Isolation of an Obligately Barophilic Bacterium and Description of a New Genus, Colwellia gen. nov.

System. Appl. Microbiol. 10, 152-160 (1988)

Isolation of an Obligately Barophilic Bacterium and Description of a New Genus, Colwellia gen. nov. w. DEMING,*1,4 LESLIE K. SOMERS,1 WILLIAM L. STRAUBE,z DAVID G. SWARTZ,3 and MICHAEL T. MACDONELL4,5

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Chesapeake Bay Institute, The Johns Hopkins University, 4800 Atwell Road, Shady Side, Maryland 20764, USA Department of Microbiology, University of Maryland, College Park, Maryland 20742, USA 3 Sea Grant College, University of Maryland, College Park, Maryland 20742, USA 4 Center of Marine Biotechnology of the Maryland Biotechnology Institute, University of Maryland, 600 East Lombard Street, Baltimore, Maryland 21202, USA 5 Present address: Molecular Biosystems, Inc., San Diego, California 92121, USA 1

2

Received September 22, 1987

Summary An obligately barophilic bacterium, designated strain BNL-1, was isolated in the laboratory, under deepsea conditions of 2 °C and 740 atm hydrostatic pressure, from a sample of particle-rich seawater collected at a hadal depth of 7,410 m in the Puerto Rico Trench of the Atlantic Ocean. Its minimum, optimum, and maximum growth pressures at 2°C were <370, 740 and >1,020 atm hydrostatic pressure, respectively. At 10 °C, ~ll cardinal growth pressures shifted upwards about 200 atm, but at no tested pressure was the organism capable of growth at 15°C. Most rapid generation time, 7 h, was observed at 10°C and 925 atm. Selected taxonomic tests and a G+C % mol ratio of 45.7 indicate the strain belongs to the vibrioenteric group of bacteria. However, evolutionary tree and principal coordinates analyses, based on the ribonucleotide sequences of 5S ribosomal RNA from BNL-1 and other bacteria, indicate its distinction at the generic level from all but one member of this group, including other known barophiIes. We recommend that a new genus, Colwellia gen. nov., be established and that the obligate barophile, strain BNL-1, be designated Colwellia hadaliensis, gen. nov. sp. nov. We further recommend the renaming of Vibrio psychroerythrus ATCC 27364, the organism with which BNL-1 shares most recent common ancestry, as Colwellia psychroerythrus comb. nov.

Key words: Vibrio - Colwellia - Deep-sea - Pressure - Barophile - Psychrophile - Phylogeny - Evolution5S rRNA

Introduction Micro£lora indigenous to abyssal depths of the ocean (4,000-6,000 m) are believed to be both psychrophilic and barophilic and, thus, uniquely adapted for optimal growth at the cold temperatures «4°C) and elevated hydrostatic pressures (400-600 atm) of their environment. This concept, though hardly new (ZoBeli and Morita, 1957), has been reinforced in recent years in two ways: 1) by the successful isolation of psychrophilic barophiles from a wide variety of abyssal samples, including decomposing amphipods (Yayanos et al., 1979; 1981), digestive tracts * Corresponding author.

of benthic fauna (Deming et al., 1981; Deming and Colwell, 1982), fecal material and sediments (Deming, 1985; Helmke and Weyland, 1986) and seawater (jannasch et aI., 1982); and 2) by the detection of barophilic responses from natural assemblages of bacteria in a similar variety of deep-sea samples (Deming and Colwell, 1982; 1985; Jannasch and Wirsen, 1982; Deming, 1985; 1986). A model that incorporates some of the latter data has led to calculations that bacteria perform a significant portion (15-30%) of the total biological recycling of organic carbon that occurs in the abyssal basins of the Atlantic Ocean (Rowe and Deming, 1985).

Barophilic Bacteria: Colwellia gen. nov.

