AN ABSTRACT OF THE THESIS OF
Michael A. Haddad for the degree of Master of Science in Microbiology
presented on August 18, 1994.
Title: Phylogenetic Characterization of the Epibiotic Bacteria Associated with
the Hydrothermal Vent Polychaete Alvinella pornpejana.
Abstract approved:
Redacted for Privacy
Stephen J. Giovannoni
Symbiotic relationships of bacteria with higher organisms are
commonly observed in nature; however, the functional role of these
relationships is only rarely understood. This is particularly evident in
epibiotic bacterial associations in the marine environment where the bacteria
are often a diverse ensemble of microorganisms, thus complicating the
identification of the functionally important members. Classical
microbiological techniques, relying primarily on culturing these organisms,
have provided an incomplete picture of these relationships. Molecular
genetic techniques, focusing on the analyses of bacterial 16S rRNA sequences
cloned directly from natural microbial populations, are now available which
allow a more thorough examination of these associated bacterial populations.
This study sought to characterize the epibiotic bacterial population associated
with a very unique organism, Alvinella pompejana, using such a molecular
approach.
Alvinella pompejana is a polychaetous annelid that inhabits active
deep-sea hydrothermal vent sites along the East Pacific Rise. This worm
colonizes the walls of actively venting high temperature chimneys and is
thought to be one of the most thermotolerant metazoans known. The
chimney environment is characterized by high concentrations of sulfide and
heavy metals in the vicinity of the worm colonies. A morphologically
diverse epibiotic microflora is associated with the worm's dorsal integument,
with a highly integrated filamentous morphotype clearly dominating the
microbial biomass. It has been suggested that this bacterial population
participates in either the nutrition of the worm or in detoxification of the
worm's immediate environment; however, previous studies have been
unable to confirm such a role. The primary goal of this study is to
phylogenetically characterize the dominant epibionts through the analysis of
16S rRNA gene sequences.
Nucleic acids were extracted from bacteria collected from the dorsal
surface of Alvinella pompejana. 16S rRNA genes were amplified with
universal bacterial primers by the polymerase chain reaction (PCR). These
genes were subsequently cloned and the resulting clone library was screened
by restriction fragment length polymorphism (RFLP) analysis to identify
unique clone types. Thirty-two distinct clone families were found in the
library. Four of these families were clearly dominant, representing over 65%
of the library. The main assumption in this study is that the numerical
dominance of the phylotypes in the starting population will be reflected in
the clone library. Thus, representative clones from the four most abundant
clone families were chosen for complete gene sequencing and phylogenetic
analysis. These gene sequences were analyzed using a variety of phylogenetic
inference methods and were found to be related to the newly established
epsilon subdivision of the Proteobacteria. In future studies, these gene
sequences will be used to construct specific oligodeoxynucleotide probes
which can be used to confirm the morphology of the clone types in the
epibiont population.
Phylogenetic Characterization of the Epibiotic Bacteria Associated with the
Hydrothermal Vent Polychaete Alvinella pompejana
by
Michael A. Haddad
A THESIS
submitted to
Oregon State University
in partial fulfillment of
the requirements for the
degree of
Master of Science
Completed August 18, 1994
Commencement June 1995
Master of Science thesis of Michael A. Haddad presented on August 18, 1994
APPROVED:
Redacted for Privacy
Major Prof ssor, re pr enting Microbiology
Redacted for Privacy
Chair
Department
icrobiology
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
Michael A. Haddad, Author
ACKNOWLEDGEMENTS
I would like to first acknowledge the Molecular and Cellular Biology
program here at Oregon State University for providing the financial support
for my first year of study. I also would like to thank everyone in my lab for
their encouragement and support. I am especially grateful to both Craig Cary
and Steve Giovannoni for giving me the opportunity to work on such an
exciting project and the guidance to help me see it through to completion.
There are so many more people whom I could acknowledge here, the
most important of which is my family. I have been extremely lucky to have
such a supportive and generous group of people as my main support system.
First my mother, Niki Ross, has been fundamental to the development of
myself as a human being. If there is one person whom I have learned the
most from about life it is her. My step-father, Bill Ross, has always
encouraged me about everything I have ever done (except maybe the
mohawk). My father, Sam Haddad, has perhaps given the gift I will treasure
the most, an education. He has financially supported my education with out
ever setting any limits. As a result, he has allowed me to pursue whatever I
have wanted. That is probably one of the most important freedoms to ever
have.
Finally, I am thankful for the tremendous amount of support I have
had from my friends: Bill Idel, Greta Wade, Becky Bierenbaum, and Jacob
Benjamin Perkinson. Without question, the most important person I have
to thank is my partner, Laura MacGregor. She is the one who has dealt with
all of my trials and tribulations first-hand, and has always been there for me.
For that enormous amount of support I am eternally grateful.
TABLE OF CONTENTS
INTRODUCTION
1
Ecology of Alvinella pompejana
The Epibiotic Microflora of Alvinella pompejana
Possible Role of the Epibionts
3
Conclusions of Previous Research
13
Use of the 16S rRNA Molecule
Experimental Rationale
14
15
METHODS
Sample Collection
6
11
18
18
Bacterial Removal and DNA Purification.
18
Amplification of 16S rRNA Genes
19
Construction and Screening of Clone Library.
RFLP Analysis.
Gene Sequencing
Phylogenetic Analysis
20
RESULTS
Clone Library Construction
Restriction Fragment Length Polymorphism Analysis
16S rRNA Gene Sequences
Phylogenetic Analysis
21
22
23
25
25
25
26
29
DISCUSSION
39
BIBLIOGRAPHY
45
APPENDICES
52
LIST OF FIGURES
Figure
1.
2.
3.
4.
5.
6.
7.
Page
Map showing active hydrothermal vent sites along the
East Pacific Rise (EPR)
5
Dorsal view of the holotype of (a) Alvinella pompejana
and (b) Alvinella caudata
8
Schematic outline of the experimental strategy used for the
phylogenetic analyses of the epibiotic microbial
community associated with the hydrothermal vent
polychaete Alvinella pompejana
17
Scaled Illustration of the restriction patterns of 16S rDNAs
representing 11 clone families identified in the alvinellid clone
library.
27
Pylogenetic tree showing the relationship of the alvinellid
clones to the other members of the Proteobacteria.
30
Proposed secondary structural model of clone APG 13B 16S
rDNA
33, 34
Proposed secondary structural model of clone APG 56B 16S
rDNA.
35, 36
LIST OF TABLES
Table
1.
2.
3.
Page
Chemical composition of vent end-member fluid @ 21° N
on the East Pacific Rise vs. ambient sea water
7
RFLP analysis results showing the numberical distribution of the
alvinellid clone families.
28
Signature base positions distinguishing the delta and epsilon
subclasses of Pro teobacteria and the alvinellid clones.
38
LIST OF APPENDICES
Page
Appendix 1: Locus APG 5A
1496 by DNA
52
Appendix 2: Locus APG 13b
1502 by rRNA
54
Appendix 3: Locus APG 44b
1502 by DNA
56
Appendix 4: Locus APG 56B
1504 by DNA
58
Phylogenetic Characterization of the Epibiotic Bacteria Associated with the
Hydrothermal Vent Polychaete Alvinella pornpejana
INTRODUCTION
Integrated bacterial associations with animals are widely distributed in
both terrestrial and marine systems. Although many of these relationships
appear obligatory, very little is known about the functional role of the
bacteria. As a result, whether these bacteria reside intracellularly
(endosymbiotic) or extracellularly (episymbiotic or epibiotic), many of these
affiliations can only roughly be defined as symbiotic. It has been impossible to
determine whether they are mutualistic, where both members are benefited
by the relationship, or merely commensal, where only a single member
profits.
Epibiotic associations of bacteria with metazoans are common in the
marine environment. Many of the affiliations are monospecific and
invariant in nature, often involving a single bacterial species with a specific
host group. Bioluminescent bacteria colonizing the light organ of the
sepiolid squid Euprymna scolopes comprise a single strain of Vibrio fischeri
(McFall-Ngai et al., 1991) However, more frequently these epibiotic
assemblages are composed of a phenotypically diverse group of
microorganisms, which further complicates resolving the contribution of
each individual member with its host. Several distinct morphological types
of bacteria are found colonizing the gills of the rock crab Cancer irroratus and
are believed to play an important role in the pollution of these organisms
(Bodammer et al., 1981). Similarly, periphytic bacteria are associated with
another crab, He lice crassa, found at tannery effluent sites, and may contribute
2
to the concentration of chromium in these environments (Johnson et al.,
1981). An unnamed limpet species inhabiting deep-sea hydrothermal vent
sites is also found with dense colonies of filamentous bacteria colonizing the
gill epithelial surface (de Burgh et al., 1984).
Probably one of the more dramatic examples of invertebrate/bacteria
associations are those found in certain sponges. In the majority of the large
marine sponges, dense populations of bacteria reside in the intercellular
spaces, comprising up to one-third of the volume of the sponge. These
bacterial populations are typically composed of between 4-7 different
morphological types (Smith et al, 1987). Although there have been several
attempts to isolate these bacteria free from their hosts, few have been
confirmed to be the symbionts. In these and other similar associations the
task of distinguishing and characterizing the important members in these
epibiotic associations from those opportunistic bacteria, has remained, until
recently, intractable.
One of the more remarkable members of deep-sea hydrothermal vent
communities is the polychaetous annelid, Alvinella pompejana, found
colonizing the walls of actively venting chimneys (Desbruyeres et al., 1980). A
highly diverse and dense assemblage of microorganisms, including an
integrated filamentous morphotype, occupies the dorsal surface of this worm
(Desbruyeres et al., 1985; Gaill et al., 1987). To date, this filamentous
morphotype has eluded all attempts at culturing (Durand et al., 1990;
Jeanthon et al., 1992; Prieur, et al. 1990). Several studies have suggested that
the bacteria participate in the nutrition of the host or in the detoxification of
the host's immediate environment (Alayse-Danet et al., 1987; Gaill et al.,
1988). However, due to the inherent difficulty in working with A.
pornpejazza, which rarely survives trips to the surface, the apparent failure to
3
culture the dominant morphotypes, these hypotheses have remained merely
suggestive.
