Abstract
Microbial community structure in natural environments has remained largely unexplored yet is generally considered to be complex. It is shown here that in a Mid-Atlantic Ridge hydrothermal vent habitat, where food webs depend on prokaryotic primary production, the surface microbial community consists largely of only one bacterial phylogenetic type (phylotype) as indicated by the dominance of a single 16S rRNA sequence. The main part of its population occurs as an ectosymbiont on the dominant animals, the shrimp Rimicaris exoculata, where it grows as a monoculture within the carapace and on the extremities. However, the same bacteria are also the major microbial component of the free-living substrate community. Phylogenetically, this type forms a distinct branch within the E-Proteobacteria. This is different from all previously studied chemoautotrophic endoand ectosymbioses from hydrothermal vents and other sulfidic habitats in which all the bacterial members cluster within the y-Proteobacteria.

epibionts growing on the MAR vent shrimp to estimate their overall importance in the surface-associated microbial community. This community is the primary food source for higher trophic levels at the MAR, as inferred on the basis of observations indicating that the shrimp, the dominating macrofaunal element, graze bacteria either from their own bodies or from the sulfide surfaces (4, 5) and show no indication of appendages modified for filter feeding (ref. 5 ; C. L. Van Dover, personal communication). Furthermore, no significant population of actively filter-feeding animals has been described at this vent site.

Since the shrimp bacteria could not be cultured (E. V. Odintsova and H. W. Jannasch, personal communication), the 16S rRNA phylogenetic framework was used to determine the relationship of the epibionts to known vent endosymbionts, which cluster with all other analyzed sulfur-oxidizing symbionts (8, 9) , and to quantify their abundance both on the shrimp body and in the free-living surface community.

Hydrothermal vents in the deep sea are among the most productive ecosystems on earth. They are known for their unusual animal communities and extremely high biomass depending on carbon fixed via chemosynthesis rather than photosynthesis (1) . At Pacific vents, this high productivity is concluded to be largely based on the activity of a number of different endosymbiotic bacteria of tubeworms and bivalves (2) . In contrast, nothing is known about the identity of the major producers at Mid-Atlantic Ridge (MAR) vents. Some of these habitats are strikingly different from the Pacific vents in that the dominant animals are the highly mobile shrimp Rimicaris exoculata, which carry dense populations of epibiotic bacteria (3) .

These shrimp cluster on solid sulfide surfaces around warm vent water emissions, where they usually form a layer several specimens thick with an estimated density of about 25,000-50,000 individuals per m2 (3, 4) . They constantly ingest small sulfide particles and their guts are often densely packed with these solids (4, 5) . One of the most unusual features of these shrimp is the extremely dense bacterial population growing inside their carapace and on their extremities ( Fig. 1 A and B) . Several studies have claimed the presence of two or three distinct ectobacterial species on the basis of morphology (4) (5) (6) . Indeed, using scanning electron microscopy (SEM), morphologically different rods and filaments ofvarious thicknesses can be detected ( Fig. 1 A and B) . They are inferred to be chemoautotrophic sulfur-oxidizers (2, 6) and may benefit from the animals' movement in the gradients formed in the mixing zone of reduced and oxidized waters, as do ectosymbiotic bacteria on some marine nematode species (7) .