Far less is known about the microbial inhabitants at hadal depths (6,000-11,000 m) in the island arc trenches of the Pacific, Atlantic, and Indian Oceans. Yayanos et aI. (1981) trapped and recovered amphipods from the bottom of the Marianas Trench of the Pacific and allowed one to decompose in the laboratory in marine broth at in situ temperature «2°C) and pressure (1050 atm). Upon decompression of this sample, bacteria were subcultured from the broth, again at in situ temperature and pressure. One of the resulting isolates, designated strain MT-41, proved in subsequent growth studies to be an obligate barophile. Unlike all other known barophiles, which have been recovered from "shallower" abyssal environments and can grow slowly at atmospheric pressure when kept cold, MT-41 cannot reproduce at pressures of 346 atm or lower and, in fact, expires when held at 1 atm and O°C (Yayanos and Dietz, 1983). This isolation of the first obligate barophile led Yayanos et aI. (1981) to speculate that microbial inhabitants of the ocean's hadal trenches might be both physiologically and taxonomically distinct from other marine microbial communities, having been isolated from them over geologic time by pressure (depth) barriers. It is clear from their pure culture work that strain MT-41, and presumably other obligate barophiles, could not compete or even survive in shallower marine environments (Yayanos et aI., 1981; Yayanos and Dietz, 1983). However, the degree to which this geographic isolation is related to a phylogenetic divergence from other marine bacteria is unknown. In search of more information about the microbiology of hadal trenches, we worked aboard the RN ISELIN in June and July of 1984 to recover samples of seawater, sinking particulates, and sediments from depths of 7,328-8,189 m in the Puerto Rico Trench of the Atlantic Ocean. Results of shipboard experiments, indicating the presence of significantly active, natural assemblages of barophilic bacteria at these depths, are described elsewhere (Deming, 1987, Abstract, Annual Meeting of the American Society for Limnology and Oceanography; manuscript in preparation). Here, we report the isolation of an obligate barophile, initially designated strain BNL-1 (Deming and Somers, 1985, Abstract, Annual Meeting of the American Society for Microbiology), from particlerich seawater at a depth of 7,410 m. Some of its growth and taxonomic characteristics are described, along with comparative analyses of the nucleotide sequence of its 5S ribosomal RNA and other known sequences (see those described by MacDonell et aI., 1986). The latter provide information relevant to the classification of this new organism and to its evolutionary relationships with the shallow-water marine psychrophile Vibrio psychroerythrus and with other members of the vibrio-enteric group, including the only other classified species of deep-sea, barophilic bacteria, Shewanella benthica.

Materials and Methods Isolation procedures

The original source of bacterial strain BNL-l was particle-rich seawater recovered in the 6-liter collection volume of a sediment

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trap, of the type described by Rowe and Gardner (1979). The trap had been moored 10 m above the seafloor in open position for 17 d at a water depth of 7410 m along the axis of the Puerto Rico Trench at 19°11.6'N by 66°14.3'W. An electronic timed release mechanism with three command options, constructed for this study in a special pressure casing (Oceanographic Instrument Systems, North Falmouth, Massachusetts), successfully closed the trap in situ and freed it from the mooring anchor. Ascent required 189 min; siting and recovery shipboard, 38 min. Due to previous indications of the negative effects of gear warming on the recovery of barophiles from deep-sea samples (Yayanos and Dietz, 1982; Deming and Colwell, 1985), we were disappointed to find that ascent through warm surface waters and recovery procedures in the Caribbean sun had warmed the trap to near 20°C. Nevertheless, we removed the particle-rich seawater sampled from the trap, returned it to hadal temperature «2°C) in an ice bath, and proceeded to measure responses of the resident bacterial population to organic enrichment at different temperatures and pressures, as described for other deep-sea samples in an earlier study (Deming, 1985). Upon return to the laboratory, bacterial strain BNL-l was cultured at 2°C and 740 atm in 2216 marine broth (Difco) from a decompressed trap aliquot that had been enriched with 0.1 % yeast extract and incubated shipboard 7 d at 2°C and 740 atm. The culture was then purified by a series of three dilution-toextinction procedures in marine broth, using incubation conditions of 2°C and 740 atm pressure at each step. Cultures were developed in sterile, disposable plastic testubes filled to capacity with chilled broth, capped with parafilm in a manner to exclude air bubbles, and pressurized in stainless steel pressure vessels (Tern-Pres Division 'of Leco Corporation, Blanchard, Pennsylvania). Distilled water at 2°C was used as hydraulic fluid and the vessels were incubated in a cold room, temperature-controlled at 2 0C. Cultures of Vibrio psychroerythrus ATCC 27364 were cultivated by, and purchased from, the American Type Tissue Culture Collection, Rockville, Maryland. Growth studies