Molecular genetic strategies now exist for the characterization of
microorganisms which have previously not been observed in culture. The
analysis of small subunit ribosomal RNA sequences (16S rRNA) cloned
directly from natural populations has expanded the limits of microbial
ecology and diversity over that which was possible with more traditional
microbiological techniques. In other studies, 16S rRNA sequence analysis has
provided a powerful means to examine the diversity of natural microbial
populations in marine and fresh water systems avoiding the reliance on
cultavatibility. These same techniques can be used to phylogenetically
characterize the microflora associated with Alvinella pompejana.
Characterizing the dominant members of the microflora would provide
essential information necessary to determine the role of these bacteria. These
same techniques can also be applied to other epibiotic microbial associations,
which will no doubt add to our limited knowledge about these other
associations.
Ecology of Alvinella pompejana
Alvinella pompejana is a tube-dwelling, polychaetous annelid that
colonizes actively venting chimneys at deep-sea hydrothermal vent sites
(Desbruyeres & Laubier 1980). A. pompejana, as well as its congener A.
caudata, are placed within their own family, the Alvinellidae (Desbruyeres et
al., 1986), along with another genus Paralvinella (Desbruyeres et al., 1985). All
members of the family appear to be restricted solely to deep-sea hydrothermal
vent sites (Desbruyeres et al., 1991). The Alvinellidae range in distribution
4
from ITS to 50°N along the East Pacific Rise (EPR) in the eastern Pacific
Ocean (Figure 1), with Alvinella spp. being restricted to the high temperature
vent sites from 17°S-21°N (Desbruyeres & Laubier, 1991; Tunnicliffe et al.,
1993). Alvinella spp. form dense colonies along the walls of actively venting
chimney-like mineral structures. These worms build very stable tubes
composed of both inorganic and organic material which are cemented to the
chimney walls (Gaill et al., 1986; Vovelle et al., 1986). It has been suggested
that these tubes assist in the stabilization of venting chimney structures by
providing additional structural substrate for the further deposition of mineral
precipitates (Tunnicliffe et al., 1990).
The immediate environment of Alvinella pompejana is one of the
most extreme environments on the planet. A. pompejana is one of the few
invertebrate species present at vent sites to live in close proximity to the hot
hydrothermal vent effluent. These worms are found colonizing basal
mounds, zinc-sulfide and black smoker chimneys where the core effluent
temperature ranges from 80°C-350°C, depending on the chimney type
(Desbruyeres & Laubier, 1991). The mixing zone of the hot hydrothermal
fluid with the cold ambient seawater produces a sharp temperature gradient,
ranging from 350°C - 1.8°C within a few centimeters (Desbruyeres et al., 1985).
In one study, temperature readings within an alvinellid colony were 20-35°C
at the tube opening, 100°C 10 cm inside the tube mass, and 249°C 20 cm inside
the tube mass (Desbruyeres et al., 1982). Recently, an actively swimming
specimen of A. pompejana was observed resting on the submersible's
temperature probe, which simultaneously registered a temperature of 105°C.
This observation lead the authors to suggest that A. pompejana may hold the
record for the upper
12'
49
N
12'
48
Figure 1. Map showing active hydrothermal vent sites along the East Pacific Rise (EPR). (a) The Alvinellidae range in
distribution from 17°S to 50'N along the EPR, with Alvinella spp. being restricted to the high temperature vent sites from
17°S-21°N. (b) Alvinella pompejana specimens used in this study were collected from the Elsa vent field at the 13°N site.
This figure was adapted from: Jollivet, D. 1993. Distribution et evolution de la faune associee aux sources
hydrothermales profondes a 13°N sur la dorsale du pacifique oriental: le cas particulier des polychetes alvinellidae. These
de doctorat de L'Universite de Bretagne Occidentale. IFREMER, Centre de Brest.
6
temperature limit of any eukaryote (Chevaldonne et al., 1992). Although
additional data are required to confirm this claim, it is believed that these
organisms exist at environmental temperatures between 20°C and 60°C
(Desbruyeres & Laubier, 1991).
Another consequence of colonizing active chimneys at deep-sea
hydrothermal vent sites is the direct exposure to high concentrations of
heavy metal compounds and hydrogen sulfide contained in the vent effluent.
Elevated concentrations of these compounds are well known to inhibit the
growth and essential biochemical activities of microorganisms and
eukaryotes (Collins et al., 1989; N.R.C., 1979). Table 1 shows concentrations of
some of these compounds at a site at 21'N on the East Pacific Rise in
comparison to average seawater. As is evident from this table, the vent water
is acidic and enriched in hydrogen sulfide and biologically toxic heavy metals,
such as arsenic, lead, and silver. Due to the proximity of the worm's tubes to
the vent effluent, A. pompejana and its associated microflora are constantly
exposed to these toxic compounds (Desbruyeres et al., 1985; Desbruyeres &
Laubier, 1991).
The Epibiotic Microflora of Alvinella pompejana
A fascinating feature of both A. pompejana and A. caudata is the
presence of an epibiotic population of bacteria associated with the worms'
dorsal surface (Figure 2). These alvinellid species differ somewhat in the
structural features that link the epibionts to their dorsal surface. A.
pompejana posses modified dorsal digiform expansions heavily colonized by
filamentous bacteria.
Similar filamentous bacterial morphotypes are linked
to modified posterior parapodia in A. caudata (Desbruyeres & Laubier, 1991).
7
Table la. Chemical composition of vent end-member fluid at 21°N on the
East Pacific Rise vs. ambient sea water.
21 °N
Ambient sea water
Li (gM)
891
Na (mM)
K (mM)
432
23.2
26
464
9.79
Rb (p.M)
28
1.3
Be (nM)
Mg (mM)
15
0.02
0
52.7
Ca (I_LM)
15.6
10.2
Sr (g.M)
81
Ba (11M)
pH
>7
3.4
87
0.14
Cl (mM)
Si (mM)
489
17.6
0.16
Al (11M)
5.2
0.01
SO4 (mM)
H2S (mM)
0
27.9
0
NH3 (mm)
Mn (gM)
7.3
0
960
<0.001
Fe (i.i.M)
1664
<0.001
Co (nM)
213
0.03
Cu (p.M)
35
106
0.007
38
0.02
155
1.0
308
247
0.01
Zn (tiM)
Ag (nM)
Cd (nM)
Pb (nM)
As (nM)
Se (nM)
72
7.8
541
0
0.01
27
2.5
a Table was taken from Edmond, J. M. and K. L. Von Damm. 1985. Chemistry
of ridge crest hot springs. In: M. L. Jones (Ed.), The hydrothermal vents of the
eastern Pacific: an overview. Bull. Biol. Sci. Wash. 6:43-47.
Figure 2. Dorsal view of the holotype of (a)Alvinella pompejana and (b) Alvinella caudata. Arrows indicate approximate
positionS of bacterial sampling for this study. This figure was adapted from: Desbruyeres, D. and L. Laubier. 1980.
Alvinella pompejana gen. sp. nov., ampharetidae aberrant des sources hydrothermales de la ride Est-Pacifique. Oceanol.
Acta 3: 267-274.
co
9
In both species the bacteria appear as a gray mat covering the majority of the
worms' surfaces and are also found lining the interior of the worms' tube
(Desbruyeres et al., 1985; Gaill & Hunt, 1986). The density of the bacterial
population seems to increase posteriorly, presumably responding to some
environmental gradient (Desbruyeres et al., 1980; Gaill et al., 1987). For the
purposes of this discussion, only the details of the epibiotic microbial
population associated with A. pompejana will be further emphasized.
The epibiotic microbial population associated with Alvinella
pompejana is morphologically diverse. Electron microscopic observations
have documented many morphotypes, including rod, coccoid, spiral, and
filamentous bacteria (Gaill et al., 1987). Although it is impossible to accurately
identify bacterial species based solely on morphology, these studies have
served to document several interesting patterns of the distribution of these
morphotypes in the population. These morphological forms have been
found in several different distributions on the host, ranging from rod-shaped
bacteria found randomly dispersed, to filamentous bacteria found colonizing
specific locations on the worm (Desbruyeres et al., 1985; Gaill et al., 1987).
These filamentous morphotypes dominate the population representing over
60% of the microbial biomass (Gaill et al., 1987). Filamentous bacteria are
found joined to small expansions of the worm's epidermis within the
intersegmentary spaces (Gaill et al., 1987). These expansions are
10 tm in
length and appear composed of glandular cells (Desbruyeres et al., 1985). The
filamentous bacteria are embedded in a polysaccharide secretion from the
worm (Gaill et al., 1988), and have been shown to be in an active state of
growth (Alayse-Danet et al., 1986). The appearance of this specific association
and abundance of the morphological form in the population has lead others
10
to suggest a specific relationship between these filamentous bacteria and A.
pompejana (Gain et al., 1988).
The high diversity of the epibiotic microbial community associated
with Alvinella pompejana is apparent not only from the morphology, but
also in the wide range of metabolic capabilities detected in the community.
Several attempts have been made at culturing the epibiotic bacteria free from
A. pompejana. A preliminary study of the heterotrophic bacteria present in
this community yielded 62 total isolates. All of the isolates were Gram-
negative and rod-shaped in their morphology (Prieur et al., 1987). In later
studies additional heterotrophs and several autotrophic bacteria were
successfully isolated. The majority of these new isolates had resistance to one
or more selected heavy metals, such as silver, copper, cadmium, zinc, and
arsenic (Jeanthon et al., 1990; Prieur et al., 1990). Numerous sulfur-oxidizing
and sulfur-reducing isolates were also obtained, as well as several isolates
involved in the nitrogen cycle, and in the oxidation of manganese (Durand et
al., 1990). Growth of these isolates was observed at temperatures ranging
from 20°C to 80°C, at both atmospheric and in situ pressure (250 atm).
Collectively, these studies complement early microscopy findings by
demonstrating the microflora of A. pompejana is both morphologically and
metabolically diverse. In all of these studies the dominant filamentous
morphotype was never confirmed to be in culture, thus illustrating the
limitations of these classical approaches.
11
Possible Role of the Epibionts
Several studies have speculated on the role of the epibiotic microbial
community in the survival of A. pompejana. The primary hypothesis has
been that the microflora in some way participates in the nutrition of the
alvinellid, either by the worm directly feeding on the bacteria, or by a
translocation of specific metabolites from the bacteria to the worm (Gaill et al.,
1988). A. pompejana has a complete digestive system similar to other
ampharetids (Saulinier-Michel et al., 1990). Analysis of both the mouth
structures and lumenal contents reveal the presence of bacterial cells
(Desbruyeres et al., 1983). This suggests that direct feeding on bacteria does
occur, but its significance to the overall nutrition of the host remains
unknown.
