in abundance. More sampling is needed to understand the basis and significance
of this variation.
The most abundant corals on Gulf of Alaska seamounts are
octocorals (9), which create a habitat structure for mobile
fauna (4). Concerns about the benthic impacts of commercial
fishing have renewed interest in habitat-forming deep-sea cor-
als (4). Studies of shallow-water scleractinian corals (12) have
revealed a diverse microflora and evidence of host-microbe
interactions. Although studies of the deep-sea octocoral mi-
croflora are under way (10), there have been no published
reports describing the microbial community composition.
Three Gulf of Alaska seamounts were visited during re-
search cruise AT7-15/16 aboard the R/V Atlantis. The biolog-
ical objectives of the cruise included sampling of deep-sea
octocorals for studies of their dispersal and reproductive strat-
egies, with a particular focus on the abundant bamboo corals
(Isididae). We took advantage of available coral specimens to
examine their associated microflora.
Coral, rock, and water column samples (Table 1) were col-
lected from the Warwick, Murray, and Chirikof seamounts
using the deep-submergence vehicle Alvin. Corals and rocks
were harvested using the submersible’s manipulators and
stored in a closed box during ascent to minimize physical dis-
turbance by surface waters. The water adjacent to coral colo-
nies was sampled using a Niskin bottle fired at depth. After
submersible recovery, freshly extruded coral exopolysaccharide
and scrapings of coral and rock surfaces were transferred to
sterile cryovials. Water samples were prefiltered through 20-
?m-pore-size Nitex, concentrated using a TFF apparatus (Mil-
lipore), and vacuum filtered (1.0-?m and 0.2-?m pore size).
The 0.2-?m filter retentate was resuspended in sterile saline
solution. Samples were frozen immediately at ?70°C and
shipped frozen for subsequent processing.
Genomic DNAs were extracted using Ultra Clean soil DNA
kits (MoBio), and 16S rRNA genes were PCR amplified using
primers 27F and 1525R (11) and PlatTaq PCR supermix (In-
vitrogen). Amplifications were performed with an initial dena-
turation of 2 min at 94°C, followed by 29 cycles of 30 s at 94°C,
30 s at 55°C, and 2 min at 72°C, with a final extension of 5 min
at 72°C. PCR products were cloned using a TOPO TA cloning
kit (Invitrogen), and primers M13F and M13R were used to
sequence positions 9 to 1545 of the 16S rRNA gene.
BLASTN (1) was used to compare our query sequences with
reference sequences from the RDP2 (3) database. Represen-
tative sequences from the BLASTN output were aligned with
our query sequences, using an RDP2-provided profile align-
ment. Neighbor-joining trees were created using PHYLIP (6)
and used to assign putative taxonomy down to the family level.
Detailed phylogenetic trees were constructed using the rele-
vant sequences from each clone library, two reference se-
quences most closely related to the query sequence, and addi-
tional reference sequences. Alignments were generated using
the RDP2 profile alignment, and bootstrapped neighbor-join-
ing trees were reconstructed using PHYLIP (6).
The clones sequenced comprised 19 phyla (see Table S1 in
the supplemental material), dominated by Proteobacteria
(classes Alphaproteobacteria and Gammaproteobacteria), Firmi-
cutes, Bacteroidetes, and Acidobacteria (Fig. 1). The relative
proportions of these groups varied widely across the five coral
samples, as did the degree to which a given library was domi-
nated by a single group (Fig. 1; see Table S1 in the supple-
mental material). At the subphylum level, families occurring in
major proportions included Rhizobiaceae, Rhodobacteraceae,
and Sphingomonadaceae (Alphaproteobacteria); Pseudomona-
daceae, Alteromonadaceae, and Halomonadaceae (Gammapro-
teobacteria); Bacillaceae, Clostridiaceae, and Mycoplasmataceae
(Firmicutes); and Flexibacteraceae and Flavobacteraceae (Bac-
Members of the family Rhodobacteraceae and the family
Pseudomonadaceae were selected for further analysis, based on
their relative abundance and on the previous finding (13) that
shallow-water corals contain significantly larger numbers of
these bacteria than the surrounding water. Members of the
family Rhodobacteraceae comprised 23 to 100% of the alpha-
proteobacterial sequences in those libraries containing at least
10% alphaproteobacteria. The majority of Rhodobacteraceae
Genomic Research, 9712 Medical Center Drive, Rockville, MD 20850.