Pacific and MAR vents harbor strikingly different animal communities, yet they are both characterized by the mass occurrence of invertebrate-bacteria associations. We investigated the phylogenetic identity and relative abundance of the
MATERIALS AND METHODS
Specimens. Shrimp and solid sulfide blocks were collected by using the DSV (deep submergence vehicle) Alvin in 1993 during dives 2610 to 2623 at the Snakepit site (3). Animals were frozen immediately on board ship for nucleic acid extraction or fixed for SEM (10) or in situ hybridizations (11) . Surfaces of solid sulfide blocks were scraped to a depth of 1-2 mm and that material was frozen for nucleic acid extraction or fixed for SEM. In total, eight patches each with an approximate radius of 10 cm were scraped on sulfide blocks collected during three different dives. The density of the sulfides was determined to average 1.9 g-ml-1. One sample from each dive was examined by SEM. Escherichia coli strain INVaF' was grown overnight in LB broth, centrifuged, washed, and stored frozen. Cells of Methanobacterium thermoautotrophicum were a generous gift of Lee Krumholz (Massachusetts Institute of Technology).
MATERIALS AND METHODS
Nucleic Acid Manipulation. Nucleic acids were extracted for two purposes: (i) PCR, cloning, and sequencing for phylogenetic analysis of the shrimp epibionts and (ii) quantitative slot-blot hybridization to determine the relative abundance of these bacteria in the total community.
MATERIALS AND METHODS
DNA for PCR was extracted from bacterial scrapings from the shrimp carapace by using enzymatic (lysozyme and proteinase K) and detergent-based (SDS) lysis (12) . This relatively gentle method avoids excessive shearing of DNA and reduces the risk of chimera formation during PCR (13) . PCR of the 16S rRNA gene using the amplification primers 27F and 1492R (14) , cloning using the TA cloning kit (Vector Laboratories), and magnetic bead sequencing were essentially as described (15) Bartlesville, OK) was used as published (16 (18) to ease the identification of homologous sequence positions. Sequence alignment was done manually in the Genetic Data Environment (GDE) (19) . All reference sequences and the basic alignment to which the epibiont sequence was added were drawn from the RDP. The final data set included 989 nucleotide positions. Phylogenetic distance and bootstrap analyses were performed with the programs DNADIST with Jukes and Cantor correction, SEQBOOT, and FITCH, all contained in the PHYLIP 3.4 package (20) implemented through the GDE. Parsimony analysis, including bootstrapping, was done in PAUP 3.0 (21).
MATERIALS AND METHODS
Probe Construction and Synthesis. To confirm the source of the dominant sequence and to determine the distribution of the corresponding organisms in the environment, an epibiontspecific probe and a negative control probe complementary to the RNA-like strand were constructed. A search against GenBank by using BLAST (22) and against the Ribosomal Database Project by using CHECK-PROBE identified a sequence stretch (5'-ATCTTCCCCTCCCAGACTCT-3', E. coli position 653-672) with which no sequence in all the data bases had less than three mismatches, two of which were always in the middle region. A negative control probe containing two mismatches (5'-ATCTTCCCCTAACAGACTCT-3') was constructed to be used as a test for unspecific labeling. Synthesis and purification of the probes were as described (15) 
MATERIALS AND METHODS
For determination of the Td values of the domain-specific probes, the respective positive control nucleic acids served as templates. For the shrimp epibiont-specific probe, nucleic acids from scrapings had to be used, since no culture of these bacteria exists. Care was taken to avoid contamination by rinsing bacteria-containing body parts with sterile water several times before gently scraping off epigrowth. Values read from the Td curve and from the standard curve (see below) may lead to an overestimation of the relative abundance of the shrimp epibionts when related to other samples caused by possible additive contribution of nonspecific hybridization. However,the scrapings were extremely pure as judged by repeated comparisons of the signal obtained by the universal probe measuring total Bacteria (25) and the epibiont-specific probe (see Table 1 ).
MATERIALS AND METHODS
Hybridizations were done overnight at 40°C (except Eukarya at 30°C). Based on the experimentally determined Td values, the wash temperatures were 420, 500, 600, and 56°C for the Eukarya (26), Archaea (25), Bacteria (25) , and the epibiont-specific probes, respectively. For qualitative analysis, blots were exposed on Reflection NEF-496 (DuPont) film. Quantification was done using a Fujix Bio Imaging Analyzer BAS2000 phosphor imager and BAS2000 Image File Manager 2.1 analysis software. Standard nucleic acids from shrimp epibiont scrapings and positive controls were spotted in different concentrations. Least-squares linear regression analysis on the standard curves yielded r2 > 0.98 in all cases. To back-calculate from total amount of nucleic acids to amount per volume of sulfide substrate, the density value of 1.9 g mlwas used.
RESULTS AND DISCUSSION
Remarkably, only one bacterial sequence or "phylotype" was recovered from nucleic acids extracted from the shrimp epibionts. A single 16S rRNA gene was evident by direct sequencing. Furthermore, of the 26 clones analyzed by partial sequencing, 23 contained the same sequence in all nucleotide positions, whereas the remaining 3 were unreadable. To test if this dominance was reflected in the makeup of the shrimpepibiont population in situ, an epibiont-specific 16S rRNAtargeted oligonucleotide hybridization probe and a negative control probe with two introduced mismatches were constructed. These probes were used in in situ hybridizations (Fig.  1) . A probe complementary to all known Bacteria 16S rRNAs (25) served as a positive control.
RESULTS AND DISCUSSION