Cultures of BNL-1 in late logarithmic growth phase at 2°C and 740 atm were decompressed, inoculated into prefiltered (0.2 !lm) marine broth at 2°C, and incubated at three temperatures (2, 10, and 15°C) and seven pressures (1, 184, 367, 551, 740, 925, and 1,020 atm) for 8-24 d. The sterile, 10-ml disposable syringes (Sarstedt) used as culturing chambers also contained sterile glass mixing beads and were housed in pressure vessels mounted on a rocking platform in a temperature-controlled water bath. Periodically, the cultures were decompressed, subsampled and, within 2 min, returned to the appropriate pressure. Decompressed subsamples were fixed immediately in prefiltered (0.2 !lm) formaldehyde at a final concentration of 2%. Bacterial densities were determined using a Coulter Counter Model ZBI (Coulter Electronics). Growth rates were calculated from 3-5 points along the logarithmic portion of the resulting growth curves, using linear regression analysis. Similar growth studies were conducted with Vibrio psychroerythrus, except that inocula were cultivated to late logarithmic growth phase in marine broth at the organism's optimal growth conditions (according to D'Aoust and Kushner, 1972, and to ATCC) of 10 °C and atmospheric pressure. Taxonomic tests

Immediately following decompression, broth cultures of strain BNL-1 that had developed at 2°C and 740 atm were tested according to conventional procedures for motility in wet mount,

J. W. Deming, L. K. Somers, W. L. Straube, D. G. Swartz, and M. T. MacDonell

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Gram reaction, and oxidase activity (Kovacs, 1956). Bacteria concentrated by centrifugation were tested for chitinase activity using the umbelliferyl conjugate of chitobiose, according to the methods of Wortman et al. (1986). DNA was extracted from a 2-liter broth culture of BNL-1, pooled from multiple small-volume cultures grown to late logarithmic phase at 2°C and 740 atm, using extraction procedures described by Marmur and Doty (1962). The crude lysate was treated with protease K (200 ~g/ml for 60 min at 37°C) to obtain an extract of sufficiently pure DNA. The DNA base ratio (mol % G+C) was then determined according to methods of Mandel et al. (1970).

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Analysis of 5S rRNA sequences The primary sequence of 5S rRNA from BNL-1 was reported earlier as part of a larger study by MacDonell and Colwell (1985). Here, we have analyzed primary and secondary structural features of the sequence and its relationship to other known 5S rRNA sequences, using more refined approaches that were not available previously. A two-dimensional evolutionary tree was constructed using the computer program KITCH (Phylogeny Inference Package; J. Felsenstein, University of Washington), based on the method of Fitch and Margoliash (1967). In order to examine the data in higher-order dimensions or "hyperspace", the first four principal coordinates were calculated from the distance matrix generated by pairwise comparisons of the 5S rRNA sequences included in this study. Theory and mathematics behind the extraction of principle coordinates from distance matrices have been described in detail by Dunn and Everitt (1982) and Pielou (1984). A discussion of principle coordinates analysis as applied specifically to 5S rRNA sequences can be found in Austin and Priest (1986); graphical examples, in MacDonell et al. (1986).

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A culture of BNL-1, grown to late logarithmic phase at 2°C and 740 atm, was decompressed, centrifuged at 2°C, and fixed in a 2% solution of EM-grade glutaraldehyde prepared in prefiltered (0.2 ~m) artificial seawater. The sample was then fixed with 1% OS04 in 0.1 M cacodylate buffer (pH 7.3) for 1 h, washed three times in distilled water, prestained in 1 % aqueous uranyl acetate for 1 h, dehydrated in a graded series of ethanol solutions, and embedded in Spurr medium (Polysciences, Inc., Warrington, Pennsylvania). Thin sections were post-stained with 0.1 % lead citrate before they were viewed on an Hitachi HU-12 transmission electron microscope at 75 kV.

