FEMS Microbiology Ecology 35 (2001) 287^293

www.fems-microbiology.org

Expansion of the geographic distribution of a novel lineage

of O-Proteobacteria to a hydrothermal vent site on
the Southern East Paci¢c Rise
a;b a;
Krista Longnecker , Anna-Louise Reysenbach *

Downloaded from https://academic.oup.com/femsec/article/35/3/287/456483 by guest on 13 September 2022

a
Department of Biology, Portland State University, 1719 10th Avenue SW, Portland, OR 97201, USA
b
College of Oceanic and Atmospheric Sciences, Oregon State University, 104 Ocean Admin Bldg, Corvallis, OR 97331-5503, USA

Received 16 October 2000; received in revised form 18 January 2001; accepted 19 January 2001

Abstract

The diversity associated with a microbial mat sample collected from a deep-sea hydrothermal vent on the Southern East Pacific Rise was
determined using a molecular phylogenetic approach based on the comparison of sequences from the small subunit ribosomal RNA gene
(16S rDNA). The DNA was extracted from the sample and the 16S rDNA was amplified by PCR. Sixteen different phylotypes were
identified by restriction fragment length polymorphism analysis; four phylotypes were later identified as putative chimeras. Analysis of the
16S rDNA sequences placed all the phylotypes within the Proteobacteria. The majority of the sequences (98%) were most closely related to a
new clade of O-Proteobacteria that were initially identified from an in situ growth chamber deployed on a deep-sea hydrothermal vent on the
Mid-Atlantic Ridge in 1995. The similarity between phylotypes identified from Atlantic and Pacific deep-sea hydrothermal vent sites
indicates that this new clade of Proteobacteria may be endemic to and widely distributed among deep-sea hydrothermal vents. ß 2001
Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.

Keywords : 16S rRNA; Phylogeny; Deep-sea bacterium ; Hydrothermal vent; Proteobacteria

1. Introduction
Over the past 20 years our knowledge about the diver-
sity and role of microorganisms at deep-sea hydrothermal
vents has expanded considerably. Within deep-sea hydro-
thermal vent ecosystems, microorganisms occupy several
di¡erent niches which include : free-living in vent £uids
and hydrothermal plumes, as symbionts with vent macro-
fauna, and as microbial mats attached to hard substrates
such as rocks, chimneys, sediments and animal surfaces
[1]. The Bacteria and Archaea cultured from vent £uids
and sul¢de chimneys primarily include organisms that ox-
idize or reduce sulfur compounds [2^4] or produce meth-
ane [5,6]. Microbiological processes such as methane oxi-
dation [7] and manganese oxidation [8] occur within
hydrothermal plumes.
Microbial mats have been observed at most, if not all,
deep-sea hydrothermal vent sites including the Galapagos
* Corresponding author. Tel. : +1 (503) 725-3864;
Fax: +1 (503) 725-8570; E-mail: reysenbacha@pdx.edu
0168-6496 / 01 / $20.00 ß 2001 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.
PII: S 0 1 6 8 - 6 4 9 6 ( 0 1 ) 0 0 0 9 9 - X
[9,10], Guaymas Basin [11], di¡erent sites on the East Pa-
ci¢c Rise [12] and Loihi Seamount [13^15]. The Beggiatoa-
like and Thiothrix-like mats from the Galapagos occupy
regions with exposure to low temperature vent £uids and
are coated with amorphous iron and manganese [9,10].
The dense mats at Guaymas Basin also contain Beggia-
toa-like ¢laments, including at least two autotrophic
strains whose CO2 ¢xation rates are stimulated by the
addition of sul¢de [11]. The microbial mats at Loihi Sea-
mount are complex communities that include Archaea and
Q-, N-, and O-Proteobacteria [14,15]. The majority of the
microorganisms present within the Loihi mats clustered
within the O-Proteobacteria [14].
This study examines the microbial diversity of a sample
collected from the Southern East Paci¢c Rise (SEPR). The
sample was a ¢lamentous mat collected via a suction sam-
pler attached to a submersible. Molecular phylogenetic
techniques based on the small subunit ribosomal RNA
molecule (16S rRNA) were used to identify the members
of the microbial community. The majority of the phylo-
types obtained from the sample belonged to a group of
uncultured O-Proteobacteria recently identi¢ed from the
Mid-Atlantic Ridge (MAR) [16].