Comparison of 513C values of A. pompejana and Rifitia pachyptila, a
gutless, vent invertebrate that harbors endosymbiotic chemoautotrophic
bacteria, suggests that these two organisms derive their organic carbon from a
similar source (Desbruyeres et al., 1983). The nutrition of Riftia pachyptila is
provided entirely by the resident chemoautotrophic endosymbionts
(Cavanaugh et al., 1981; Felbeck, 1981). At this time it is unknown whether
the endosymbionts provide nutriment via some translocation product or are
merely selectively digested by the host. Examinations of the worm's anatomy
reveals disorganized collagenous fibers underlying the worm's cuticle at the
intersegmentary spaces (Gaill et al., 1987) as well as highly vascularized
underlying epithelium (Desbruyeres et al., 1985). Some of the rod-shaped
bacteria present in the population have thin filamentous structures which
attach to the worm's cuticle (Gaill et al., 1987). These observations have led
12
the authors to suggest the potential for the passage of metabolites from the
bacteria to the worm (Alayse-Danet et al., 1987; Gain et al., 1988).
A second hypothesis for the role of the epibionts is in the detoxification
of the immediate environment of Alvinella pompejana (Alayse-Danetet al.,
1987; Gain et al., 1988). As stated previously, this alvinellid lives in an
environment with high concentrations of biologically toxic heavy metals and
hydrogen sulfide. Many bacteria are known to possess strategies for toxic
heavy metal resistance, such as sequestering metals in the cell wall or in
intracelluar compartments, enzymatic modification of these metals to less
toxic forms, or by rapidly expelling these metals from the cytoplasm (Silver et
al., 1989). It is thought the epibiotic microflora of A. pompejana could
participate in the detoxification the worms' environment through the
concentration of toxic heavy metals. A similar function for the epibiotic
bacteria of the crab He lice crassa has also been proposed (Johnson et al., 1981).
These bacteria colonize the carapace and gills of the crab and participate in the
concentration of chromium.
Hydrogen sulfide, at concentrations less that one-thousandth that
found in vent habitats, is highly toxic to almost all living systems. Sulfide
poisons certain respiratory proteins, such as cytochrome c oxidase (N.R.C.,
1979), involved in the use of molecular oxygen in aerobic respiration. Several
novel mechanisms exist in certain invertebrates to cope with the elevated
sulfide levels in the vent systems. Riftia pachyptila possesses a sulfide
binding protein in the blood which tightly binds free sulfide thus preventing
access to sulfide sensitive cellular constituents (Arp et al., 1983; Powell et al.,
1983). Marine organisms that are routinely exposed to toxic levels of locally
produced sulfide often possess high activities of sulfide oxidizing systems
(Somero et al., 1983). Although the blood of A. pompejana possesses
13
extremely high binding efficiencies for oxygen in order to deal with the
elevated temperatures and hypoxic conditions (Toulmond et al., 1990), no
specific sulfide binding capability has been reported. It is conceivable that the
microbial community associated with A. pompejana oxidizes available
sulfide within the tube, thereby detoxifying the immediate environment of
the worm.
Conclusions of Previous Research
The majority of epibiotic associations of bacteria with metazoans are
poorly understood. Deciphering the functional role of the bacteria in these
relationships is often confounded by the complexity of the associated bacterial
communities. Nowhere is this problem more evident than with the epibiotic
bacterial community associated with the deep-sea hydrothermal vent
polychaete A. pompejana. This epibiotic microflora has been shown to be
both morphologically and metabolically diverse; however, the exact
relationships between the bacteria and the polychaete have yet to be
definitively established. It has been suggested that the bacteria participate in
the nutrition of the worm, and/or in the detoxification of the worms'
environment. Previous experiments have been unable to prove either of
these hypotheses. One of the main difficulties in researching such a
relationship is the viability of the microorganisms in culture. The
filamentous bacterial form, which has been suggested to play an important
symbiotic role, has not been observed in previous culture attempts. It is
conceivable that these bacteria are pleiomorphic and might already be in
previously established culture collections, however the reliance on
morphology for the characterization of this population is a major weakness
14
in these studies. Even if the pleiomorph of the filamentous bacteria did
appear in culture, it would be difficult to identify its spatial distribution in the
worm's environment. In order to circumvent these difficulties and begin to
understand the phylogenetic diversity of the alvinellid epibiont community,
an experimental strategy developed for the analysis of mixed populations of
free-living microorganisms was chosen. This approach is based the
philosophy that, like organelles, microorganisms can be studied without
cultivation. The approached used in this study is based on the sequence and
phylogenetic analysis of bacterial 16S rRNA genes cloned directly from
natural microbial communities.
Use of the 16S rRNA Molecule
Nucleic acid sequence comparisons have provided an invaluable
perspective on the evolutionary relationships of microorganisms. Microbial
systematics had previously relied on certain phenotypic characters of
microorganisms, which were later found to be poor phylogenetic indicators.
The most utilized molecule for nucleic acid sequence comparisons among
microorganisms is the small subunit ribosomal RNA (16S rRNA). The 16S
rRNA molecule is functionally and evolutionarily homologous among all
organisms, due to its essential role in protein synthesis, and is not laterally
transferred between microorganisms (Olsen et al., 1986). Thus, the
phylogenetic relationships between the 16S rRNAs are reflective of the
evolutionary relationships of the organisms. The sequence of the 16S rRNA
molecule, or its gene, are mosaics of both highly conserved and variable
regions, which extends the utility of this molecule to a wide phylogenetic
range (Woese, 1987).
15
The utility of 16S rRNA sequence analysis has extended into the study
of the ecology and diversity of natural populations of microorganisms. By
linking the sequence analysis approach with current cloning technologies, a
more complete view of the structure and composition of microbial
communities is beginning to emerge. Novel 16S rRNA genes from
bacterioplankton samples collected from the Sargasso Sea do not resemble
genes from known bacterial species (Britschgi et al., 1991; Giovannoni et al.,
1990). In addition, unique 16S rRNA genes belonging to the domain Archaea
have recently been detected in the Pacific Ocean (Fuhrman et al., 1992). These
analyses have also been extended to the analysis of microorganisms associated
with marine snow (De Long et al., 1993) and to the examination of the
endosymbiotic bacteria associated with the gutless bivalve Solemya reidi
(Cary, 1994). These molecular techniques are ideally suited to analyze the
microbial community associated with Alvinella pompejazza.
Experimental Rationale
The primary goal of this study is to characterize the evolutionary
relationships of the predominant bacteria present in the epibiotic population
of microorganisms associated with Alvinella pornpejana. The dominant
member(s) of this population are filamentous bacteria. Due to the abundance
of these bacteria in the population and the intimate association with A.
pornpejana (see previous section), this bacterial form is believed to play an
important symbiotic role. Therefore, its further characterization may be
important in defining the nature of this symbiosis.
In this study, cloning and sequencing methodologies were matched to
genetically dissect the bacterial community associated with Alvinella
16
pompejana (Figure 3). A specimen of A. pompejana was collected from an
active hydrothermal vent site at 13'N on the EPR and was preserved frozen
to maintain the integrity of the nucleic acids. A small patch of bacteria was
removed from the dorsal integument of the animal. DNA was purified from
this sample. The 16S rRNA genes were amplified using the polymerase chain
reaction (PCR) with general bacterial primers. These primers are known to
hybridize to the 5' and 3' ends of the 16S rRNA genes of all known bacteria.
Thus, this provides a representation of the complete bacterial 16S rRNA
genes present in an environmental DNA sample. These primers have
proven successful in amplifying diverse assemblages of bacterial 16S rRNA
genes from natural populations. The amplified 16S rRNA genes were
subsequently cloned into a plasmid vector to separate and propagate the
amplified genes. In order to identify unique phylotypes and determine their
numerical distribution in the library, each cloned 16S rRNA gene was
screened using restriction fragment length polymorphism (RFLP) analysis.
Assuming the numerical dominance of the phylotypes in the library is
reflective of the actual distribution in the natural community, representatives
from the four most dominant clone types were chosen for complete gene
sequencing and phylogenetic analysis.
17
EXPERIMENTAL APPROACH
Alvinella pompejana
Isolate bacteria
Extract and purify DNA
f
Amplify 16S rRNA genes
Construct clone library
Group clones using RFLP analysis
Sequence and phylogenetically analyze representatives of the
numerically dominant clone types
Figure 3. Schematic outline of the experimental strategy used for the phylogenetic
analysis of the epibiotic microbial community associated with the hydrothermal vent
polychaete Alvinella pompejana.
18
METHODS
Sample Collection.
Specimens of Alvinella pompejana were collected in October 1991
during the joint French-US HERO (Hydrothermal Environment Research
Observatory) expeditions at the Elsa vent site 13°N (2620m) on the East Pacific
Rise (12°48'N, 103°56'W). Individuals were collected from worm colonies
along the walls of active chimneys using the mechanical arm of the Deep Sea
Research Vessel Alvin. The specimens were kept cold (<4°C) in a thermally
insulated container until surfacing. Once on board, the animals were
immediately frozen in liquid nitrogen and kept at -80°C until needed.
Bacterial Removal and DNA Purification.
A large patch of bacteria, including a portion of worm integument, was
removed from a frozen A. pompejana individual using sterile forceps. The
bacterial sample was completely homogenized in 5M guanidinium
isothiocyanate, 50mM Tris (pH 7.4), 25mM EDTA, 0.8% 2-mercaptoethanol,
using a Dounce homogenizer. The homogenate volume was doubled with
50mM Tris-Cl (pH 8.0), 25mM EDTA, and centrifuged at 10,000 rpm in a
tabletop centrifuge for 15 minutes at room temperature to remove cellular
debris. Nucleic acids were precipitated with 0.8 volumes of 100% isopropanol
at room temperature for 1 hour. The precipitate was collected by
centrifugation at maximum speed in a tabletop centrifuge for 30 minutes,
washed with ice cold 70% ethanol, resuspended in TE buffer (10mM Tris -Cl,
1mM EDTA pH 8.0). The extracted bulk nucleic acids were digested with
proteinase K (a final concentration of 500µg /ml at 50°C for 1 hour). The
19
sample was then extracted twice with an equal volume of phenol/chloroform
(4:1) and once with an equal volume of chloroform/isoamyl alcohol (24:1).