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Fig. 2. Growth rate of strain BNL-1 at 2°C (solid line) and 10 °C (dotted line) as a function of elevated hydrostatic pressure. Individual rates were calculated, using linear regression analysis, from the logarithmic portion of growth curves, such as those shown in Fig. 1. Five growth rate determinations were made for each pressure tested at 2°C; error bars indicate S. D. of the mean. Values below dashed line indicate decreases in bacteria ml-I with time of incubation.

Growth studies Representative growth curves for BNL-1 at 2°C and a range of pressures are shown in Fig. 1. Mean growth rate (± S.D.) at 2°C as a function of pressure (n = 3-5 for each pressure tested) is shown in Fig. 2. Of the pressures tested, the approximate in situ pressure at depth of collection (740 atm) afforded most rapid growth at 2°C. Under those conditions, the mean generation time of BNL-1 was 13 (± 2) h and the maximum yield, 2x 107 bacteria per ml. No growth was observed at tested pressures below 370 atm; in fact, bacterial numbers decreased with time at both 184 and 1 atm. Maximum growth pressure appeared to be very near the maximum pressure tested (1,020 atm), where the generation time was 102 (± 61) h.

Barophilic Bacteria: Colwellia gen. nov.

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Fig. 3. Transmission electron micrograph of obligate barophile BNL-l, cultured at 2°C and 740 atm hydrostatic pressure and fixed immediately upon decompression. Bar indicates 1 ~m.

Growth rates of BNL-1 at 10°C are also shown in Fig. 2. Although based on fewer ~ete.rminations (n = 2 for each pressure tested), the results mdicated that a temperature increase of 8°C (from 2 to 10 0c) shifted the organism's cardinal growth pressures upwards by approximately 200 atm. No growth (decreases in bacterial number with time) was observed at tested pressures below 551 atm; most rapid growth, at 925 atm (generation time of 9 h or a growth rate 1.4 times the maximal rate observed at 2 °C). In deference to laboratory safety, we have not attempted to determine the maximum growth 'pressure of BNL-1 at 10 °C, but it would appear from FIg. 2 to .approach 1,150 atm. Maximum yields at 10°C were eqUivalent to those typically observed at 2 °C. No growth was detected at 15 °C under any of the seven pressures tested. Growth of Vibrio psychroerythrus was barosensitive at all temperatures tested. For example, at 10°C, where the optimal generation time of 9 (± 3) h (n = 4) was observe.d at 1 atm, application of even a modest level of hydrostatIC pressure (184 atm, a pressure too low for growth of BNL1) reduced the organism's growth rate by a factor of sev~n (generation time of 62 [± 20] h [n = 4]). Decreas.es m bacterial number over time were observed at all hIgher pressures, regardless of the temperature tested (2, 10, 15°C). We did not test for growth at elevated pressures and temperatures higher than 15 DC, but no growth o~­ curred at room temperature (about 25°C) and 1 atm, m keeping with the 0-19°C growth range for V. psychroerythrus, previously reported by D'Aoust and Kushner (1972).

Taxonomic tests and TEM

Routine Gram stains and phase microscopy of unstained cultures revealed BNL-1 to be a Gram negative, but highly refractile, slightly curved rod, typicalJy 0.8 !l~ wide and 3.0-5.0 !lm long. Freshly decompressed bactena in wet mount (unstained, unfixed) appeared motile by polar flagella. However, within about 15 min of sample decompression and slide preparation, directed movement ceased and individual bacteria were observed to form "round bodies". BNL-1 reacted positively to oxidase and chitinase assays. The G+C mol % of its DNA was 45? Although the results of similar taxonomic analyses ~f VIbrio psychroerythrus are available in the literature, It apparently had not been tested for chitinase activity (D'Aoust arid Kushner, 1972); our tests of an ATCC culture were positive. Efforts to apply traditional fermentative tests to BNL-1 at the elevated pressures it requires for optimal growth have been problematic to date (see Straube and O'Brien, 1986, Abstract, American Society for Microbiol~gy), but have allowed a general assessment of the orgamsm as a facultative anaerobe. Transmission electron microscopy revealed the typical structure of a Gram negative bacterial rod (Fig.3). No flagella were observed in thin section, but our TEM procedures were not designed specifically to detect them.