FEMSEC 1225 5-4-01 Cyaan Magenta Geel Zwart

288 K. Longnecker, A.-L. Reysenbach / FEMS Microbiology Ecology 35 (2001) 287^293
2. Materials and methods
2.1. Sample collection and storage
A ¢lamentous mat sample was collected from a hydro-
thermal vent ¢eld at 17³S, 113³W on the SEPR (Fig. 1) at
a depth of 2592 m with a suction sampler attached to
DSRV Alvin on January 3, 1999. The mat completely cov-
ered an extinct chimney next to a high temperature
(Tmax = 317³C) smoker named Spike. The ¢laments ap-
peared as a white homogeneous mat. Unfortunately the
temperature within the microbial mat is unknown and
points to the importance and di¤culty of obtaining phys-
ical data associated with samples from deep-sea hydrother-
mal vents. Once shipboard, the samples were immediately
frozen in 70% (v/v) ethanol at 380³C.
2.2. DNA extraction, ampli¢cation, cloning, and restriction
fragment length polymorphism (RFLP) analysis
DNA was extracted following a modi¢ed extraction
protocol using cetyltrimethylammonium bromide
(CTAB) [17]. A 50-Wl aliquot of the sample was centri-
fuged for 5 min, the supernatant removed, and the pellet
resuspended in 467 Wl of TE bu¡er (10 mM Tris^HCl and
1 mM EDTA) and an equal volume of extraction bu¡er
(100 mM EDTA, 0.5% SDS, 3.25% Chelex-100, 0.25 mg
ml31 Proteinase K). The sample was incubated at 37³C for
1 h. The sample was spun brie£y to separate the Chelex-
bound metals and cell lysate. NaCl (0.8 M ¢nal concen-
tration) and a CTAB/NaCl solution (1% CTAB and 0.7 M
NaCl ¢nal concentration) was added. The sample was in-
cubated at 65³C for 30 min. The DNA was ¢rst extracted
with chloroform :isoamyl alcohol (24:1) and then with
phenol:chloroform:isoamyl alcohol (25:24:1). DNA was
precipitated with an equal volume of 100% isopropanol
overnight at 320³C.
Primers used to amplify 16S rDNA were the universal
primer 1492R (5P-GGT TAC CTT GTT ACG ACT T-3P),
the bacterial speci¢c primer 27F (5P-AGA GTT TGA
TCC TGG CTC AG-3P), and the archaeal speci¢c primer
21F (5P-TCC GGT TGA TCC TGC CRG-3P where R = A
or G). Conditions for PCR were an initial denaturation at
94³C for 5 min, 30 cycles of 94³C, 30 s; 50³C, 30 s; 72³C,
90 s; and a ¢nal 10 min at 72³C. The PCR product was
puri¢ed with the Wizard Prep kit (Promega) following the
manufacturer's instructions.
PCR products were cloned into the pCR4 vector (Invi-
trogen). Plasmid DNA was extracted following the proto-
col of Sambrook et al. [18]. Plasmid DNA was screened
for appropriate-sized inserts with the vector-speci¢c M13
primers: M13F (5P-GTA AAA CGA CGG CCA G-3P)
and M13R (5P-CAG GAA ACA GCT ATG AC-3P). The
PCR-ampli¢ed inserts were digested with 1 U of the re-
striction endonucleases MspI and HinPI following the
manufacturer's instructions (New England Biolabs). The
FEMSEC 1225 5-4-01
resulting products were separated by gel electrophoresis
on a 3.5% NuSieve (FMC Bioproducts) agarose gel run
in TBE bu¡er at 4³C. The clones were separated into
di¡erent phylotypes based on the RFLP banding patterns.
2.3. Sequencing and phylogenetic analysis
Both strands of the 16S rDNA were fully sequenced by
cycle sequencing as described previously [16]. Sequences
were assembled using AutoAssembler (Applied Biosystems
Inc.). The sequences were checked for the presence of chi-
meras using the Ribosomal Database Project's (RDP)
CHIMERA_CHECK program [19]. The CHIMERA_-
Downloaded from https://academic.oup.com/femsec/article/35/3/287/456483 by guest on 13 September 2022
CHECK analysis was run with and without the addition
of the sequences obtained from this experiment. Potential
chimeras were removed from further analysis. Sequence
accession numbers are given in Table 1.
Sequences were manually aligned in the Genetic Data
Environment (GDE) [20]. The alignment included close
representatives that were identi¢ed based on data from
BLAST (basic logic alignment search tool) [21] and RDP's
Similarity Matrix tool [19]. Distance matrices were con-
structed by pairwise analysis using the Kimura-2 parame-
ter model [22]. PAUP version 4.0b4a was used for parsi-
mony analysis [23]. Approximately 1500 nucleotides were
obtained from sequencing and about 1250 nucleotides in
evolutionarily conserved regions were used for the maxi-
mum likelihood analysis. Maximum likelihood trees were
constructed using fastDNAml [24]. The optimal T value
(T = 1.25) was determined by comparing the likelihood
score of trees constructed with a range of T values
(T = 1.0^3.0) to a tree constructed with T = 2.0. Once the
T value was determined, multiple maximum likelihood
analyses were conducted with the sequence addition order
jumbled to produce the optimum tree. Bootstrap data rep-
resent the result of 500 replicates.
2.4. Fluorescent in situ hybridization (FISH)
An oligonucleotide probe labeled with the Cy3 £uoro-
chrome was designed to target the O-Proteobacteria phy-
lotypes identi¢ed in this study. The probe was a 100%
match to nucleotides 112^120 (Escherichia coli numbering)
and is 5P-GTA GCT ACG TGT TAC TCA CC-3P. To
prevent cross reaction with organisms with some mis-
matches, the conditions for FISH were as previously de-
scribed [25] except with a more stringent hybridization
temperature (55³C). The samples were viewed on an
Olympus BX60 microscope.
3. Results and discussion
3.1. Phylogenetic analysis
Analysis of the sequences from the mat sample collected
Cyaan Magenta Geel Zwart
 K. Longnecker, A.-L. Reysenbach / FEMS Microbiology Ecology 35 (2001) 287^293 289