For each extraction the phases were allowed to completely separate and the
aqueous layer was retained. Nucleic acids were precipitated overnight with
0.1 volumes of 3M sodium acetate (pH 5.2) and 2 volumes of cold 100%
ethanol at -20°C. The precipitate was collected following centrifugation as
above, washed with 70% ethanol, and resuspended in TE.
To remove contaminating RNA's the sample was treated with RNase
(33 ng/1_11) for 10 minutes at 65°C, and phenol purified as above. The integrity
of the DNA sample was determined by agarose gel electrophoresis through a
0.8% agarose gel in 1X TAE (40mM Tris-acetate, 1mM EDTA). The final DNA
concentration was determined spectrophotometrically.
Amplification of 16S rRNA Genes.
Bacterial 16S rRNA genes were amplified from the environmental
sample using the polymerase chain reaction (PCR) using two general bacterial
16S rRNA primers: EubB (5'-AGA GUU UGA UCM UGG CUC AG-3') and
EubA (5'-ACR CCN ACC TAG TGG AGG AA-3'); (Giovannoni, 1991). PCR
amplifications were performed on a Coy thermal cycler (Coy Corporation,
Ann Arbor, MI). The reaction conditions were: 50mM KC1, 10mM Tris-HC1
(pH 8.4); 2.5mM MgC12; 0.2mM each dATP, dCTP, dGTP, dTTP; 0.21.1M of each
amplification primer; 1Ong of purified template DNA; 2.5U Taq polymerase
(Promega), in a total volume of 1004 Amplification profile conditions were
95°C for 1 min., 55°C for 1 min., and 72°C for 3 min. for 30 cycles. The
resulting PCR products were ethanol precipitated and analyzed by gel
electrophoresis as described above.
20
Construction and Screening of Clone Library.
Two separate clone libraries were constructed from a single A.
pompejana specimen. The amplified genes were cloned using a plasmid
vector (pCRII, Invitrogen) specifically designed to accommodate PCR products
that contain single non-template dependent additions of deoxyadenosines at
the 3' ends of the DNA duplexes. The only significant difference between the
two libraries was that in the first attempt the host cells were less competent
and resulted in a low transformation efficiency.
The clone libraries were constructed using the TA Cloning System
version 1.3 (Invitrogen) following the manufacturer's instructions, with the
following modifications. The ligation reaction was performed with PCR
products obtained from a reaction with a reduced number of cycles (30) and
that had been ethanol precipitated prior to ligation. The transformants were
plated onto LB agar plates (10g tryptone, lOg NaC1, 5g yeast extract, 15g bacto­
agar/L, pH 7.0) containing the antibiotic kanamycin (50µg /ml) with 25111 of X-
Gal (40mg/m1) spread evenly across the agar plates. White colonies were
scored and picked from all available plates and confirmed by restreaking. Stab
cultures of positive clones were made in LB agar for long-term storage.
Plasmid DNA was prepared from each of the confirmed transformants
using an alkaline lysis preparation protocol (Sambrook et al., 1989).
Confirmed white colonies were grown in 5m1 of LB media containing
kanamycin (50ug/m1) overnight at 37°C with vigorous shaking. Two
milliliters of this culture was used in the plasmid preparation. The purified
plasmids were resolved on a 1% agarose gel in IX TAE and visualized with
ethidium bromide staining. A plasmid known to contain the correct size
insert and one lacking an insert (blue) were used as size standards.
21
Transformant plasmid DNAs which co-migrated with the 1.5 kb-insert
control were scored as having a full-length 16S rRNA gene.
RFLP Analysis.
In order to sort the clones into similar clone types, clones containing
full-length inserts were subjected to RFLP analysis. The cloned insert was re-
amplified using the same PCR conditions and the primers used previously to
establish the library. This amplification step was required to generate a
sufficient amount of isolated product for the restriction analysis. 1111 of a 1:10
dilution of the each of the alkaline lysis plasmid DNA preparations was used
as the starting template.
Each PCR product was digested using two restriction endonucleases,
Hae III and Mbo I (Promega), each of which recognize a four-base pair
restriction site. 5111 of each PCR reaction was digested with 2.5U of each
enzyme in 0.5X restriction buffer (5mM Tris-C1, 5mM MgC12, 25mM NaC1,
0.5mM DTT pH 7.9) at 37°C for 2 hours. The final reaction volume was 10 111.
The reaction was stopped by the addition of 0.5p1 of 500mM EDTA (pH 8.0).
The entire reaction volume was electrophoresed through a 3% Nu Sieve
agarose gel (FMC) in IX TAE using a standard 6X loading buffer (Sambrook et
al., 1989). Nu Sieve agarose was used in these analyses because it has been
specifically developed for resolving DNA products less than 1000 bp. A third
tracking dye, orange G, was added to the mixture (0.35% w/v final
concentration) to aid in the running consistency between gels.
22
Gene Sequencing
Representative clones from each of the four numerically dominant
clone families resolved by the RFLP analyses were bidirectionally sequenced
using a combination of both automated and manual plasmid sequencing.
Automated sequencing was performed on an ABI model 373A automated
gene sequencer at the Center for Gene Research and Biotechnology (Oregon
State University).
Manual sequencing was performed by standard dideoxynucleotide­
terminated sequencing protocols using a Sequenase v2.0 DNA sequencing kit
(U.S. Biochemical Corp.) Sequencing reactions were performed following the
supplier's instructions. Template plasmid DNA was prepared using Magic
Mini plasmid purification kits (Promega). Two fig of purified DNA were
denatured for each sequencing reaction. Template was denatured as follows:
2 lig of plasmid DNA were brought to a final concentration of 0.18M
Na0H/0.18mM EDTA and incubated for 5 min. at room temperature. The
DNA was precipitated with 0.1 volumes of 3M sodium acetate and 2 volumes
of ice cold 100% ethanol for 30 min. at -70°C. The precipitate was collected by
centrifugation at 20,000 rpm at 4°C for 30 min. in a Beckman TL-100
refrigerated tabletop ultracentrifuge. The pellet was washed in ice cold 70%
ethanol and centrifuged. The pellet was allowed to air dry and was stored at
-70°C until used for sequencing. Sequencing reactions were resolved by
electrophoresis through a 7% polyacrylamide gel using Long-Ranger
acrylamide (AT Biochem).
23
Phylogenetic Analysis
The alvinellid clone 16S rDNA sequences were manually aligned to a
subset of other aligned bacterial 16S rRNA sequences obtained from the
Ribosomal Database Project (RDP) (Larsen et al., 1993). In addition, the 16S
rRNA sequence for Desulfurella ncetivorans (EMBL accession number
X72768) was aligned and included in the analysis. The conserved regions and
established secondary structural models of the 16S rRNA sequence were used
as guides to insure a correct alignment of the homologous regions of the
sequences. Sequence data were manipulated using the Genetic Data
Environment (GDE) v2.0 sequence analysis software package (Smith et al.,
1992). Regions of ambiguous alignment i.e. positions 1-11, 69-102, 128-132,
176-226, 265-269, 451-480, 840-869, 999-1045, 1124-1185, 1262-1298, 1357-3'
terminus (using the E. co/i numbering system) were excluded from the
analysis. A total of 1061 nucleotide positions were used in the analysis.
Phylogenetic trees were edited using the Treetools program (provided by
Mike Maciukenas from the University of Illinois at Urbana-Champaign for
the RDP) Programs used to infer phylogenetic trees are contained in the
Phylip v3.5c software package (Felsenstein, 1993). DNADIST was used to
calculate evolutionary distances with the Kimura 2-parameter model for
nucleotide change and a transition/transversion ratio of 2.0 (Kimura, 1980).
Phylogenetic trees were reconstructed from evolutionary distance data using
the neighbor-joining method (Saitou et al., 1987), implemented through the
program NEIGHBOR. Parsimony trees were reconstructed using DNAPARS.
A total of 100 bootstrapped replicate resampling data sets for both DNADIST
and DNAPARS were generated using SEQBOOT, with random sequence
addition and global rearrangement. Secondary structural models for the
24
cloned 16S rDNA sequences were obtained with the gRNAID v1.4 program
(provided by Shannon Whitmore) and optimized by comparison with other
established 16S rRNA secondary structures (Gutell, 1993).
25
RESULTS
Clone Library Construction
DNA was isolated from a sample of the epibiotic microflora obtained
from an A. pompejana individual and the bacterial 16S rRNA genes were
amplified from this preparation. Two separate attempts were made at cloning
these amplified genes. Both attempts used the TA Cloning System v1.3
(Invitrogen); however the first attempt utilized a version in which the
competency of the cells was significantly lower. In the earlier attempt a total
of 40 white transformants were obtained and all were screened for correct
sized (1.5kb) inserts. Only 4 transformants was found to contain full-length
inserts. The second attempt using more competent host cells resulted in 249
total white transformants. A total of 162 white transformants was screened
and 135 of these contained the correct size inserts. Clones containing full-
length inserts from both attempts were grouped into one library resulting in a
final total of 139 clones. Clones from the first attempt are designated "APG
#A", while those from the second library are designated "APG #B".
Restriction Fragment Length Polymorphism Analysis
In order to identify unique clone types, clones confirmed to contain a
full-length insert were subjected to RFLP analysis. The insert was first
amplified from purified plasmid DNA and the resulting amplification
product was digested with two restriction endonucleases, Hae III and Mbo I.
The clones were then grouped into "families" based on their restriction
pattern; that is, clones with identical restriction patterns were placed into the
26
same family. The restriction analysis of the 139 full-length clones identified
32 distinct clone families. Figure 4 is a schematic representation of the
restriction patterns of 11 representative clone families. This figure illustrates
some of the diversity of restriction patterns seen in the library.
Table 2 shows the numerical distribution of each of the clone families.
Four of these families, designated Fl, F2, F9, and F13, dominated the library,
representing 16.4% (23 clones), 43.2% (60 clones), 5.8% (8 clones), and 4.3% (6
clones) of the library, respectively. The remaining families were represented
by fewer members, with the majority of the families containing only one
member. Based on the assumption that the numerical dominance of the
bacterial species will be reflected in the clone library, representative clones
from the four most dominant families (Fl, F2, F9, and F13) were chosen for
complete sequencing of the cloned insert. Because there is no evidence at this
time to support this assumption, it has merely served as a starting point in
the analysis of the microbial population associated with A. pompejana. The
clones representing these four families are as follows: APG 5A (F1), APG 13B
(F2), APG 56B (F9), and APG 44B (F13).
