Psychroserpens burtonensis gen. nov., sp. nov., and Gelidibacter algens gen. nov., sp. nov., psychrophilic bacteria isolated from Antarctic lacustrine and sea ice habitats

INTERNATIONAL JOURNAL OF SYSTEMATIC BACTERIOLOGY, July 1997, p. 670-677

Copyright 0 1997, International Union of Microbiological Societies 0020-7713/97/$04.00 + 0

Vol. 47, No. 3

Psychrosepens burtonensis gen. nov., sp. nov., and Gelidibacter algens ~

gen. nov., sp. nov., Psychrophilic Bacteria Isolated from Antarctic Lacustrine and Sea Ice Habitats

JOHN P. SHAREE A. McCAMMON,' JANELLE L. BROWN,' PETER D. NICHOLS,1'3 AND TOM A. McMEEKINl.'

Antarctic CRC' and Department of Agiicultural Science, University of Tasmania, and CSIRO Division of Marine Research, Hobart, Tasmania 7001, Australia

Psychrophilic, yellow-pigmented, seawater-requiring bacteria isolated from the pycnocline of meromictic Burton Lake and from sea ice cores obtained in the Vestfold Hills (68"S, 78"E) in eastern Antarctica were characterized. Phenotypic analysis showed that the strains isolated formed two distinct taxa. The first taxon included nonmotile, nutritionally fastidious strains that were isolated from the pycnocline of Burton Lake. The cells of these strains were morphologically variant, ranging from vibrioid to ring shaped to coiled and filamentous; in addition, the strains were unable to metabolize carbohydrates or polysaccharides and had DNA G+C contents of 27 to 29 mol%. The strains of the second taxon, which were isolated from sea ice cores and from ice algal biomass, were saccharolytic, exhibited rapid gliding motility, were rodlike to filamentous, and had DNA G+C contents of 36 to 38 mol%. A 16s ribosomal DNA (rDNA) sequence analysis revealed that the two Antarctic taxa formed related but distinct lineages within the [Flexibacter] maritimus rRNA branch of the family Flavobacteriaceae. The levels of 16s rDNA sequence similarity between the taxa were 90.5 to 91.3%, while the levels of similarity to other members of the [F.] maritimus rRNA branch were 85 to 90%. The whole-cell lipid profiles of the Antarctic strains were mainly comprised of branched and unbranched monounsaturated C,, to C,, fatty acids. The presence of significant levels of the lipids al5:lwlOc and a17:lm7c appeared to be useful biomarkers for the new Antarctic taxa and for differentiating these organisms from other members of the family Flavobacteriaceae. On the basis of polyphasic taxonomic data we propose that the new taxa are novel bacterial species designated Psychroserpens burtonensis gen. nov., sp. nov. (type strain, ACAM 188) and Gelidi- bacter algens gen. nov., sp. nov. (type strain, ACAM 536).

The coastal sea ice and pack ice habitats of Antarctica are colonized by bacteria (20) and support a predominantly psychrophilic bacterial population (11, 23, 38) that is most active within the ice following the annual spring bloom of sea ice diatoms (26). Sympagic bacteria are responsible for the major- ity of the secondary productivity in sea ice, and the populations peak with the highest levels of sea ice diatoms (27). During winter most biological activity in sea ice is of bacterial origin, and the polar winter darkness forces ice diatoms into a state of decline and dormancy (23). The dominant heterotrophic bacterial taxa in sea ice determined in a recent biodiversity survey (6) include Shewanella, Colwellia, Psychrobacter, and Pseudo- alteromonas species, gram-positive micrococci, and many pigmented marine Cytophaga-like and Flavobacterium rod-shaped and filamentous bacteria. Many of the sea ice bacteria that have been isolated appear to represent novel species and genera. There are some indications that many marine psychrophiles are closely associated with or are frequently epiphytes of the sea ice and pelagic phytoplankton (4,6, 18) that proliferate in Antarctic coastal sea ice, pack ice, and ice edge zones.