lotypes (a total of 10 sequences) were eliminated from

further analysis. Phylogenetic analysis of the SEPR sample
clustered the phylotypes into three groups: two with the O-
Proteobacteria and one with the Q-Proteobacteria (Fig. 2).
The majority of the sequences (30 sequences, V75%)
are part of a new clade within the Proteobacteria (Fig.
2) that includes sequences identi¢ed from an in situ growth
chamber deployed on a hydrothermal vent on the MAR
[16] and CHA3-122, a sequence from a hydrothermal
chimney collected from the same site where the in situ
growth chamber had been deployed (V. Cilia and D.
Prieur, sequence submitted to GenBank, #AJ132722).
The distance matrices (Table 2) indicate that the Group

Downloaded from https://academic.oup.com/femsec/article/35/3/287/456483 by guest on 13 September 2022

Fig. 1. Map of the SEPR indicating the location of the sampling site
for this study. The map was originally published by Macdonald et al.
[38] and was modi¢ed by R. Vrijenhoek.
at the deep-sea hydrothermal vent site at 17³S on the
SEPR indicated that the sequences were all Proteobacte-
ria. No archaeal 16S rDNA was ampli¢ed. RFLP analysis
of the sequences identi¢ed 16 di¡erent phylotypes from the
50 sequences analyzed. A conservative analysis of the re-
sults from CHIMERA_CHECK indicated that four phy-
lotypes may have been chimeric and therefore those phy-
I phylotypes are closest to the sequences identi¢ed from
the MAR [16].
The Group II is represented by nine sequences (V23%)
that clustered into three phylotypes (Table 1). The phylo-
types within this group (S17sBac16, -17, -25) are intention-
ally de¢ned separately from the phylotypes in Group I.
Group II is either the deepest branch within Group I as
shown in Fig. 2 or the deepest lineage within the described
O-Proteobacteria. With the choice of sequences used for
the tree in Fig. 2, the Group II sequences grouped with
previously described O-Proteobacteria in 13% of the boot-
strap replicates. While this bootstrap result is not signi¢-
cant, using di¡erent outgroups in the analysis did result in
consensus trees placing the Group II sequences within the
O-Proteobacteria. Parsimony analysis of the entire 16S
rRNA gene clustered the Group II sequences within the
described O-Proteobacteria. However parsimony analysis
using only the evolutionarily conserved regions that were
used for the maximum likelihood analysis resulted in the
same tree topology as the maximum likelihood analysis.
The distance matrices indicated that the Group II sequen-
ces are equally distant from the sequences identi¢ed from
the in situ growth chamber as they are to described Bac-
teria (Table 2), further supporting the di¡erences between
the Group I and Group II sequences.

Table 1
Summary of sequences obtained from the microbial mat collected from 17³S on the East Paci¢c Rise
Phylogenetic a¤liationa Sequenced Clone `phylotype' Accession number Number of clones according to RFLP analysisb
Group I S17sBac3 AF299127 5
S17sBac4 AF299129 12
S17sBac5 AF299130 3
S17sBac8 AF299131 1
S17sBac14 AF299124 1
S17sBac19 AF299125 2
S17sBac21 AF299126 3
S17sBac34 AF299128 3
Group II S17sBac16 AF299121 1
S17sBac17 AF299122 3
S17sBac25 AF299123 5
Q-Proteobacteria S17sBac13 AF299120 1
Chimerasc S17sBac12, -31, -38, -44 10
a
According to the results of the maximum likelihood analysis.
b
A total of 50 clones were analyzed.
c
As determined following the CHIMERA_CHECK analysis.

FEMSEC 1225 5-4-01 Cyaan Magenta Geel Zwart

290 K. Longnecker, A.-L. Reysenbach / FEMS Microbiology Ecology 35 (2001) 287^293

Downloaded from https://academic.oup.com/femsec/article/35/3/287/456483 by guest on 13 September 2022

Fig. 2. Phylogenetic analysis of the 16S rDNA sequences obtained from the sample collected at 17³S, 113³W on the SEPR. The tree is the result of
maximum likelihood analysis using Bacillus subtilis as the outgroup. The numbers at the nodes represent percent of bootstrap values obtained from 500
samplings. Scale bar represents 0.1 nucleotide substitutions per sequence position. SEPR sequences are marked in bold type. In addition to the SEPR
sequences, the following sequences were obtained from GenBank: Campylobacter jejuni (Z29326), Sulfurospirillum arcachonense (Y11561), PVB_63
(U15102), symbiont of A. pompejana (L35521), symbiont of R. exoculata (U29081), Desulfonema limicola (U45990), Desulfovibrio desulfuricans
(M34113), P. putida (Z76667), Neisseria gonorrhoeae (X07714), Spirillum volutans (M34131), Azospirillum amazonense (X79735), Agrobacterium tumefa-
ciens (D14500), CHA3-122 (AJ132722), VC2.1Bac17 (AF068795), and VC2.1Bac7 (AF068788).