J. W. Deming, L. K. Somers, W. L. Straube, D. G. Swartz, and M . T. MacDonell

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Analysis of 5S rRNA sequence

Results of evolutionary tree analysis and principal coordinates analyses are shown in Fig. 4. They confirm the specific relationship of obligate barophile BNL-1 to Vibrio psychroerythrus (MacDonell and Colwell, 1985) and protray its more general relationship to other members of the vibrio-enteric group, including the only other barophilic bacteria that have been classified, Shewanella benthica strains W145 and UM40. Proposed secondary structures of 5S rRNA from BNL-1 and V. psychroerythrus are shown in Fig. 5. Features from each of these figures are discussed and interpreted below. Discussion Successful isolations of barophilic bacteria from abyssal ocean samples (depths of 4,000-6,000 m) have almost always involved selective enrichment at temperatures and pressures approximating those encountered in situ (see

Deming et aI., 1984, and Helmke and Weyland, 1986, for exceptions). At in situ temperatures, all known abyssal barophiles (Yayanos et ai., 1982; Deming et aI., 1984; Jannasch and Wirsen, 1984) grow over a pressure range of about 800 atm (including 1 atm), with most rapid growth occurring at or near the original isolation pressure (typically, 400-600 atm). From this kind of information came the prediction that obligate barophiles, unable to grow at atmospheric pressure, might be found at still greater, hadal depths (> 6,000 m) in the ocean (Yayanos et aI., 1981). Indeed, the first obligate barophile, strain MT-41, was recovered from a sample collected at 10,476 m (Yayanos et aI., 1981). Our strain BNL-1, recovered from a sample collected at 7,410 m, also failed to grow at atmospheric pressure (or other tested pressures below 367 atm at 2°C and below 551 atm at 100C). Therefore, BNL-1, like MT-41, is an obligate barophile, uniquely adapted to outcompete other marine bacteria that may sink to hadal depths in the ocean. Its generation time of 13 h in marine broth at 2°C

J. W. Deming,

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L. K. Somers, W. L. Straube, D. G. Swartz, and M. T. MacDonell

tion on these samplers (Yayanos and Dietz, 1982) considered the (irrelevant) negative effects of elevated temperature on barophiles at 1 atm, instead of the positive effects at elevated pressure. Whether recovery of BNL-1 from a warmed and decompressed seawater sample was significant, or simply fortuitous, is unknown. Like other barophiles, BNL-1 tolerates brief decompression/recompression cycles at cold temperatures, but appears to expire if left decompressed, especially at warmer temperatures (negative values in Fig.2; observation of rapid development of aberrant morphology in wet mount preparations at room temperature). We can only speculate that survival properties of BNL-1 in its natural milieu (hadal seawater in the sediment trap) may differ from those observed in rich laboratory media. Although the cardinal growth temperatures and pressures of all known barophiles suggest their ecological isolation from other marine bacteria by ocean depth, information about their taxonomic or evolutionary relatedness to other bacteria remains sparse. Only the two strains W-145 and UM-40, isolated from abyssal invertebrate guts, have been characterized sufficiently for a taxonomic identification (Deming et aI., 1984). Their ultimate classification as Shewanella benthica (MacDonell and CoLwell, 1985) was made on the basis of degree of sequence homology between their rRNAs and those of other bacteria, as described by Fox et al. (1980) and Stackebrandt and Woese (1984). In this study, we relied on comparative analysis of 55 rRNAs once again, along with conventional