16S rRNA Gene Sequences
The 16S rRNA gene sequences for the four alvinellid clones have been
deposited in Gen Bank under the following accession numbers: APG 5A,
L35523; APG 13B, L35520; APG 44B, L35521; and APG 56B, L35522.
27
M
1
Fl F2
1
1
F4 F9
1
1
111 III
F10 F11 F13 F16 F17 F19
F20 M
I
1018
1000
800
600
506
500
396
400
344
298
220
300
200
201
154
134
100
75
Figure 4. Scaled illustration of the restriction patterns of 16S rDNAs
representing 11 clone families identified in the alvinellid done library. Each
of these families contain 2 or more representative clones. Molecular weight
standards (M) are included for comparison. The fragment sizes are given in
base pairs.
28
Table 2. RFLP analysis results showing the numerical distribution of the
alvinellid clone families.
Clone Family
Number of Clones
,t,"
% of Library
23
F
F3
F4
F5
F6
F7
F8
F14
F15
F16
F 17
1
3
1
1
1
1
2
3
F18
1
F 19
F 20
5
2
F21
F22
1
F 23
F 24
F 25
F 26
F 27
F 28
F 29
F 30
F 31
F 32
1
1
1
1
1
1
1
1
0.7
2.2
0.7
0.7
0.7
0.7
0.7
1.4
2.2
0.7
3.6
1.4
0.7
0.7
0.7
0.7
0.7
0.7
0.7
0.7
0.7
0.7
0.7
0.7
a Highlighted rows indicate the four numerically dominant clone families
chosen for phylogenetic characterization.
29
Phylogenetic Analysis
Three different types of analyses were performed to determine the
correct phylogenetic placement of the four alvinellid clones: 1) phylogenetic
tree reconstruction inferred from both evolutionary distance and character
based calculations, 2) comparison of secondary structural models of the clone
16S rRNA to other established 16S rRNA secondary structures, and 3)
comparison of established signature base positions in the 16S rRNA
molecule.
Figure 5 is a phylogenetic tree inferred by the neighbor-joining method
of phylogenetic tree reconstruction. This analysis includes the four alvinellid
clone 16S rRNA sequences, a subset of reference sequences from the RDP, and
the 16S rRNA sequence from a newly characterized bacterium, Desulfurella
acetivorans (Rainey et al., 1993). Bootstrap values corresponding to 100
replicate re-samplings are shown above the lines leading to each branch,
unless otherwise designated. This analysis included a total of 1061 nucleotide
positions.
The four alvinellid clone 16S rRNA sequences form their own cluster
within the epsilon subdivision of the Proteobacteria. The alvinellid clones
cluster together to the exclusion of other species, supported by a bootstrap
value of 100%. This cluster clearly groups within the epsilon Proteobacteria,
which is also supported by a bootstrap value of 100%. An identical tree
topology was also obtained with the parsimony method, with bootstrap
values of 99% and 100%, respectively, showing the robustness of these two
branches (data not shown). Clones APG 5A and APG 13B are more closely
related within this Glade, as are APG 44B and APG 56B.
30
Myxococcus xanthus
o
Desulfuromonas acetoxidans
Desulfovibrio desulfuricans
Flexispira rappini
Helicobacter pylori
Wolinella succinogens
Arcobacter cryaerophilus
88
100
100
Campylobacter jejuni
E
Thiovulum sp.
APG 5A
APG 13B
APG 44B
APG 56B
100
100
100
78
Desulfurella acetivorans
100
87
100
98
79
95
Rhodospirillum rubrum
Agrobacterium tumifaciens
Nitrosomonas europae
Pseudomcmas testosteroni
Thiobacillus hydrothermalis
a
Escherichia coli
93
100 J
Bathymodiolus thermophilus symbiont
Calyptogena rnagmfica symbiont
Thiomicrospira L-12
0.10
Figure 5. Phylogenetic tree showing the relationship of the alvinellid clones
to the other members of the Proteobacteria. This tree was inferred from 16S
rRNA sequence data using the neighbor-joining method. Molecular
sequences for all reference strains (exceptDesulfurella acetivorans) was
obtained from the RDP. Boldface type indicates 16S rRNA sequences cloned
from the epibiotic microbial population of an Alvinella pornpejana
individual. A total of 1084 nucleotide positions were included in the analysis.
The tree was rooted with the sequence of Bacillus subtilis. Scale bar indicates
0.1 fixed mutations per nucleotide position. Numbers refer to boostrap
values for each node out of a total of 100 replicate resamplings (values below
75 are not shown).
31
Despite the high confidence placed in the branch associating the
alvinellid cluster with the epsilon Proteobacteria, the position of the
alvinellid cluster within this subdivision is not supported as strongly. In the
tree shown in Fig. 5, the alvinellid cluster appears as a sister group to the
Campylobacter /Thiovulum group. This branch is supported by bootstrap
values of only 57% by the neighbor-joining method, and 66% by the
parsimony method. Examination of the alternative branching orders
obtained with these analyses showed the alvinellid cluster varied in its
position within the epsilon subdivision. The alvinellid cluster also appeared
as a deeper group to the epsilon Proteobacteria (data not show). This
variation was dependent on the nucleotide positions included in the
analyses; however, the affiliation of the alvinellid cluster with the epsilon
Proteobacteria was consistently recovered.
Higher-order structural features of the 16S rRNA molecule are useful
in the phylogenetic characterization of microbial groups (Woese, 1987b).
Comparison of the 16S rRNA secondary structural models between distantly
related groups, i.e. at the phyla and subdivision levels, can reveal structural
features which support their phylogenetic relatedness. Comparison of these
structural features between closely related groups is usually not as
informative. Because 16S rRNA sequences for the alvinellid clones APG
5A/APG 13B and APG 44B/APG 56B are very similar it would be unlikely
that reconstruction of a 16S rRNA secondary structural model for all four
clone sequences would reveal significant defining characters. For this reason
secondary structural models for only two of the alvinellid clones were
generated. The alvinellid clones APG 13B and APG 56B were chosen to
represent their respective groups.
32
Figures 6 and 7 show the proposed secondary structures of two
alvinellid clone sequences, APG 13B and APG 56B. Both of these structures
are consistent with the general topological features of other established
bacterial 16S rRNA secondary structures. Several structural features were
observed which support the placement of the alvinellid clones within the
epsilon Proteobacteria. Three secondary structural features characteristic of
the epsilon Proteobacteria (then referred to as the Campylobacter Thiovulum
subdivision) had been previously reported (Lane et al., 1992). All three of
these structural features have been observed in our alvinellid clones
(position numbers are those of the 16S rRNA sequence of E. coli): 1) a
conserved deletion of a helix corresponding to positions 455-477, 2) a unique
compensatory base pair change at position 921 (U ) A) and 1396 (A
U), and
3) a "long-short" stem arrangement involving stem structures at positions
184-193 and 198-219 respectively (Figure 6 and 7). In addition to these, another
feature was observed which has been reported among the epsilon
Proteobacteria (Rainey et al., 1993): an insertion of a (C) in a conserved loop
structure corresponding to positions 1357-1365. This was observed
independently of the discovery by Rainey and colleagues (1993).
In order to confirm the results from the previous two phylogenetic
analyses, an examination of the signature base positions distinguishing the
subdivisions of the Proteobacteria was undertaken. Signature base positions
are specific positions within the 16S rRNA molecule which have been used to
distinguish various members of the bacterial, archace, and eukaryal domains
(Woese, 1987). Because the epsilons are a newly recognized subdivision of the
Proteobacteria, the signature base positions for these members have only
recently been compiled (Rainey et al., 1993). In order to confirm the
composition of these nucleotide positions, complete 16S rRNA
33
Figure 6. Proposed secondary structural model of clone APG 13B 16S rRNA.
Numbers indicate regions which are discussed in text. This structural model
was obtained using the gRNAID v 1.4 program and comparative analysis with
other bacterial 16S rRNA structures (Gutell 1993).
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37
sequences representing 51 available epsilon Proteobacteria obtained from the
RDP were examined. The signature base positions for the four alvinellid
clones were compiled separately for comparison.
Table 3 shows the signature base positions compiled for the epsilon and
delta Proteobacteria. A total of 72 signature positions were examined. Out of
these 72 positions, 48 were found to differ between the delta and epsilon
subdivisions. These signature base positions were also examined in the
alvinellid clones. Out of the 48 positions distinguishing the deltas from the
epsilons, only four were found to differ between the epsilons and the
alvinellid clones (see highlighted positions in Table 3). In order to rule out
the possibility that these differences were due to experimental artifacts, these
same positions were re-examined in the clone 16S rRNA secondary structural
models. The discrepancies at positions 370, 391, and 640 were seen in two of
the alvinellid clones, APG 44B and APG 56B. Secondary structure analysis for
clone APG 56B supports these three discrepancies, revealing compensatory
base pair changes at the adjoining positions in the stem structures of the
molecule (Figure 7). Compensatory base pair changes are changes in variable
nucleotides which preserve the conserved secondary structure of the 16S
rRNA molecule(Gutell et al., 1990). This type of comparison is not possible
with the difference at position 108 because it occurs in a non-stem structure.
Table g. Signature base positions distinguishing the delta and epsilon subclasses of the Proteobacteria a and the alvinellid clones.
Position
Delta
6
Gb
Epsilon
C
C
C
7
44
A
A
50
APG clones
N. D.
N. D.
107
..
124
129
129:1
233
236
237
242
284
..37.
C
A
C
871
A:u
A
A
C
C
C
C
C
U
C
U
A
Y
C
C
C:g
G:c
U
U
A
A
C:R:u
G:a
A
U
U:c
A
U
U
Ig.V.PagERIFAPPIRIEBRE
390
C:u
g:a
A:c:u
Vn
689
690
698
722
760
823
825
kg
U
a
564
812
822
875
877
878
916
929
947
948
976
1015
1024
1026
1116
1120
1153
1219
1233
1234
1246
1252
1260
.