The Group I and II phylotypes together represented the
majority (98%) of the sequences identi¢ed from the SEPR
sample. Although the percentage of sequences within each
phylotype is not necessarily indicative of its abundance in
an ecosystem, FISH analysis using probes designed specif-
ically to target the O-Proteobacteria phylotypes identi¢ed
in this study indicate that these phylotypes are the domi-
nant ( s 90%) members of the community. The O-Proteo-
bacteria phylotypes were long, ¢lamentous rods or shorter
(about 2 Wm), curved rods (Fig. 3). In addition, non-£uo-
rescent single and dividing cocci were noted under phase
contrast microscopy. Due to a 10% mismatch between the
probe and certain distantly related phylogenetic groups,
such as members of the Aqui¢cales, FISH conditions re-
quired hybridization temperatures greater than 55³C (un-
published results).
A single clone, S17sBac13, is most closely related to
Pseudomonas putida. As part of another study on micro-
bial diversity in hydrothermal vent water, Pseudomonas
species were also identi¢ed despite the apparent absence
of contamination in the negative controls [26]. Our clone is
likely another example of this group from deep-sea vents,

Table 2
Summary of distance matrices constructed using pairwise distances of conserved regions and the Kimura-2 parameter model [22]
Sequences Distance from the MAR sequences (%) Distance from other bacterial 16S rRNA sequences (%)
S17sBac3, -4, -5, -8, -14, -19, -21, -34 (Group I) 3.5^13.0 14.5^18.2 (A. pompejana epibionts)
S17sBac16, -17, -25 (Group II) 10.2^16.0 10.1^15.3 (S. arcachonense)
S17sBac13 3.9 (P. putida)