By comparison, the seawater environment underneath the sea ice exhibits lower bacterial biodiversity, and the bacteria are more metabolically quiescent (20, 23). The cultivable heterotrophic microbial community is dominated by psychrotol- erant Psychrobacter, Pseudoalteromonas, and Halomonas species and other primarily oxidative gram-negative bacteria (4, 10, 53). Psychrophilic bacteria are fairly uncommon and ac-

* Corresponding author. Mailing address: Antarctic CRC, GPO Box 252-80, Hobart, Tasmania 7001, Australia. Phone: 61 002 26 2776. Fax: 61 02 26 2973. E-mail: [email protected].

count for less than 10% of the total population (11). Antarctic meromictic lakes, fjords, and partially enclosed bays and inlets exhibit relatively stable hydrography and therefore offer more appealing habitats for psychrophiles (38); this is due in part to the increased availability of nutrients from continental inputs, pelagic algal blooms, and breakout of sympagic algae into the water column from ice melting in the spring and summer. Burton Lake, a meromictic body of water connected by a narrow inlet to Ellis Fjord in the Vestfold Hills ice-free zone in eastern Antarctica, harbors a relatively high proportion of psychrophiles (17, 32). A number of psychrophilic strains isolated from the pycnocline (depth, 10 to 11 m) of Burton Lake have been characterized; these organisms include [Flectobacillus] glomeratus (names in brackets are generically misclassified) (32, 33). Other psychrophilic, gas-vacuolate bacteria have also been isolated from congelation ice and seawater in McMurdo Sound, Antarctica (19, 25).

Strains isolated previously from Burton Lake (32) and more recently from the sea ice of Ellis Fjord and ice diatom biomass (6) include a variety of yellow-pigmented, rod-shaped or filamentous, seawater-requiring bacteria. A polyphasic taxonomic analysis (50) showed that these Antarctic strains represent two distinct and novel taxa related to [Flexibacter] maritimus and [Flectobacillus] glomeratus in the family Flavobacteriaceae. In this paper we describe Antarctic strains belonging to two novel taxa, Psychroseipens burtonensis gen. nov., sp. nov., and Geli- dibacter algens gen. nov., sp. nov.

MATERIALS AND METHODS

Strain cultivation. The strains studied are listed in Table 1. Strains ACAM 167, ACAM 181, and ACAM 18gT were originally isolated by McGuire (32). The remaining strains were isolated by procedures described by Nichols et al. (37).

670

VOL. 47, 1997 NEW PSYCHROPHILIC BACTERIA 671

TABLE 1. Strains investigated in this studv

Strain" Source

ACAM 167 ............................... Burton Lake pycnocline, Antarctica ACAM 181 ............................... Burton Lake pycnocline, Antarctica ACAM 18gT ............................. Burton Lake pycnocline, Antarctica ACAM 536T ............................. Sea ice, Ellis Fjord, Antarctica EFDBMS3 ................................ Sea ice, Ellis Fjord, Antarctica

GFSIOTS5 ............................... Sea ice, Ellis Fjord, Antarctica ACAM 550 ............................... Ice alga biomass, Ellis Fjord, Antarctica

HFSIOMB ................................ Sea ice, Ellis Fjord, Antarctica

A297 .......................................... Ice alga biomass, Ellis Fjord, Antarctica A373 .......................................... Ice alga biomass, Ellis Fjord, Antarctica

ACAM 171T ......................... Burton Lake pycnocline, Antarctica

ACAM 74T ........................... Beach mud, Limon, Costa Rica

ACAM 75T ........................... Seawater, Aberdeen, Scotland

ACAM 538T ......................... Marine sediment

ACAM 551 ............................... Ice alga biomass, Ellis Fjord, Antarctica [Flectobucillus] glomerutus

[Cyrophugu] hticu

[Cyfophugu] murinofluvu

[Cytophugu] uliginosu

ACAM, Australian Collection of Antarctic Microorganisms, Antarctic Cooper- ative Research Center, University of Tasmania, Hobart, Tasmania, Australia.