371
398
438
449
485
496
502
513
538
543
554
Position
G:u
A
C
C:a
U:c
U:a
U:c
U
A
U
A
C
C
A
C
C
Delta
C
R:u
R:u
A:g
U
U:c
Epsilon
APG Clones
C
C
Y:a
Y:a
G
G
U
A:U:c:g
G:u
Y
C
G
GA
A:g
G:a
C
A:U:g:c
A
R
U
Y
R
A
C
U
C
U
G
Y
G
A
A
G
G
C
G
G:a
C
C
C
C
N
Y:A
R:U
A:C
U:G
A
R
R
R
C:a
C
C
G:c
G
Y
Y:C
G:u
G:u
R
C
C
G
A
A:U
A:u
C:Y
kg
1291
C:g
Y
1297
1298
1325
Y
C:A
C
Y
1421
Y
C
C:g
A:g
C
U
C:a
C
U
U
C
G
1426
U:R
C
1431
Y:a
U
U
Y
C
C
C
C:g:a
A
U:g
A
1437
1441
1443
G:u
C
A
A
R
C
The information for the delta subclass was taken from Table 4 of Woese (1987) and Table I of Rainey et al (1993). The information for the epsilon subclass was
compiled from an alignment of 51 16S rRNA sequences of epsilon Proteobacterla. Y, pyrimidine; R, purine; N, any nucleotide; N. D., not determined.
b Upper case, major base; if no other specified, it accounts for >90% of assayable cases. Lower case, minor occurrence base; found In <15% of assayable cases (or in only
one sequence).
c Shaded positions indicate nucleotide differences between the epsilon Proteobacteria and the alvinellid clones.
39
DISCUSSION
Bacterial/invertebrate symbioses are well recognized in marine
systems. However, defining the fidelity of these relationships is frequently
problematic. Differentiating relationships that are commensal, where only
one partner gains benefit from the relationship, from those that are truly
mutualistic, where both partners profit, is difficult. The partners are usually
so intricately tied that resolving the level of interaction has been impossible.
Although considerably more is known about monospecific endosymbiotic
associations in certain marine invertebrates (Cavanaugh, 1985; Fisher, 1990),
there are numerous cases of diverse microbial assemblages, found obligately
associated with the external surfaces of certain invertebrates, which have
eluded sufficient explanation (de Burgh & Sing la, 1984; Johnson et al., 1981;
Richards et al., 1982). Molecular genetic techniques now provide an avenue
by which to study these tightly associated symbioses.
The relationship between the hydrothermal vent polychaetes
Alvinella spp. and their associated microflora has intrigued investigators
since the discovery of these unique organisms (Desbruyeres & Laubier, 1980b).
Both A. pornpejana and A. caudata possess morphologically and
metabolically diverse assemblages of microorganisms associated with the
worms' dorsal surfaces, some of which appear specifically attached (Gaill et
al., 1987; Jeanthon & Prieur, 1990; Prieur et al., 1990b; Prieur et al., 1987a);
Desbruyeres, 1985 #1636. It has been suggested that the dominant integrated
morphotype, a filament, plays a significant role in the either the nutrition of
its host or in the detoxification of the worms' immediate environment (Gaill
et al., 1988). The primary focus of this study was to examine the diversity of
40
this unique microflora and, through the approach presented here,
phylogenetically characterize several of the dominant phylotypes.
The initial RFLP analysis of the alvinellid clone library grouped the 139
clones into similar clone types or families. The analysis identified 32 distinct
clone families. Four of these clone families collectively represented over 70%
of the total number of clones, with the remaining families consisting of only
one or a few members. There is no evidence to suggest that the assumption
that the distribution of clone types in the library represents the actual
distribution in the natural population. However, the numerical distribution
of the dominant clone families is in agreement with previous electron
microscopic observations, which demonstrated that a filamentous
morphotype dominated the microbial biomass (Gaill et al., 1987). The choice
of clone types to phylogenetically characterize was based on their numerical
dominance in the library.
The 16S rRNA sequences of individual alvinellid clones representing the
four dominant clone families and a subset of other representative bacterial 16S
rRNA sequences were used to infer their evolutionary relationships. Both
parsimony and distance phylogenetic inference methods placed the four
alvinellid clones as a unique cluster within the epsilon Proteobacteria. Both
methods gave a bootstrap value of 100%, illustrating the robustness of this
placement. In the phylogenetic tree presented here (Figure 5) the alvinellid
cluster is shown as a sister lineage to the Campylobacter/Thiovulum Glade.
However, this association is not strongly supported; the corresponding bootstrap
values are 57% and 66%, obtained with the neighbor-joining and parsimony
methods, respectively. The next most strongly supported placement of the
alvinellid cluster is as a deeper branching lineage within the epsilon subdivision.
It is clear that the alvinellid clones form their own cluster within the epsilon
41
Proteobacteria to the exclusion of other species; however this analysis is not able
to resolve the precise placement of the cluster within the epsilon subdivision
with a high degree of confidence.
Two additional comparative analyses of 16S rRNA sequences are
commonly used to provide support for the phylogenetic affiliations of
organisms: 1) the comparison of specific structural features in 16S rRNA
secondary structural models and 2) the identification of specific signature base
positions in the 16S rRNA sequence (Woese, 1987b). Both of these analyses
were carried out with the sequences of the alvinellid clones, in order to lend
additional support to the phylogenetic position obtained with the tree
inference methods.
The comparison of completed secondary structural models for two of
the alvinellid clone 16S rRNAs with other established bacterial 16S rRNA
secondary structures revealed several features that supported the inclusion of
the alvinellid clones within the epsilon Proteobacteria. Lane and colleagues
(1992), reported three important structural features which were conserved
among several members of the epsilon subdivision (then referred to as the
Thiovulum-Campylobacter subdivision). All three of these structural
features were present in the alvinellid clones. In addition, an insertion of a
cytosine residue in the conserved loop structure corresponding to nucleotide
positions 1357-1363 (E. coil numbering system) was found in all of the
alvinellid clones. The significance of this insertion was first reported in
Desulfurella acetivorans, a more distant relative of the epsilon Proteobacteria
(Rainey et al., 1993). A thorough analysis of other members of the epsilon
subdivision supports this insertion as being highly conserved. Examination
of both the 16S rRNA secondary structure of another epsilon proteobacterial
member, Campylobacter sputorurn subsp. sputorum (Gutell, 1993). and of a
42
general alignment of epsilon 16S rRNA sequences obtained from the RDP
suggests an equivalent insertion at this position. Examination of other
proteobacterial 16S rRNA sequences showed that an insertion at this position
occurs in only 10 out of 925 available 16S rRNA sequences from the four
other established proteobacterial subdivisions. Thus, this insertion has the
potential to become a defining character of the epsilon Proteobacteria.
The presence of certain 16S rRNA signature nucleotides of the
Proteobacteria (Woese, 1987a) provides the final criterion with which to
evaluate the phylogenetic position of an organism within the phylum.
Because the epsilons are a newly recognized member of the Proteobacteria,
the signature base positions for this group have only recently been compiled
(Rainey et al., 1993). A total of 72 positions were examined in 51 available
epsilon 16S rRNA sequences from the RDP. Comparison of these signatures
to the alvinellid clones was consistent with their placement in the epsilon
Proteobacteria. Only four positions were found to differ significantly between
the alvinellid clones and the epsilons, and at least three of these differences
appear to be real characteristics of the clone sequences. In each of these cases
the nucleotide differences were supported by compensatory base pair changes
in adjoining nucleotide positions of the clone secondary structure in which
they occur. This type of analysis has been used previously to rule out
nucleotide differences which might be due to sequencing errors or PCR
artifacts (De Long et al., 1993).
Recently a newly isolated bacterium, Desulfurella acetivorans, was
phylogenetically characterized as a distant relative to the epsilon
Proteobacteria, possibly representing a new subclass of this phylum (Rainey et
al., 1993). These investigators used phylogenetic tree reconstruction and
comparison of signature nucleotides of 16S rRNA sequences to infer the
43
evolutionary relationship of this thermophilic sulfur-reducing bacterium.
The analysis presented in the current study confirms the phylogenetic
position of D. acetivorans proposed by Rainey and co-workers; however, it
has identified several discrepancies between a number of the key signature
nucleotides for the epsilon Proteobacteria used in their analysis (data not
shown). Although it is not possible to resolve these inconsistencies at this
time, further examination of additional epsilon representatives will more
firmly establish the epsilon nucleotide signatures for use in future studies.
It is often difficult to infer the conserved phenotypic characters of a
particular group of microorganisms based solely on their phylogenetic
relatedness. However, phenotypic qualities that appear in ancestral branches
and within more closely related species may provide some insight into the
metabolic potential and ecology of unknown but related organisms.
Currently there are seven known genera which form the two clusters
comprising the epsilon subdivision of the Proteobacteria (Eaton et al., 1993;
Lau et al., 1987; Lee et al., 1992; Paster et al., 1988; Paster et al., 1991; Romaniuk
et al., 1987; Sly et al., 1993; Thompson et al., 1988; Vandamme et al., 1991)
.
A
survey of the conserved phenotypic characters among these epsilon genera
reveals that the majority are adapted to environments low in oxygen; they
range from microaerophiles to some which are able to grow anaerobically
(Holt et al., 1994; Kreig et al., 1984; Vandamme et al., 1991)
.
In addition, all of
these genera, except the sulfur-oxidizing marine bacterium Thiovulum sp.
have been found associated with a eukaryotic organism as a host, occurring in
digestive tracts, reproductive organs, or oral cavities of higher vertebrates.
These conserved phenotypic characters are consistent with the environment
of the alvinellid epibionts. As well as being associated with a metazoan host,
44
these bacteria would also be adapted to the hypoxic conditions within the
tubes of A. pompejana (Chevaldonne et al., 1991).
In conclusion, the novel molecular approach described here has
allowed the assessment of the microbial diversity of the epibiotic bacterial
population associated with A. pompejana. From an established 16S rRNA
clone library containing 32 distinct clone variants, several dominant
phylotypes were characterized as members of the epsilon subdivision of the
Proteobacteria. Future studies are currently underway to design and test
oligodeoxynucleo tide probes specifically targeting regions of the Alvinella
cloned 16S rRNA's. Previous studies have established the utility of such
probes in identifying the morphology of a cell type through in situ
hybridization techniques (Amann et al., 1991; Amann et al., 1990; Reysenbach
et al., 1994); Giovannoni, 1988 #533. These experiments will determine the
morphology of the clone types. The probes can then be used to screen large
insert DNA libraries established directly from epibiont nucleic acid samples.