FEMSEC 1225 5-4-01 Cyaan Magenta Geel Zwart

 K. Longnecker, A.-L. Reysenbach / FEMS Microbiology Ecology 35 (2001) 287^293
Fig. 3. FISH of the 17³S community. a: Images with the Cy3-labeled oligonucleotide probe speci¢c for the O-Proteobacteria phylotypes. b: Phase con-
trast micrograph of the same ¢eld. The scale bar is 20 Wm.
although the possibility that it represents a laboratory
contaminant [27] cannot entirely be ruled out.
3.2. O-Proteobacteria at deep-sea hydrothermal vents
Proteobacteria have been identi¢ed from a variety of
marine and coastal ecosystems [28^30]. In marine systems,
O-Proteobacteria have thus far been restricted to deep-sea
hydrothermal vents [14,16,31,32] and shallow coastal ma-
rine water [33]. At deep-sea hydrothermal vents, the
O-Proteobacteria can exist as epibionts of the vent shrimp
Rimicaris exoculata [31] and the polychaete Alvinella pom-
pejana [32]. The epibionts have been hypothesized to be a
food source for their invertebrate hosts [34^36]. Further-
more A. pompejana's epibionts have been hypothesized to
detoxify the local environment for their invertebrate hosts
by removal of sul¢de compounds from the vent water [37].
The epibionts can also be a component of the free-living
microbial community attached to sul¢de edi¢ces [31].
Given the diversity of metabolic types within the O-Pro-
teobacteria, hypothesizing about the metabolic capacity of
the new group of Bacteria identi¢ed from 17³S is not
possible. However, the white sulfur-like coloration of the
¢laments may indicate these phylotypes from the SEPR
represent another group of deep-sea hydrothermal vent
microorganisms involved in sulfur cycling.
O-Proteobacteria at deep-sea hydrothermal vents have
also been identi¢ed as members of the free-living microbial
community at Loihi Seamount [14] and associated with an
in situ colonization experiment at Snakepit on the MAR
[16]. The results of the phylogenetic analysis of the Loihi
Seamount microbial mats indicated that one group,
FEMSEC 1225 5-4-01
291
Downloaded from https://academic.oup.com/femsec/article/35/3/287/456483 by guest on 13 September 2022
PVB_OTU2, was 65% of the bacterial clones (based on
percent of bacterial sequences grouped by RFLP analysis).
This group clustered within the O-Proteobacteria and was
most closely related to Thiovulum sp. [14]. The O-Proteo-
bacteria identi¢ed from the in situ growth chamber de-
ployed on the MAR clustered into three di¡erent groups:
two groups closely related to existing O-Proteobacteria and
one group that was only distantly related to existing
O-Proteobacteria [16]. The latter group from the MAR
was the largest group of Bacteria (28% of the bacterial
sequences according to RFLP analysis) identi¢ed from
the in situ growth chamber [16].
The Group I and Group II phylotypes identi¢ed in this
study from 17³S cluster with the novel O-Proteobacteria
phylotypes from the MAR. Based on the 16S rDNA se-
quences and the FISH analysis, these O-Proteobacteria
clearly represent dominant members of the community.