and 740 atm suggests that BNL-1 could even outcompete MT -41, which doubles only once every 25 h under the same conditions (Yayanos et aI., 1982). The most rapid doubling time of BNL-1 (9 h; Fig. 2) was measured not at bottom water conditions of the Puerto Rico Trench, but at a temperature (10°C) and pressure (925 atm) higher than the organism could encounter in its environment. Aside from speculation about the influence of hydrothermal activity on psychrophilic barophiles (Yayanos et ai., 1981) (no evidence yet exists for such activity at hadal ocean depths) or the evolution of barophily in a warm Archaean ocean (Deming et aI., 1984) (difficult to test), simple physical-chemical explanations can be invoked to explain this phenomenon. Pressure is known to oppose the adverse effects of elevated temperature on enzyme systems (reviewed by Morita, 1976), facilitating growth of strict psychrophiles at mesophilic temperatures (e. g., Deming et ai., 1984). It may also exert a QlO effect on growth or activity, wholly apart from any absolute temperature increase, by the fact that it lowers the freezing point of seawater. We favor consideration of such physical-chemical explanations, if only because they can be tested in future experiments. One practical ramification of the opposing effects of temperature and pressure is that deep-sea water samplers designed to maintain in situ pressure during recovery !Jannasch et aI., 1976; 1982; Tabor et aI., 1981) may not require heavy insulation against temperature increases in order to recover barophiles. Past criticism of poor insula-

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taxonomic tests that could be applied readily to an obligate barophile, to assess the phylogenetic position of strain BNL-1. Sequence information available two years ago indicated that BNL-1 was a member of the gamma 3 subdivision of the Rhodobacteria (vibrio-enteric group) (Stackebrandt and Woese, 1984) and that it shared closest common ancestry with Vibrio psychroerythrus (MacDonell and Colwell, 1985). A common identifying characteristic of 5S rRNAs of the vibrio-enteric group is an eight base-pair helix 5 with a 3 pyrimidine loop "e". The 5S rRNAs from BNL-1 and V. psychroerythrus have primary structures that imply a seven base-pair helix 5 with a 4 mixed-base (pyrimidine, purine) loop "e" (Fig. 5). This shortened helix 5 and mixed loop "e" have been found in only one other genus of the vibrio-enteric group, i. e., in Aeromonas species (MacDonell and Colwell, 1985). However, earlier sequence comparisons (MacDonell and Colwell, 1985) and those made by the more refined approaches of this study (Fig. 4) preclude a specific relationship between the aeromonads and BNL-1 and V. psychroerythrus. The evolutionary tree analysis of this study (Fig.4a) indicates that 5S rRNA sequences of BNL-1 and Vibrio psychroerythrus are sufficiently distinct from all other known groups, including the genus Vibrio (MacDonell and Colwell, 1985), to establish them in a new genus. We propose that this new genus be named Colwellia, that obligate barophile BNL-1 be designated Colwellia hadaliensis, gen. nov. sp. nov., and that V. psychroerythrus be renamed Colwellia psychroerythrus comb. nov. (see descriptions below). What significance can be inferred from the observation that an obligately barophilic psychrophile from the bottom of the Puerto Rico Trench shares closest common ancestry with a barosensitive psychrophile from shallow Norwegian waters? Perhaps the genetic "distance" between barophily and psychrophily is not great: the strongest common trait expressed by both BNL-1 and "V." psychroerythrus is a strict growth requirement for low temperature « 15°C for BNL-1; < 20°C for "V." psychroerythrus). Or perhaps "V." psychroerythrus is (or was) barophilic, but failed to express barophily in our tests. In the complete absence of information on the molecular-genetic basis of true barophily, these possibilities remain unresolved. However, at this time, with 5S rRNA sequence information from two genera encompassing barophilic bacteria (Shewanella and Colwellia) , it does not appear that the barophilic trait itself represents a fundamental divergence of deep-sea bacteria from all other marine and non-marine bacteria, as has been hypothesized (Yayanos et ai., 1981). No specific relationship between BNL-1 and S. benthica is apparent (Fig.4a) and no uniquely shared structural feature of the 5S rRNA molecule, potentially related to preferential functioning under elevated pressure (Deming et ai., 1984), has yet presented itself. Of course, this view (and that of current relationships among barophiles and non-barophiles) may become refined as additional strains, sequence information, and other data become available for analysis. For example, the advanced treeing algorithm