Phone: (301) 795-7813. Fax: (301) 838-0208. E-mail: firstname.lastname@example.org.
† Supplemental material for this article may be found at http://aem
sequences obtained in this and previous (12, 13) studies fall
within the marine roseobacters (see Fig. S1 in the supplemen-
tal material), a major clade of culturable marine heterotrophs
(7), many of which play a role in sulfur cycles (e.g., see refer-
ence 8). One clade of six CGOF sequences is most closely
related to NAC11-6 from a dimethylsulfoniopropionate-pro-
ducing algal bloom (8), while CGOCA38 groups closely with
NAC11-7 (from the same algal bloom study ) and an uncul-
tivated marine bacterium, ZD0207, associated with dimethyl-
sulfoniopropionate uptake (15). CGOAB33 is most similar to
one (slope strain EI1*) of a group of thiosulfate-oxidizing
bacteria from marine sediments and hydrothermal vents (14).
Members of the family Pseudomonadaceae comprised 23 to
69% of the gammaproteobacterial sequences in those samples
FIG. 1. Histogram showing percentages of composition (by taxon) for 16S rRNA gene libraries generated for this study, showing only taxa
comprising at least 20% of sequences in at least one clone library.
TABLE 1. Summary of Gulf of Alaska samples from which 16S rRNA gene sequences were obtained
Sample OriginSeamountAlvin dive no.
(no. of members)
(no. of sequences)b
PhylaClasses OrdersFamiliesSing DoubTrip Cluster
aDepths covered during entire dive (specific sample collection depth not available).
bResults of sorting sequences using FastGroup. Sing, unique sequences; Doub, doubletons (two identical sequences); Trip, tripletons (three identical sequences);
Cluster, cluster of more than three identical sequences.
VOL. 72, 2006BACTERIAL COMMUNITIES ON ALASKAN SEAMOUNT CORALS1681
containing at least 10% gammaproteobacteria. Sequences fall-
ing within the pseudomonad tree (see Fig. S2 in the supple-
mental material) appear most closely related to the oligotro-
phic marine gammaproteobacteria (OMG) (2). The lack of a
close phylogenetic relationship between representatives of the
described major OMG clades and our coral sequences suggests
that the latter represent new OMG clades.
Rarefaction analysis of our data and of sequence data from
shallow-water scleractinian coral communities (12) suggested
that our accumulated deepwater octocoral samples showed
less diversity than their shallow-water counterparts (Fig. 2);
with 350 sequences sampled, the shallow-water data set con-
tained approximately twice as many observed operational tax-
onomic units (97% threshold for operational taxonomic unit
definition) as the deepwater set.
This study provides a first glimpse of the deep-sea octocoral
microflora. The results suggest that these populations are dom-
inated by several major groups but that the relative propor-
tions of these groups vary (bearing in mind that known meth-
odological biases  limit the extent to which clone library
compositions reflect community compositions). Phylotypes
clustered according to sample origin, and we did not observe
much overlap between coral-associated phylotypes and those
recovered from the water column and rock surfaces (see Fig.
S1 and S2 in the supplemental material), suggesting character-
istic coral-associated assemblages with minimal influence of
transient water-column microbes. Future sampling of multiple
individuals and their immediate environment is clearly needed
to perform a more comprehensive survey and to address ques-
tions regarding the nutritional relationships, evolution, and
biogeography of these populations.
We thank R/V Atlantis and DSV Alvin personnel, NOAA’s Ocean
Exploration Program, and Brad Stevens, Randy Keller, Tom Shirley,
and Tom Guilderson for help with data acquisition.
Phylogenetic analysis was supported in part by NSF Assembling the
Tree of Life grant 0228651 to J.A.E. and N.W.