In situ hybridization and epifluorescence microscopy confirmed the genetic identity of all three morphotypes of shrimp symbionts on the 16S rRNA level (Fig. 1) . The specific probe gave signal patterns identical to those of the universal bacterial probe (Fig. 1 D and F) with no background staining from the negative control probe (Fig. 1 I and J) . Both thin and thick filaments were stained ( Fig. 1 C and D) . Regions of active growth within these filaments are discernible because the strength of the signal is correlated to cellular RNA content, which is a measure of its physiological state (27) (Fig. 1 C and D and G and H). The small rods are less visible, yet staining in a region where only these cells grow showed an intense fluorescence with the specific probe ( Fig. 1 E and F) . These results are supported by the quantitative hybridizations, which gave essentially identical signals between the universal bacterial probe and the epibiont-specific probe when applied to nucleic acids extracted from shrimp bodies ( Table 1) .
RESULTS AND DISCUSSION
The results of the hybridizations indicate a high degree of specificity in the shrimp-epibiont association with a single bacterial phylotype, suggesting that a single species or multiple species with identical 16S rRNA are growing on the shrimp body. The probing region for the specific oligonucleotide and the negative control were chosen in a highly variable area of (Fig. 2) . This is distinctly different from previously analyzed ecto-and endosymbionts of animals from sulfidic habitats, including hydrothermal vents, which all cluster within the y-Proteobacteria (Fig. 2) (8, 9) . However, association with animals is the best-known mode of living in the s-Proteobacteria, although all examples are parasitic. Sulfur metabolism and microaerophily are also common in this group and there exists a moderate relationship between the R exoculata symbiont and the sulfide-gradient organism Thiovulum sp. (29) .
RESULTS AND DISCUSSION
To address the question of numerical importance of the ectosymbionts in the surface microbial community, oligonucleotide slot-blot hybridization on nucleic acids from different sources using probes specific for the shrimp epibiont and for the three domains Bacteria, Archaea, and Eukarya was performed (Fig. 3, Table 1 ). Most surprisingly, the quantification indicates that the shrimp epibiont is also the dominant phylotype on the sulfide surface, contributing half of the detectable bacterial rRNA (Table 1 ). This is supported by SEM observation -of the characteristic filaments on the sulfide surfaces (Fig. 4) . Qualitative slot-blot analysis showed a complete absence of signals from the Archaea and Eukarya probes (Fig. 3) , which parallels results obtained by using lipid biomarker analysis of surface sulfides from a Pacific hydrothermal vent site (30) .
RESULTS AND DISCUSSION
The main biomass of bacteria at these vents appears to be associated with the shrimp bodies. Assuming that the 16S rRNA content in an average bacterial cell is about 23% of the total nucleic acids and 15 fg (31), we can calculate from the quantitative probing (Table 1) (unpublished results) and the observed ingestion of sulfides by the massive shrimp population (4, 5) is likely to remove significant numbers of attached bacteria. Assuming that all bacteria on the sulfides are affected indiscriminately, the free-living population of the epibionts may achieve high densities through its symbiotic life stage. While bacterial species growing exclusively on the sulfides can increase their numbers only through growth in situ, symbionts falling off the shrimp bodies likely serve as a continuous inoculum replenishing their substrate population. Thus, an indirect yet mutually favorable interaction between the two partners arises, further enabling them to reach high biomass under otherwise adverse conditions.
RESULTS AND DISCUSSION
One of the most intriguing results of the phylogenetic approach to microbial ecology (36, 37) hlas been the discovery of the overwhelming diversity of microorganisms, which had been previously unrecognized. For example, it was inferred from DNA reassociation kinetics that the equivalent of 4000 bacterial species was present in 1 g of forest soil (38) . In addition, cloning and sequencing of 16S rRNA genes from natural communities have produced extensive lists of uncultured microorganisms, many of which represent unknown phylogenetic lineages (for reviews see refs. 39 and 40). In the light of these findings, the extreme dominance of one bacterial phylotype in the microbial surface community at the MAR vents appears highly unusual. A similar phenomenon has so for been measured only once, in short-lived bloom situations in a pelagic environment (41) . However, quantitative studies assessing the importance of single species in a total community are still rare, and more examples from diverse environments will have to be collected before unifying principles of microbial diversity, dominance, and community structure will emerge.
RESULTS AND DISCUSSION
Symbiotic association appears to be a fundamental characteristic of hydrothermal vent communities. In both Pacific and Atlantic vents the driving forces behind the close interaction between bacteria and invertebrates (35) may be similar, yet result in the respective dominance of two phenomenologically very different types of association, ecto-and endosymbiosis. Information about bacterial community structure in natural environments is still scarce. We have shown that limited diversity and dominance of one species on the macrofaunal level may be reflected in a microbial community. Given how common symbiotic interactions are in many environments, comparative measurement of the numerical representation of symbiotic species may shed light on the true significance of neutral and beneficial interactions among organisms in nature.
RESULTS AND DISCUSSION
Note Added in Proof. Recently, 16S rDNA sequences derived from E-Proteobacteria have been detected in clone libraries obtained from a bacterial mat at a hydrothermal vent (42) and from an epibiotic community of a vent polychaete (43) .
RESULTS AND DISCUSSION
We thank Chief Scientist C. L. Van Dover, the captain and crew of the RIVAtlantis II and DSVAlvin, J. Robinson for help on board, and L. Krumholz for generously providing cells of M. thermoautotrophicum. In addition, we thank anonymous reviewers for helpful comments. This work was supported by a grant from the National Science Foundation.

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