The expansion of the geographic range of this new group
of O-Proteobacteria from its initial discovery on the MAR
to a Paci¢c deep-sea hydrothermal vent indicates this
group may be endemic to deep-sea hydrothermal vents.
Acknowledgements
We thank Robert Vrijenhoek and John Lupton for the
invitation to participate on the cruise and for guiding the
science during the SEPR cruise. Special thanks to the R/V
Atlantis and DSRV Alvin crews for their hard work and
dedication throughout this entire cruise. Paula Aguiar was
instrumental in the FISH analysis. We thank the members
of the Reysenbach lab for critically reading the manu-
Cyaan Magenta Geel Zwart
292 K. Longnecker, A.-L. Reysenbach / FEMS Microbiology Ecology 35 (2001) 287^293
script. This work partially supported by National Science
Foundation-LExEn Grant (OCE9729784) to A.L.R.,
OCE9910799 and OCE9633131 to R. Vrijenhoek and R.
Lutz.
References
[1] Karl, D.M. (1995) Ecology of free-living, hydrothermal vent micro-
bial communities. In: The Microbiology of Deep-Sea Hydrothermal
Vents (Karl, D.M., Ed.), pp. 35^124. CRC, New York.
[2] Huber, R., Sto«hr, J., Hohenhaus, S., Rachel, R., Burggraf, S., Jan-
nasch, H.W. and Stetter, K.O. (1995) Thermococcus chitonophagus sp.
nov., a novel, chitin-degrading, hyperthermophilic archaeum from a
deep-sea hydrothermal vent environment. Arch. Microbiol. 164, 255^
264.
[3] L'Haridon, S., Cilia, V., Messner, P., Raguëne©s, G., Gambacorta, A.,
Sleytr, U.B., Prieur, D. and Jeanthon, C. (1998) Desulfurobacterium
thermolithotrophum gen. nov., sp. nov., a novel autotrophic, sulfur-
reducing bacterium isolated from a deep-sea hydrothermal vent. Int.
J. Syst. Bacteriol. 48, 701^711.
[4] Burggraf, S., Jannasch, H.W., Nicolaus, B. and Stetter, K.O. (1990)
Archaeoglobus profundus sp. nov., represents a new species within the
sulfate-reducing archaebacteria. Syst. Appl. Microbiol. 13, 24.
[5] Kurr, M., Huber, R., Ko«nig, H., Jannasch, H.W., Fricke, H., Trin-
cone, A., Kristjansson, J.K. and Stetter, K.O. (1991) Methanopyrus
kandleri, gen. and sp. nov. represents a novel group of hyperthermo-
philic methanogens, growing at 110³C. Arch. Microbiol. 156, 239^
247.
[6] Jones, W.J., Leigh, J.A., Mayer, F., Woese, C.R. and Wolfe, R.S.
(1983) Methanococcus jannaschii sp. nov., an extremely thermophilic
methanogen from a submarine hydrothermal vent. Arch. Microbiol.
136, 254^261.
[7] de Angelis, M.A., Lilley, M.D., Olson, E.J. and Baross, J.A. (1993)
Methane oxidation in deep-sea hydrothermal plumes of the Endeav-
our segment of the Juan de Fuca Ridge. Deep-Sea Res. 40, 1169^
1186.
[8] Cowen, J.P., Massoth, G.J. and Baker, E.T. (1986) Bacterial scaveng-
ing of Mn and Fe in a mid- to far-¢eld hydrothermal particle plume.
Nature 322, 169^171.
[9] Jannasch, H.W. (1983) Microbial processes at deep sea hydrothermal
vents. In: Hydrothermal Processes at Sea£oor Spreading Centers
(Rona, P.A., Bostro«m, K., Laubier, L. and Smith, K.L., Jr., Eds.),
pp. 677^709. Plenum, New York.