All strains were cultivated routinely on R2A agar (Oxoid Ltd., Basingstoke, United Kingdom) prepared with 3.5% (wt/vol) Ocean Nature seawater salts (Aquasonic, Levington, New South Wales, Australia) or on marine 2216 agar (Difco Laboratories, Detroit, Mich.) and incubated at 10°C.

Phenotypic characterization. Phase-contrast microscopy was utilized to deter- mine cellular morphological features, the presence of gliding motility, and the presence of gas vesicles (25). Phenotypic tests used to further characterize the strains have been described previously (5). Additional tests included the dextran hydrolysis test, which was performed on blue dextran agar (34) supplemented with 0.2% yeast extract and seawater salts. The bathochromic shift test with 20% (wt/vol) KOH was performed to detect flexirubin pigmentation (14). Carbon source utilization tests were performed by using a medium containing 1 g of ammonium chloride, 2 g of Tris-HC1,2 ml of 1 M sodium phosphate buffer (pH 6.8), 2 g of yeast extract, 2 ml of SL-10 trace element mineral salts (40), 10 ml of vitamin solution no. 6 (47), and 1% purified agar (Oxoid) prepared in seawater.

Pigment analysis. Cells of strains grown on marine 2216 agar plates in the dark were harvested and extracted with ethanol, and the cellular residue was removed by centrifugation. Extracts were then dried under a stream of nitrogen, and the residue was redissolved in n-hexane and scanned with a model GBC 916 spec- trophotometer (GBC Scientific, Ltd., Dandenong, Victoria, Australia) to deter- mine absorbance maxima. The contents of the extracts were also compared by silica gel thin-layer chromatography as described by Lewin and Lounsbery (29) by using a 9:l mixture of acetone and petroleum ether (boiling point, 30 to 60°C) as the solvent system.

Genotypic characterization. Genomic DNAs were extracted from the isolates and purified (30). The DNA G+C contents were then determined by using the thermal denaturation procedure (46). Genomic DNAs were also sheared by sonication (to produce an average fragment size of 1 kb) for DNA-DNA hybridization experiments in which the spectrophotometric renaturation rate procedure was used (24). The buffer solution used for the DNA-DNA hybridization experiments was 3X SSC (0.45 M NaCl plus 0.045 M trisodium citrate, pH 7.0).

Whole-cell lipid profiles. The modified one-phase chloroform-methanol-water method was employed for quantitative lipid extraction (51). The chloroform phase was brought to dryness with a rotatory evaporator and then saponified with 5% KOH in 80% (vol/vol) methanol at 60°C. The nonsaponifiable neutral lipid fraction was recovered by extraction with hexane-chloroform (4:l). Acidification of the remaining extract liberated fatty acids, which were then extracted with hexane-chloroform (4:l) and methylated to form fatty acid methyl esters (FAME) by incubation in methanol-HC1-chloroform (1O:l:l) at 100°C for 1 h. FAME samples were then diluted with chloroform containing a CZ3 FAME internal injection standard. Samples were also treated with bis(trimethylsily1)tri- fluoroacetamide to convert hydroxy fatty acids to their o-trimethylsilyl derivatives (45). FAME samples were analyzed by gas chromatography and gas chromatography-mass spectrometry. Gas chromatography was performed with a Hewlett- Packard model 5890A gas chromatography equipped with a Hewlett-Packard model 7673A autosampler, a cross-linked methyl silicone fused silica capillary column (50 m by 0.32 mm), and a flame ionization detector; hydrogen was used as the carrier gas. Selected FAME samples were also analyzed with a Hewlett- Packard model 5970 mass selective detector fitted with a direct capillary inlet. Identification of fatty acids was achieved by using retention time data and molecular ion-fragment data obtained by gas chromatography and gas chroma-

tography-mass spectrometry, respectively. The locations and configurations of double bonds in monounsaturated FAME were determined by gas chromatography-mass spectrometry of their dimethyldisulfide adducts as described by Ni- chols et al. (39). Below, double bond positions are indicated from the methyl (0)

end of the fatty acid. The prefixes i and a indicate is0 and anteiso branching, respectively, and the suffixes c and t indicate cis and frans isomer configuration, respectively.