1. Altschul, S., T. Madden, A. Schaffer, J. Zhang, Z. Zhang, W. Miller, and
D. J. Lipman. 1997. Gapped BLAST and PSI-BLAST: a new generation of
protein database search programs. Nucleic Acids Res. 25:3389–3402.
2. Cho, J. C., and S. J. Giovannoni. 2004. Cultivation and growth characteristics
of a diverse group of oligotrophic marine Gammaproteobacteria. Appl. En-
viron Microbiol. 70:432–440.
3. Cole, J., B. Chai, T. Marsh, R. Farris, Q. Wang, S. Kulam, S. Chandra, D.
McGarrell, T. Schmidt, G. Garrity, and J. Tiedje. 2003. The Ribosomal
Database Project (RDP-II): previewing a new autoaligner that allows regular
updates and the new prokaryotic taxonomy. Nucleic Acids Res. 31:442–443.
4. Etnoyer, P., and L. Morgan. December 2003, posting date. Occurrences of
habitat-forming deep sea corals in the northeast Pacific Ocean: a report to
NOAA’s Office of Habitat Conservation. [Online.] http://www.mcbi.org
5. Farrelly, V., F. A. Rainey, and E. Stackebrandt. 1995. Effect of genome size
and rrn gene copy number on PCR amplification of 16S rRNA genes from a
mixture of bacterial species. Appl. Environ. Microbiol. 61:2798–2801.
6. Felsenstein, J. 1989. PHYLIP—phylogeny inference package (version 3.2).
7. Giovannoni, S., and M. Rappe ´. 2000. Evolution, diversity, and molecular
ecology of marine prokaryotes, p. 47–84. In D. L. Kirchman (ed.), Microbial
ecology of the oceans. John Wiley & Sons, New York, N.Y.
8. Gonzalez, J. M., R. Simo, R. Massana, J. S. Covert, E. O. Casamayor, C.
Pedros-Alio, and M. A. Moran. 2000. Bacterial community structure associ-
ated with a dimethylsulfoniopropionate-producing North Atlantic algal
bloom. Appl. Environ. Microbiol. 66:4237–4246.
9. Heifetz, J. 2002. Coral in Alaska: distribution, abundance, and species asso-
ciations. Hydrobiologia 471:19–27.
10. Kellogg, C., and R. Stone. 2004. A pilot study of deep-water coral microbial
ecology. Presented at the ASLO/TOS Ocean Research Conference, Hono-
11. Rainey, F. A., N. Ward-Rainey, R. M. Kroppenstedt, and E. Stackebrandt.
1996. The genus Nocardiopsis represents a phylogenetically coherent taxon
FIG. 2. Rarefaction curves for the accumulated coral-associated 16S rRNA gene sequences generated for this study (CGOA, -C, -D, -F, and
-G) and the sequences of Rohwer et al. (12, 13). Bars indicate 95% confidence intervals. Statistical resampling was performed using EstimateS.
1682 PENN ET AL.APPL. ENVIRON. MICROBIOL.
and a distinct actinomycete lineage: proposal of Nocardiopsaceae fam. nov.
Int. J. Syst. Bacteriol. 46:1088–1092.
12. Rohwer, F., M. Breitbart, J. Jara, F. Azam, and N. Knowlton. 2001. Diversity
of bacteria associated with the Caribbean coral Montastraea franksi. Coral
13. Rohwer, F., V. Seguritan, A. Farooq, and N. Knowlton. 2002. Diversity and
distribution of coral-associated bacteria. Mar. Ecol. Prog. Ser. 243:1–10.
14. Teske, A., T. Brinkhoff, G. Muyzer, D. P. Moser, J. Rethmeier, and H. W.
Jannasch. 2000. Diversity of thiosulfate-oxidizing bacteria from marine sed-
iments and hydrothermal vents. Appl. Environ. Microbiol. 66:3125–3133.
15. Zubkov, M. V., B. M. Fuchs, S. D. Archer, R. P. Kiene, R. Amann, and P. H.
Burkill. 2001. Linking the composition of bacterioplankton to rapid turnover
of dissolved dimethylsulphoniopropionate in an algal bloom in the North
Sea. Environ. Microbiol. 3:304–311.
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