[10] Jannasch, H.W. and Wirsen, C.O. (1981) Morphological survey of
microbial mats near deep-sea thermal vents. Appl. Environ. Micro-
biol. 41, 528^538.
[11] Nelson, D.C., Wirsen, C.O. and Jannasch, H.W. (1989) Character-
ization of large autotrophic Beggiatoa abundant at hydrothermal
vents of the Guaymas Basin. Appl. Environ. Microbiol. 55, 2909^
2917.
[12] Juniper, S.K. and Fouquet, Y. (1988) Filamentous iron^silica depos-
its from modern and ancient hydrothermal sites. Can. Mineral. 26,
859^869.
[13] Karl, D.M., Brittain, A.M. and Tilbrook, B.D. (1989) Hydrothermal
and microbial processes at Loihi Seamount, a mid-plate hot-spot
volcano. Deep-Sea Res. 36, 1655^1673.
[14] Moyer, C.L., Dobbs, F.C. and Karl, D.M. (1995) Phylogenetic diver-
sity of the bacterial community from a microbial mat at an active,
hydrothermal vent system, Loihi Seamount, Hawaii. Appl. Environ.
Microbiol. 61, 1555^1562.
[15] Moyer, C.L., Tiedje, J.M., Dobbs, F.C. and Karl, D.M. (1998) Di-
versity of deep-sea hydrothermal vent Archaea from Loihi Seamount,
Hawaii. Deep-Sea Res. II 45, 303^317.
[16] Reysenbach, A.-L., Longnecker, K. and Kirshtein, J. (2000) Novel
FEMSEC 1225 5-4-01 Cyaan Magenta Geel Zwart
bacterial and archaeal lineages from an in situ growth chamber de-
ployed at a Mid-Atlantic Ridge hydrothermal vent. Appl. Environ.
Microbiol. 66, 3788^3797.
[17] Ausubel, F.M., Brent, R., Kingston, R.E., Moore, D.D., Seidman,
J.G., Smith, J.A. and Struhl, K. (1994) Current Protocols in Molec-
ular Biology. John Wiley and Sons, New York.
[18] Sambrook, J., Fritsch, E.F. and Maniatus, T. (1989) Molecular Clon-
ing: A Laboratory Manual. Cold Spring Harbor Laboratory Press,
Plainview, NY.
[19] Maidak, B.L., Cole, J.R., Lilburn, T.G., Parker, C.T., Saxman, P.R.,
Stredwick, J.M., Garrity, G.M., Li, B., Olsen, G.J., Pramanik, S.,
Schmidt, T.M. and Tiedje, J.M. (2000) The RDP (Ribosomal Data-
base Project) continues. Nucleic Acids Res. 28, 173^174.
[20] Smith, S.W., Overbeek, R., Woese, C.R., Gilbert, W. and Gillevet,
P.M. (1994) The genetic data environment an expandable GUI for
multiple sequence analysis. Comput. Appl. Biosci. 10, 671^675.
Downloaded from https://academic.oup.com/femsec/article/35/3/287/456483 by guest on 13 September 2022
[21] Altschul, S.F., Madden, T.L., Scha«¡er, A.A., Zhang, J., Zhang, Z.,
Miller, W. and Lipman, D.J. (1997) Gapped BLAST and PSI-
BLAST : a new generation of protein database search programs. Nu-
cleic Acids Res. 25, 3389^3402.
[22] Kimura, M. (1980) A simple model for estimating evolutionary rates
of base substitutions through comparative studies of nucleotide se-
quences. J. Mol. Evol. 16, 111^120.
[23] Swo¡ord, D.L. (1998) PAUP*, Phylogenetic Analysis Using Parsi-
mony (*and other methods), Version 4. Sinauer Associates, Sunder-
land, MA.
[24] Olsen, G.J., Matsuda, H., Hagstrom, R. and Overbeek, R. (1994)
fastDNAml: A tool for construction of phylogenetic trees of DNA
sequences using maximum likelihood. Comput. Appl. Biosci. 10, 41^
48.
[25] Reysenbach, A.-L., Wickham, G.S. and Pace, N.R. (1994) Phyloge-