16s rDNA sequencing and analysis. The 16s ribosomal DNA (rDNA) gene sequences of strains ACAM 188T, ACAM 181, ACAM 167, ACAM 536T, ACAM 550, and ACAM 551 were determined. The PCR conditions used, the cloning procedures used with the T-pGem vector system (Promega, San Diego, Calif.), and the plasmid purification procedure used subsequently have been described previously (12). A PRISM Ready Reaction dideoxy termination kit or an Ampli-Taq dye M13 primer cycle sequencing kit (Applied Biosystems, Foster City, Calif.) and a model 377A automated DNA sequencer (Applied Biosystems) were utilized to obtain DNA sequence information. The 16s rDNA sequences obtained were nearly complete and 1,450 to 1,470 bp long.

The 16s rDNA sequences determined for the Antarctic strains were compared to the sequences in the GenBank nucleotide database by using BLAST searches available through the United States National Institutes of Health internet site. Sequences were downloaded from the GenBank database and were manually aligned with the sequence data determined in this study. A potentially ambiguous alignment of hypervariable sequence regions was minimized by using only the most closely related 16s rDNA sequences available and by confirming align- ments with previously published 16s rDNA secondary structures (21). PHYLIP (version 3.57~) (16) was used for subsequent analyses. For each 16s rDNA sequence, 1,470 to 1,490 nucleotides from nucleotide positions 7 to 1498 (Esch- erichiu coli numbering) were compared. Similarity values were determined with the DNADIST program by using the maximum-likelihood algorithm option. The program FITCH was then used to obtain the best unrooted phylogenetic tree, with the [Cytophugu] uliginosu sequence serving as the outgroup sequence. For a bootstrap analysis with 1,000 sample replications the programs SEQBOOT and CONSENSE were used. A phylogenetic tree was generated by using the program DRAWGRAM.

Nucleotide sequence accession numbers. The 16s rDNA sequences used for the phylogenetic analysis included the sequences of [Cyfophugu] laferculu (Gen- Bank accession no. D12665), [Cyfophugu] hficu (D12666), [Cyfophugu] rnurino- flavu (D12668), [Cytophugu] uliginosu (D12674), [Fluvobucteriurn] gondwunense (M92278), [Fluvobacteriurn] sulegens (M92279), [Flectobucillus] glornerutus (M58775), [Flexibucter] rnurifirnus (D14023), gas-vacuolate strain 23-P (M61002), and gas-vacuolate strain 301 (U14586). The 16s rDNA sequences generated in this study have been deposited in the GenBank nucleotide database under accession no. U62911 to U62916.

RESULTS

Phenotypic characteristics. Colony and cellular morphological characteristics readily distinguished the Antarctic strains investigated into two groups. The first group included strains isolated from Burton Lake, while the second group included strains isolated from sea ice and sea ice algal biomass samples (Table 1). On marine 2216 and R2A agar media, the Burton Lake isolates formed yellow-orange- to tan-pigmented colonies that lacked spreading edges and had a viscid texture. The strains were nonmotile. Gliding motility was not observed on any medium tested. Very young cultures formed compact mi- crocolonies with predominantly rod-shaped and curved cells that were 2 to 6 pm long and 0.5 to 0.6 pm wide. As the cultures aged, the cells became more pleomorphic, with vibrioid and sometimes ring-shaped cells predominating (Fig. 1). Longer, highly flexible, coiling cells which were sometimes helical also frequently occurred, and some of these cells were up to 20 p,m long. The bacteria in older cultures tended to transform into nonrefractile, coccoid cells that were 0.5 to 2.0 p,m in diameter. Microscopic investigations revealed no gas vesicles in the cells of any of the Burton Lake strains. The strains were psychrophilic, grew at o"C, grew best at 10 to 12"C, and were unable to grow at temperatures of 20°C or higher. The Burton Lake strains were stenohaline, with no growth occurring on media supplemented with NaCl alone, and thus possibly required divalent cations. The strains also required vitamins for growth and an organic nitrogen source. Yeast extract at a concentration of about 0.2% was sufficient to pro- vide these growth requirements and strongly stimulated growth.