This approach has been applied before to locate functional genes in free-living
marine archebacteria (Stein, 1994). The functional genes can then be related
to specific clone types, contributing additional information about the
metabolic potential of these epibionts. Through these molecular approaches
the function of the relationship between the Alvinella spp. and their
associated bacterial population can be determined without the need to culture
the organisms.
45
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APPENDICES
52
APPENDIX 1
LOCUS
APG5A
1496 by DNA
11-AUG-1994
DEFINITION Epibiont of Alvinella pompejana of 16S rRNA gene encoding
16S ribosomal RNA.
KEYWORDS 16S ribosomal RNA.
SOURCE
Epibiont of Alvinella pompejana, 5A from APG.
ORGANISM Epibiont of Alvinella pompejana
Bacteria /Prokaryota; Proteobacteria; subgroup: epsilon.
REFERENCE 1 (bases 8 to 1542)
AUTHORS Haddad,M.A., Camacho,F. and Cary,S.C.
TITLE
Phylogenetic characterization of the epibiotic bacteria
associated with the hydrothermal vent polychaete, Alvinella
pompejana
JOURNAL in prep, 0-0 (1994)
STANDARD full automatic
COMMENT
Data kindly submitted in computer readable form by:
S. Craig Cary Ph.D
Microbiology
Oregon State University
Corvallis Oregon 97331
USA
Phone: (503) 737-1837
Email: caryc@ava.bcc.orst.edu
Fax:
(503) 737-0496
[1] Author requested hold until 01-OCT-1994
Location/Qualifiers
1..1496
rRNA
/gene="16S rRNA"
iproduct="16S ribosomal RNA"
FEATURES
BASE COUNT 415 a 321 c 446 g 311 t 3 others
ORIGIN
1 AGAGTTTGAT CCTGGCTCAG AGTGAACGCT GGCGGCGTGC
TTAACACATG CAAGTCGAAC
61 GGTAACAGAT CTTCGGAGTG CTGACGAGTG GCGAACGGGT
GAGTAAGGTA TAGCTAACTT
121 GCCTCTTGGA GAGGGATAGC CACTGGAAAG GGTGATTAAT
ACCTCATACT CCTCTTAACT
181 ATAAGGTTAA GAGGGAAATG GTTTATTCCG CCAAGGGATA
GGGCTATATG GTATCAGTTA
241 GTTGGTGGGG TAAAGGCCTA CCAAGACAAT GACGCCTAGC
TGGTCTGAGA GGATGATCAG
53
301 CCACACTGGA ACTGAGACAC GGTCCAGACT CCTACGGGAG
GCAGCAGTGG GGAATATTGC
361 ACAATGGGGG AAACCCTGAT GCAGCGACGC CGCGTGGAGG
AAGAAACCCT TAGGGGCGT A
421 AACTCCTTTT ATAGTCGAAG AAGATGACGG TAGACTATGA
ATAAGCACCG GCTAACTCCG
481 TGCCAGCAGC CGCGGTAATA CGGAGGGTGC AAGCGTTACT
CGGAATCACT GGGCGTAAAG
541 CGCATGTAGG CGGTTAATTA AGTTAGAAGT GAAAGCCCAC
AGCTTAACTG TGGAACAGCT
601 TCCAAAACTG ATTAACTAGA GTCTGGGAGA GGAAGATGGA
ATTAGTAGTG TAGGGGTAAA
661 ATCCGTAGAG ATTACTAGGA ATACCGAAAG CGAAGGCGAT
CTTCTGGAAC AGGACTGACG
721 CTGAGATGCG AAAGCGTGGG GAGCAAACAG GATTAGATAC
CCTGGTAGTC CACGCCCTAA
781 ACGATGAATG TTAGTCGTTG GAGTGCTAGC CACTTCAGTG
ATGCAGCTAA CGCATTAAAC
841 ATTCCGCCTG GGGAGTACGG CCGCAAGGTT AAAACTCAAA
GGAATAGACG GGGACCCGCA
901 CAAGTGGTGG AGCATGTGGT TTAATTCGAA GATACGCGAA
GAACCTTACC TGGCCTTGAC
961 ATATACCAGA ACCCACCAGA GATGGTGGGG TGCCTTCGGG
AGCTGGTATA CAGGTGCTGC
1021 ACGGCTGTCG TCAGCTCGTG TCGTGAGATG TTGGGTTAAG
TCCCACAACG AGCGCAACCC
1081 TCGTGACTAG TTACTAACAG CATAGGCTGA GGACTCTAGT
CAGACTGCCT TCGTAAGGAG
1141 GAGGAAGGTG GGGACGACGT CAAGTCATCA TGGCCCTTAC
GGCCAGGGCG ACACACGTGC
1201 TACAATGGGG AGGACAATGA GAAGCGAAAT CGCGGGATGG
AGCAAATCTA TAAACCTTCT
1261 CTCAGTTCGG ATTGCAGTCT GCAACTCGAC TGCATGAAGC
TGGAATCACT AGTAATCGTG
1321 AATCAGCCAT GTCACGGTGA ATACGTTCCC GGGTCTTGTA
CTCACGCCCG TCACACCATG
1381 GGAGTTGATT TCACCCGAAG CGGGGATGCT AAATAGGCTA
CCCGCCACGG TGGAAGACTG
1441 GGGTGAAGTC GTAACAAGGT AACCGTAGGA GNACCTGTGG
NTGGATCACC NCCTTA
54
APPENDIX 2
LOCUS
APG13b
1502 by rRNA
11-AUG-1994
DEFINITION Epibiont of Alvinella pompejana of 16S rRNA gene encoding
16S ribosomal RNA.
KEYWORDS 16S ribosomal RNA.
SOURCE
Epibiont of Alvinella pompejana, 13B from APG.
ORGANISM Epibiont of Alvinella pompejana
Bacteria/Prokaryota; Proteobacteria; subgroup: epsilon.
REFERENCE 1 (bases 8 to 1542)
AUTHORS Haddad,M.A., Camacho,F. and Cary,S.C.
TITLE
Phylogenetic characterization of the epibiotic bacteria
associated with the hydrothermal vent polychaete, Alvinella
pompejana
JOURNAL in prep, 0-0 (1994)
STANDARD full automatic
COMMENT
Data kindly submitted in computer readable form by:
S. Craig Cary Ph.D.
Microbiology
Oregon State University
Corvallis Oregon 97331
USA
Phone: (503) 737-1837
Email: caryc@ava.bcc.orst.edu
Fax:
(503) 737-0496
[1] Author requested hold until 01-OCT-1994
Location/Qualifiers
rRNA
1..1502
/gene="16S rRNA"
/product="16S ribosomal RNA"
FEATURES
BASE COUNT 424 a 319 c 437 g 320 t
2 others
ORIGIN
1 AGAGTTTGAT CATGGCTCAG AGTGAACGCT GGCGGCGTGC
TTAACACATG CAAGTCGAAC
61 GGTAACAGGT CTTCGGAGTG CTGACGAGTG GCGAACGGGT
GAGTAATGTA TAGTTAATTT
121 TCCCCTTGGA GAGGGATAGC CACTGGAAAC GGTGATTAAT
ACCTCATACT CCTTTTAACC
181 AAAAGGTTGA AAGGGAAATG GTTTATTCTG CCAAGGGATA
AGACTATATC CTATCAGCTA
241 GTTGGTAGTG TAAGGGACTA CCAAGGCAAT GACGGGTAGC
TGGTCTGAGA GGATGATCAG
55
301 CCACACTGGA ACTGAGACAC GGTCCAGACT CCTACGGGAG
GCAGCAGTGG GGAATATTGC
361 ACAATGGGGG AAACCCTGAT GCAGCAACGC CGCGTGGAGG
AAGAAGCATT TAGGTGTGTA
421 AACTCCTTTT ATCAAGGAAG AAGACGACGG TACTTGATGA
ATAAGCACCG GCTAACTCCG
481 TGCCAGCAGC CGCGGTAATA CGGAGGGTGC AAGCGTTACT
CGGAATCACT GGGCGTAAAG
541 CGCATGTAGG CGGTTGATTA
GGCTCAACCG TAGAACAGCT
601 TCTAAAACTG ATTAACTAGA
ATTAGTAGTG TAGGGGTAAA
661 ATCCGTAGAG ATTACTAGGA
CTTCTGGAAC AGGACTGACG
721 CTGAGATGCG AAAGCGTGGG
CCTGGTAGTC CACGCCCTAA
AGTTAGAAGT GAAAGCCTAC
GTCTGGGAGA GGAAGATGGA
ATACCGAAAG CGAAGGCGAT
GAGCAAACAG GATTAGATAC
781 ACGATGAATG 'TTAGTCGTTG GAGTGCTAGC CACTTCAGCG
ATGCAGCTAA CGCATTAAAC
841 ATTCCGCCTG GGGAGTACGS CCGCAAGGTT AAAACTCAAA
GGAATAGACG GGGACCCGCA
901 CAAGTGGTGG AGCATGTGGT TTAATTCGAA GATACGCGAA
GAACCTTACC TGGCCTTGAC
961 ATATATCAGA ACCCACCAGA GATGGTGGGG TGCCTTCGGG
AGCTGATATA CAGGTGCTGC
1021 ACGGCTGTCG TCAGCTCGTG TCGTGTGATG TTGGGTTAAG
TCCCGCAACG AGCGCAACCC
1081 TCGTGACTAG TTACTAACAG CTTAGGCTGA GGACTCTAGT
CAGACTGCCT TCGTAAGGAG
1141 GAGGAAGGTA AGGACGACGT CAAGTCATCA TGGCCCTTAC
GGCCAGGGCG ACACACGTGC
1201 TACAATGGGA AGGACAAAGA GAAGCAAAAC TGCGAGGTGG
AGYAAATCTA TAAACCTTCT
1261 CTCAGTTCGG ATTGCAGTCT GCAACTCGAC TGCATGAAGC
TGGAATCACT AGTAATCGTG
1321 AATCAGCCAT GTCACGGTGA ATACGTTCCC GGGTCTTGTA
CTCACCGCCC GTCACACCAT
1381 GGGAGTTGAT TTCACCCGAA GTGGGGATGC TAAATAGGCT
ACCCACCACG GTGGAATTAG
1441 CGACTGGGGT GAAGTCGTAA CAAGGTAACC GTAGGAGAAC
CTGCGGCTGG ATCACCTCCT
1501 TA
56
APPENDIX 3
LOCUS
APG44b
1502 by DNA
11-AUG-1994
DEFINITION Epibiont of Alvinella pompejana of 16S rRNA gene encoding
16S ribosomal RNA.