netic analysis of the hyperthermophilic pink ¢lament community in
Octopus Spring, Yellowstone National Park. Appl. Environ. Micro-
biol. 60, 2113^2119.
[26] Takai, K. and Sako, Y. (1999) A molecular view of archaeal diversity
in marine and terrestrial hot water environments. FEMS Microbiol.
Ecol. 28, 177^188.
[27] Tanner, M.A., Goebel, B.M., Dojka, M.A. and Pace, N.R. (1998)
Speci¢c ribosomal DNA sequences from diverse environmental set-
tings correlate with experimental contaminants. Appl. Environ. Mi-
crobiol. 64, 3110^3113.
[28] DeLong, E.F., Franks, D.G. and Alldredge, A.L. (1993) Phylogenetic
diversity of aggregate-attached vs. free-living marine bacterial assem-
blages. Limnol. Oceanogr. 38, 924^934.
[29] Suzuki, M.T., Rappë, M.S., Haimberger, Z.W., Win¢eld, H., Adair,
N., Stro«bel, J. and Giovannoni, S.J. (1997) Bacterial diversity among
small-subunit rRNA gene clones and cellular isolates from the same
seawater sample. Appl. Environ. Microbiol. 63, 983^989.
[30] Cottrell, M.T. and Kirchman, D.L. (2000) Community composition
of marine bacterioplankton determined by 16S rRNA gene clone
libraries and £uorescence in situ hybridization. Appl. Environ. Mi-
crobiol. 66, 5116^5122.
[31] Polz, M.F. and Cavanaugh, C.M. (1995) Dominance of one bacterial
phylotype at a Mid-Atlantic Ridge hydrothermal vent site. Proc.
Natl. Acad. Sci. USA 92, 7232^7236.
[32] Cary, S.C., Cottrell, M.T., Stein, J.L., Camacho, F. and Desbruye©res,
D. (1997) Molecular identi¢cation and localization of ¢lamentous
symbiotic Bacteria associated with the hydrothermal vent annelid
Alvinella pompejana. Appl. Environ. Microbiol. 63, 1124^1130.
[33] Taylor, C.D., Wirsen, C.O. and Gaill, F. (1999) Rapid microbial
production of ¢lamentous sulfur mats at hydrothermal vents. Appl.
Environ. Microbiol. 65, 2253^2255.
[34] Van Dover, C.L., Fry, B., Grassle, J.F., Humphris, S. and Rona,
P.A. (1988) Feeding biology of the shrimp Rimicaris exoculata at
hydrothermal vents on the Mid-Atlantic Ridge. Mar. Biol. 98, 209^
216.
[35] Gebruk, A.V., Pimenov, N.V. and Savvichev, A.S. (1993) Feeding
 K. Longnecker, A.-L. Reysenbach / FEMS Microbiology Ecology 35 (2001) 287^293 293

specialization of bresiliid shrimps in the TAG site hydrothermal com-
munity. Mar. Ecol. Prog. Ser. 98, 247^253.
[36] Gaill, F., Desbruye©res, D. and Laubier, L. (1988) Relationships be-
tween the `Pompeii worms' and their epibiotic bacteria. Oceanol.
Acta 8, 147^154.
[37] Alayse-Danet, A.M., Desbruyeres, D. and Gaill, F. (1987) The pos-
sible nutritional or detoxi¢cation role of the epibiotic bacteria of
Alvinellid polychaetes: Review of current data. Symbiosis 4, 51^62.
[38] Macdonald, K.C., Haymon, R.M., Miller, S.P., Sempere, J.-C. and
Fox, P.J. (1988) Deep-Tow and Sea Beam studies of dueling prop-
agating ridges on the East Paci¢c Rise near 20³40PS. J. Geophys. Res.
93, 2875^2898.

Downloaded from https://academic.oup.com/femsec/article/35/3/287/456483 by guest on 13 September 2022

FEMSEC 1225 5-4-01 Cyaan Magenta Geel Zwart