672 BOWMAN ETAL. INT. J. SYST. BACTERIOL.

FIG. 1. Phase-contrast micrograph of P. burtonensis ACAM lMT grown on marine 2216 agar for 3 days at 10°C. The small arrows indicate coccoid cells, while the large arrow indicates a helical cell. Bar = 10 km.

In addition, growth of the strains was stimulated by the presence of Tweens, especially Tween 20 and Tween 40, which were actively degraded. The metabolism of the strains was strictly aerobic, and carbohydrates were not utilized. The strains were weakly proteolytic but were unable to attack uric acid, xanthine, tyrosine, DNA, esculin, or any polysaccharide tested. Additional phenotypic characteristics are shown in Ta- ble 2.

The phenotypic characteristics of yellow-pigmented strains isolated from ice diatoms (primarily Thalassiosira spp. and Nitzschia stellata) that had been filter concentrated from sea ice cores or directly from melted ice cores are shown in Table 2. The sea ice strains formed mucoid, sticky, yellow colonies with spreading, rhizoid edges indicative of gliding motility. Phase-contrast microscopy confirmed that all of these strains glided rapidly on both marine 2216 agar and R2A seawater agar. The cells were generally rodlike, 1 to 4 pm long, and 0.5 pm wide. In young cultures a number of cells were filamentous (with lengths up to 50 Fm), and these cells appeared to fragment into shorter rods (Fig. 2). As the cultures aged, the cells became progressively shorter, with nonrefractile, coccoid cells (0.5 to 2.0 pm in diameter) eventually predominating. Microscopic examination revealed no gas vesicles or ring- shaped or helical cells. The sea ice strains were psychrophilic, grew at O'C, grew best at 15 to 18"C, and were unable to grow at temperatures of 25°C or higher. Seawater was required for growth, but vitamins were not stimulatory or required for growth. No growth occurred in media containing inorganic nitrogen sources, such as nitrate or am- monia. The sea ice strains were also strictly aerobic and were able to hydrolyze starch and dextran. A limited number of carbohydrates were utilized by all of the sea ice isolates (Table 2), and this utilization was accompanied by acid production detectable on Leifson marine carbohydrate medium (28).

Genotypic characteristics. The G+ C contents of the Burton Lake strains ranged from 27.5 to 28.6 mol%. The sea ice strains had G+C contents which ranged from 36.4 to 38.1 mol%. The

DNA-DNA hybridization analysis in which renaturation kinet- ics was used revealed high levels of relatedness among the Burton Lake strains, with DNA reassociation values ranging from 81 to 95% (Table 3). Similarly, high levels of DNA-DNA hybridization (>85%) were found among the sea ice strains (Table 3). No significant DNA reassociation between the Bur- ton Lake and sea ice isolates was detected, which is consistent with the differences in phenotype and DNA G+C content between the taxa. No significant DNA reassociation between the Antarctic isolates and [Flectobacillus] glomeratus ACAM 171T was detected.