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used to generate Figure 4a has revealed Shewanella benthica and the non-barophilic bacterium, S. putrefaciens, to be less closely related than suggested originally by conventional UPG cluster analysis (MacDonell and Colwell, 1985). Though the evolutionary distances, or lengths of "branches," on an evolutionary tree are calculated values, the rotation at any given branch point is unknown. How a tree is depicted on a flat sheet of paper (what species names, or "leaves" on the different branches, appear next to each other) depends on drafting decisions to avoid branch-crossings and super-position of one cluster of leaves over another. Principal coordinates analysis is an ordination procedure that enables examination of a "swarm" of data, such as comparative sequence information, in higher order space, thus revealing potential rel:itionships not otherwise evident in a two-dimensional treatment (please see theoretical discussions and examples in Dunn and Everitt, 1982; Pielou, 1984; Austin and Priest, 1986; and MacDonell et ai., 1986). The hyperspatial plots of Fig. 4b and c, based on the first four principal coordinates, (necessarily) portray the same generic clusters observed by evolutionary tree analysis (Fig.4a), but new relationships betweeri clusters are evident. Of the 23 species included in this ordination procedure, the one plotting most closely to obligate barophile BNL-1 (besides its nearest common ancestor, "V." psychroerythrus) in the most significant of the vector comparisons (P1 versus P2 in Fig. 4b and c; see Dunn and Everitt, 1982) was the only other barophilic species, Shewanella benthica. Since BNL-1 and S. benthica do not share a close common ancestry, we interpret these results as visual evidence of the convergent evolution of barophily in the two genera. If correct, then future barophilic bacteria, regardless of genus or species, will cluster in hyperspace with BNL-1 and S. benthica, making ordination analysis by prin~iple coordinates a powerful approach to this type of mqUIry. Description of the Genus Colwellia gen. nov.

(Colwel'li'a. M. L. ending -ia. M. L. fem.n. Colwellia; named after Rita Colwell to honor her work in the systematics of marine bacteria). Curved or straight ro~s 0.5-1.0 !lm wide and 2.5-5.0 !lm long, Gram-negative, asporogenous, motile by polar flagella, oxidase and chitinase positive, chemo-organotrophic, facultatively anaerobic, associated with cold marine habitats. G+C mol% 40-46. Type species: Colwellia psychroerythrus, comb. nov., the red-pigmented marine psychrophile formerly known as Vibrio psychroerythrus (D'Aoust and Kushner, 1972). Genus also includes Colwellia hadaliensis sp. nov. Description of Colwellia hadaliensis sp. nov.

(ha'dah'en'sis. M. L. adj. ending -iensis. Eng. adj. hadal; used in oceanographic terminology; of, or pertaining to, the greatest depths of the ocean; derived from Gr. n. Hades, god of the underworld). Curved rods 0.8 !lm wide and 3.0-5.0 !lm long, Gram negative, highly refrac-

160

]. W. Deming, L. K. Somers, W. L. Straube, D. G. Swartz, and M. T. MacDonell

tile, asporogenous, motile by polar flagella. Oxidase and chitinase positive, chemo-organotrophic, facultatively anaerobic, G+C mol % 45.7. Growth psychrophilic, requiring temperatures less than 15 °C, and obligately barophilic, requiring hydrostatic pressures of at least 300-1,020 atm atrC and 500-1,020 atm at 10°C. Cell lysis at 2°C and < 200 atm and at 10 °C and < 400 atm, absolute upper pressure limits for growth and survival unknown. Associated with cold « 4 0q, hadal (depths of 6,000-11,000 m) marine waters. Type strain: BNL-l. Acknowledgements. Primary support for this research was provided by US National Science Foundation Grant OCE-8300371 UWD) with additional funds from the Johns Hopkins University UWD and LKS) and University of Maryland Graduate School (WLS), Sea Grant College (DGS), and Center of Marine Biotechnology within the Maryland Biotechnology Institute UWD and MTM). We thank Gil Rowe for use of his sediment traps and, along with Philippe Crassous, Alexis Khripounoff, Steve Macko, Chuck Somerville, John Tietjen, Rick Wilke, and the officers and crew of the RN ISELIN, cheerful assistance at sea. We are also grateful to Tim Maugel at the University of Maryland, Department of Zoology, for expert assistance with transmission electron microscopy, and to John Baross and two anonymous reviewers for thoughtful comments on an earlier version of the manuscript. This is COMB contribution No. 100.

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Dr. Jody W. Deming, Chesapeake Bay Institute, The Johns Hopkins University, 4800 Atwell Road, Shady Side, Maryland 20764, USA