KEYWORDS 16S ribosomal RNA.
SOURCE
Epibiont of Alvinella pompejana, 5A from APG.
ORGANISM Epibiont of Alvinella pompejana
Bacteria / Prokaryota; Proteobacteria; subgroup: epsilon.
REFERENCE 1 (bases 8 to 1542)
AUTHORS Haddad,M.A., Camacho,F. and Cary,S.C.
TITLE Phylogenetic characterization of the epibiotic bacteria
associated with the hydrothermal vent polychaete, Alvinella
pompejana
JOURNAL in prep, 0-0 (1994)
STANDARD full automatic
COMMENT
Data kindly submitted in computer readable form by:
S. Craig Cary Ph.D
Microbiology
Oregon State University
Corvallis Oregon 97331
USA
Phone: (503) 737-1837
Email: caryc@ava.bcc.orst.edu
Fax: (503) 737-0496
[1] Author requested hold until 01-OCT-1994
FEATURES
Location/Qualifiers
rRNA
1..1502
/gene="16S rRNA"
/product="16S ribosomal RNA"
BASE COUNT 399 a 344 c 449 g 310 t
ORIGIN
1 AGAGTTTGAT CCTGGCTCAG AGTGAACGCT GGCGGCGTGC
TTAACACATG CAAGTCGAAC
61 GGTAACAGCA GCTTGCACTG GGCTGACGAG TGGCGAACGG
GTGAGTAGTG TATAGCTAAT
121 ATGCCCTTTG GAGGGGGATA GCCACTAGAA ATGGTGATTA
ATACCCCATA CTACTTCTTC
181 TCACAAGAGT TGATGGGAAA TCTTTTTTGG CCAAAGGATT
GGGCTATACG GTATCAGCTT
241 GTTGGTGAGG TAACTGCTCA CCAAGGCTAT GACGCCTAGC
TGGTCTGAGA GGATGATCAG
57
301 CCACACTGGA ACTGAGACAC GGTCCAGACT CCTACGGGAG
GCAGCAGTGG GGAATATTGG
361 GCAATGGGCG AAAGCCTGAC CCAGCAACGC CGCGTGGAGG
AAGACACCCT TCGGGGCGTA
421 AACTCCTTTT ATAGCCGAAG TTATGACGGT AGGCTATGAA
TAAGCACCGG CAAACTCCGT
481 GCCAGCAGCG CGGTAATACG GAGGGTGCAA GCGTTACTCG
GAATCACTGG GCGTAAAGAG
541 CGCGCAGGCG GTATTGTAAG CTAGATGTGG AAGCCTACAG
CTCAACTGTA GAACTGCATC
601 TAGAACTGCG ATACTAGAGT CTGGGAGAGG TAACTGGAAT
TAGTGGTGTA GGGGTAAAAT
661 CCGTAGAGAT CACTAGGAAT ACCGAAAGCG AAGGCAAGTT
ACTGGAACAG TACTGACGCT
721 GAGGCGCGAA AGCGTGGGGA GCAAACAGGA TTAGATACCC
TGGTAGTCCA CGCCCTAAAC
781 GATGGATGTT AGTCGTTGGG AAGCTAGTCT TCTCAGCGAT
GCAGCTAACG CATTAAACAT
841 CCCGCCTGGG GAGTACGGCC GCAAGGTTAA AACTCAAAGG
AATAGACGGG GACCCGCACA
901 AGTGGTGGAG CATGTGGTTT AATTCGAAGA TACGCGAAGA
ACCTTACCCA GCCTTGACAT
961 CGATGGAACC CGCTAGAAAT AGTGGGGTGC CCCTTTTGGG
GAGCCTGAAG ACAGGTGCTG
1021 CACGGCTGTC GTCAGCTCGT GTCGTGAGAT GTTGGGTTAA
GTCCCGCAAC GAGCGCAACC
1081 CTCATGCTTA GTTACTAACA GTTCGGCTGA GGACTCTAAG
CAGACTGCCC GGGCAACCGG
1141 GAGGAAGGTG GGGACGACGT CAAGTCATCA TGGCCCTTAC
GGCTGGGGCG ACACACGTGC
1201 TACAATGGGT AGGACAGAAA GATGCAATAC TGCGAAGTGG
AGCAAAACTC TAAACCTACT
1261 CTCAGTTCGG ATTGTAGTCT GCAACTCGAC TACATGAAGC
TGGAATCACT AGTAATCGCG
1321 AATCAGCCAT GTCGCGGTGA ATACGTTCCC GGGTCTTGTA
CTCACCGCCC GTCACACCAT
1381 GGGAGTTGAG TTCACCCGAA GCGGGGATGC TAAAACAGCT
ACCCTCCACG GTGGGCTCAG
1441 CGACTGGGGT GAAGTCGTAA CAAGGTAACC GTAGGAGAAC
CTGTGGCTGG ATCACCTCCT
1501 TA
58
APPENDIX 4
LOCUS
APG56B
1504 by DNA
11-AUG-1994
DEFINITION Epibiont of Alvinella pompejana of 16S rRNA gene encoding
16S ribosomal RNA.
KEYWORDS 16S ribosomal RNA.
SOURCE
Epibiont of Alvinella pompejana, 5A from APG.
ORGANISM Epibiont of Alvinella pompejana
Bacteria /Prokaryota; Proteobacteria; subgroup: epsilon.
REFERENCE 1 (bases 8 to 1542)
AUTHORS Haddad,M.A., Camacho,F. and Cary,S.C.
TITLE Phylogenetic characterization of the epibiotic bacteria
associated with the hydrothermal vent polychaete, Alvinella
pompejana
JOURNAL in prep, 0-0 (1994)
STANDARD full automatic
COMMENT
Data kindly submitted in computer readable form by:
S. Craig Cary Ph.D
Microbiology
Oregon State University
Corvallis Oregon 97331
USA
Phone: (503) 737-1837
Email: caryc@ava.bcc.orst.edu
Fax:
(503) 737-0496
[1] Author requested hold until 01-OCT-1994
FEATURES
Location/Qualifiers
rRNA
1..1504
/gene="16S rRNA"
/product="16S ribosomal RNA"
BASE COUNT
397 a 341 c 459 g 305 t 2 others
ORIGIN
1 AGAGTTTGAT CATGGCTCAG AGTGAACGCT GGCGGCGTGC
TTAACACATG CAAGTCGAAC
61 GGAAACAGGC CCTTCGGGGT GCTGTCGAGT GGCGCACGGG
TGAGTAATGT ATAGCTAACA
121 TGCCCTTTGG AGGGGGATAG CCACTGGAAA CGGTGATTAA
TACCCCATAC TCCTCGAGAA
181 CTTAAGTTCT TGAGGGAAAT CTTTTTTGGC CAAAGGATTG
GGCTATATAG TATCAGCTTG
241 TTGGTGAGGT AACGGCTCAC CAAGGCTATG ACGCTTAGCT
GGTCTGAGAG GATGACCAGC
59
301 CACACTGGAA CTGAGACACG GTCCAGACTC CTACGGGAGG
CAGCAGTGGG GAATATTGGG
361 CAATGGGCGG AAGCCTGACC CAGCAACGCC GCGTGGAGGA
AGAAACCCTT AGGGGCGTAA
421 ACTCCTTTTA TCAGGGAAGA TAATGACGGT ACCTGATGAA
TAAGCACCGG CTAACTCCGT
481 GCCAGCAGNC GCGGTAATAC GGAGGGTGCA AGCGTTACTC
GGAATCACTG GGCGTAAAGA
541 GCGCGCAGGC GGTTTGGCAA GCTAGATGTG AAAGCCTGTA
GCTCAACC AC AGAACTGCAT
601 CTAGAACTGC AGACTAGAGT CTGGGAGGGG TAGCTGGAAT
TAGTGGTGTA GGGGTAAAAT
661 CCGTAGAGAT CACTAGGAAT ACCGAAAGCG AAGGCGAGCT
ACTGGAAC AG TACTGACGCT
721 GAGGCGCGAA AGCGTGGGGA GCAAACAGGA TTAGATACCC
TGGTAGTCCA CGCCCTAAAC
781 GATGGATGTT AGTCGTTGGA GAGCAAGTCT CTCCAGTGAT
GCAGCTAACG CATTAAACAT
841 CCCGCCAGGG GAGTACGGTC GCAAGACTAA AACTCAAAGG
AATAGACGGG GACCCGCACA
901 AGTGGTGGAG CATGTGGTTT AATTCGAAGA TACGCGAAGA
ACCTTACCCA GTCTTGACAT
961 CGATGGAACC TGCTAGAGAT AGCGGGGTGC CCCTTTTGGG
GAACCTGAAG ACAGGTGCTG
1021 CACGGCTGTC GTCAGCTCGT GTCGTGAGAT GTTGGGTTAA
GTCCCGCAAC GAGCGCAACC
1081 CTCATCCTTA GTTGCTAACA GGTTTGGCTG AGAACTCTAA
GGAGACTGCC CGGGCAACCG
1141 GGAGGAAGGT GGGGATGACG TCAAGTCATC ATGGCCCTTA
CGGCTGGGGC GACACACGTG
1201 CTACAATGGG CAGGACAGAG AGAAGCAATA CCGCGAGGTG
GAGCAAAGCT GTTAAACCTG
1261 CTCTCAGTTC GGATTGTAGT CTGCAACTCG ACTACATGAA
GTTGGAATCA CTAGTAATCG
1321 CGAATCAGCC ATGTCGCGGT GAATACGTTC CCGGGTCTTG
TACTCACCNC CCGTCACACC
1381 ATGGGAGTTG AG'TTCACCCG AAGCGGGGAT GCTAAAATAG
CTACCCTCCA CGGTGGGCTC
1441 AGCGACTGGG GTGAAGTCGT AACAAGGTAA CCGTAGGAGA
ACCTGTGGTT GGATCACCTC
1501 CTTA