Chemotaxonomic properties. Both taxa had fatty acid profiles which featured high proportions of branched-chain fatty acids. Polyunsaturated fatty acids were not detected (Table 4). The presence of a wide variety of C,, fatty acids was particularly evident in all profiles. The Burton Lake strains were distinguished from the sea ice strains by having higher levels of 15:106c, 15:1w4c7 i16:lwllc7 and 16:1w5c7 whereas the sea ice strains had much higher levels of i16:lw6c7 i16:0, 16:1w7c7 and a17:1w7c. The lipid profiles were similar in containing equiv- alent levels of i15:1w1Oc7 a15:lw10c7 i15:0, a15:0, 15:lwllc, and 15:O (Table 4). [Flectobacillus] glomeratus ACAM 171T was readily differentiated from the Antarctic strains by its higher levels of i15:O and 3-OH i15:O. [Flexibacter] maritimus was distinguished from the Antarctic strains by its high levels of 2-OH i15:O and 3-OH i15:O. In addition, [Flectobacillus] glomeratus and [Flexibacter] maritimus lacked a15:1~10c7 i17:1w7c7 and 15:lwllc.

The yellow carotenoid pigments of the Antarctic strains, [Flectobacillus] glomeratus, and reference strain [C. ] lytica ACAM 74T had similar wavelength absorbance spectra. The absorbance spectra included peaks at 445 to 450 and 470 to 477 nm and a shoulder at 420 to 425 nm. The silica gel thin-layer chromatography profiles for the same strains included two yellow to orange spots at Rf values of 0.25 and 0.40 when acetone-petroleum ether (9:l) was used as the solvent system. These profiles closely match the carotenoid profiles found for Flexithrrjc dorotheae, which has been shown to contain predominantly zeaxanthin (29). The bathochromic shift test with 20% KOH revealed no flexirubin pigments in any of the Antarctic isolates.

16s rDNA sequences and phylogeny. The almost complete 16s rDNA sequences of six Antarctic strains indicated that members of the Flavobacteiiaceae which group in the vicinity of [Flexibacter] maritimus are the closest relatives of the Antarctic isolates. Levels of 16s rDNA sequence similarity and an unrooted phylogenetic tree (Fig. 3) were determined by using PHYLIP (version 3.57~). Burton Lake strains ACAM 167, ACAM 181, and ACAM 188T formed a cluster and were related at levels of sequence similarity of 98.6 to 99.2%. Sea ice strains ACAM 536T, ACAM 550, and ACAM 551 were very closely related as determined by the 16s rDNA sequences (Fig. 3). The two Antarctic strain 16s rDNA clusters were related at levels of sequence similarity of 90.5 to 91.3%. The 16s rDNA sequences of members of the [Flexibacter] maritimus 16s rRNA branch were 84.5 to 90.1% similar to the 16s rDNA sequences of the Antarctic isolates.

DISCUSSION

From the phenotypic analysis results it is clear that the Burton Lake strains can be distinguished from the strains isolated from sea ice and ice diatoms (Table 2). The sea ice strains are more versatile metabolically overall and have the ability, albeit limited, to utilize a range of carbohydrates and polysaccharides. The ability to hydrolyze dextran is an unusual feature


TABLE 2. Phenotypic characteristics that differentiate the Antarctic isolates and [Flectobacillus] glomeratus

Burton Sea ice [Flectobacillus]

isolates isolates glorneratus ACAM 171T Lake Characteristic

Cell morphology Ring-shaped cells Helical or coiled cells

Gliding motility Growth requirements

Vitamins Amino acids

Growth with 0.5X seawater Growth at 20°C Growth on 1% peptone-

Catalase Oxidase ONPG testb Hydrolysis of

Gelatin Casein L-Tyrosine Esculin Starch Dextran Tween 40 Tween 80

D-Glucose Starch

Utilization of D-Glucose Maltose L-Rhamnose D-Xylose D-Mannitol D-Sorbitol D-Gluconate Glycogen a-Glycerol phosphate Butyrate Isobutyrate Malonate Succinate Glutarate Pimelate Azelate trans- Aconitate Citrate Fumarate Pyruvate DL-Lactate Oxaloacetate L-Cystine 2-Aminobutyrate

G + C content (mol%)d

seawater agar

Acid produced from:

-

-

+ -

+ + V

+ + - -

V

V

+ + + V

V

V

+ c

+ + - - -

+ + + V

V

+ + V

V

+ V

+ V

V

+ + + + -

V

36-38

+, positive for all strains; (+), growth is weak; v, variable among strains; -, negative for all strains. The following traits were positive for all strains: yellow pigmentation; gram-negative staining reaction; growth in 1 X to 1.5X seawater; growth at 0 to 15°C; hydrolysis of Tween 20; and alkaline phosphatase activity. The following traits were negative for all strains: flexirubin pigments; anaerobic growth with or without nitrate; tolerance to or stimulation by ox bile; growth in Ox to 3 x seawater; growth at temperatures of 28°C or higher; nitrate reduction; hydrogen sulfide production (from thiosulfate); hydrolysis of agar, chitin, urate, and xanthine; acid production from L-rhamnose, D-fructose, D-galactose, cello- biose, lactose, maltose, D-melibiose, D-raffinose, sucrose, trehalose, N--acetylglucosamine, D-xylose, adonitol, glycerol, D-mannitol, myo-inositol, and D-sorbitol; arginine dihydrolase; lysine and ornithine decarboxylase; phenylalanine and typ- tophan deaminase; indole production; Voges-Proskauer test; and utilization of eellobiose, D-fructose, D-galactose, lactose, D-mannose, D-melibiose, D-raffinose, sucrose, trehalose, N-acetylglucosamine, glycerol, D-glucuronate, saccharate, acetate, valerate, isovalerate, caproate, heptanoate, caprylate, nonanoate,

expressed by some of the sea ice strains (ACAM 536T, ACAM 550, HFSIOMB, and GFSIOTSS), and this is the first report of dextranolytic activity occurring in marine microorganisms. Dextranase expression by sea ice bacteria suggests that the sea microbial community may harbor microbiota capable of forming dextran. Dextran synthesis is usually confined to plant- and food-associated lactobacilli and oral streptococci (8), and there has been no confirmation that there are dextran-forming strains in marine environments.

Phylogenetic results based on 16s rDNA sequence data support a generalized relationship among the Burton Lake strains, the sea ice strains, [Flaibacter] maritimus, and [Flectobacillus] glomeratus. This conclusion is consistent with the fact that these organisms share some phenotypic and ecophysiological characteristics. All of these bacteria have a yellow carotenoid pigment provisionally identified as zeaxanthin, require seawater, and are relatively restricted metabolically, and several require an organic nitrogen source and/or vitamins. Flexirubin pigments are absent. The marine habitats and lifestyles also roughly correspond as the bacteria actively colonize or probably rely on marine organisms for nutrients. It is likely that the Burton Lake strains, including [Flectobacillus] glomeratus strains, because of their lack of motility and their presence at the pycnocline of Burton Lake, are epiphytes of marine diatoms and dinoflagellates, which frequently bloom in this lake, in Ellis Fjord, and at other coastal Antarctic sites (7, 42). Filamentous bacteria have been observed colonizing the surfaces of diatoms and dinoflagellates common in Antarctic coastal waters (31, 48). The gas-vacuolate strains (18, 25) are probably also alga associated, although are most likely not epiphytic. Instead, they apparently use their gas vesicles to stay close to phytoplankton in the water column, particularly at the sea ice-seawater interface, where diatoms concentrate (18). Sea ice strains ACAM 550, EFDBMS3, HFSIOMB, and GFSIOTS5 were directly isolated from algal material obtained from melted sea ice. In contrast, [Flaibacter] aurantiacus subsp. copepodarum, [Flexibacter] maritimus, and [Flaibacter] ovolyticus tend to colonize the surfaces of copepods, fish eggs, and the skin and extremities of marine fish, respectively (1,22).

Fatty acid profiles have been useful for defining a number of genera within the Flavobacteriaceae. This is because of the diversity of branched-chain fatty acids in members of this group (15,41, 44). The fatty acid profiles of the two groups of Antarctic strains correlate well with the moderately close phylogenetic association of these taxa. The Antarctic strains contain some fatty acids which make them stand out from other marine gliding and filamentous bacteria. The fatty acid a15: 101Oc (and a17:107c in the sea ice strains) appears to be a useful signature fatty acid for the novel taxa as it is quite rare among members of the Flavobacteriaceae (2,3,9,15,35,41,43, 44, 49, 52).

The Antarctic strains are related to members of the family Flavobacteriaceae on the basis of phylogenetic data; this family, particularly the genera Flavobacterium and Cytophaga, has recently undergone substantial reclassification (3, 36). Phyloge-

3-hydroxybutyrate, 2-oxoglutarate, L-malate, L-alanine, L-asparagine, L-aspartate, L-histidine, L-leucine, L-ornithine, L-phenylalanine, hydroxy-L-proline, L-threo- nine, L-tyrosine, putrescine, and urate. The following traits were variable for the sea ice strains and were negative for the Burton Lake strains: acid production from L-arabinose, D-mannose, and dextran; and utilization of L-arabinose, rn- inositol, propionate, adipate, L-glutamate, and L-proline. Utilization of L-serine was variable for the Burton Lake strains and was negative for the sea ice strains.

ONPG, o-nitrophenyl-P-D-galactopyranoside. Acid production was delayed andlor weak for some strains. Determined by the thermal denaturation method.

674 BOWMAN ET AL. INT. J. SYST. BACTERIOL.

FIG. 2. Phase-contrast micrograph of G. algens ACAM 53tjT grown on marine 2216 agar for 3 days at 10°C. Bar = 10 pm. The arrows indicate coccoid cells.

netic studies and RNA probing studies have also shown that [Flectobacillus] glomeratus is related to the fish pathogen [Flai- bacter] maritimus, while Antarctic gas vesicle-forming strains 23-P and 301 are closely related to [Flectobacillus] glomeratus (19). The 16s rDNA-based phylogenetic analysis performed in this study showed that the Antarctic strains form two distinct clusters related at similarity levels of 90.5 to 91.3%. From this it can be concluded that the Burton Lake isolates and the sea ice isolates form related but distinct monophyletic lineages. Lower similarity values, ranging from 85 to 90%, were found for other members of the [Flexibacter] maritimus branch (Table 5), which indicates that the Antarctic isolates are not related at the genus level to any of the previously described valid or invalid species belonging to the Flavobacteriaceae.

TABLE 3. DNA-DNA hybridization data for the Antarctic strains and [Flectobacillus] glomeratus

Strain % DNA-DNA reassociation with":

ACAM 188T ACAM 536T

Burton Lake isolates ACAM 18ST ACAM 181 ACAM 167

Sea ice isolates ACAM 536T EFDBMS3 HFSIOMB ACAM 550 A373 ACAM 551

[Flectobacillus] glomeratus ACAM 171T

100 95 81

27

ND 19 21 18

14

N D ~

27 22 23

100 95 98 85 92 88

8

" DNA-DNA hybridization values are the averages of the values from three

' ND, not determined. replicates. Standard deviations ranged from ?5 to +-15%.

TABLE 4. Comparison of whole cell fatty acid profiles for representatives of the Antarctic isolates and related

members of the [Flexibacter] rnan'tirnus 16s rDNA branch

% of total fatty acids"

Fatty acid Burton Lake Sea ice [Flectobacillus] [Flexibacter] isolates isolates glomeratus maritimus

(3 strains) ( 3 strains) ACAM 171Tb (6 strainsy

14:O 15:O 16:O l8:O i13:O i14:O i15:O a15:O il5:lwlOc a 15 : 1 w 1Oc i16:O i16: l w l l c i16:lw6c a17:O i17: lw7c a17:lw7c a17:l or i17:3 15:lwllc 15:lw6c 15: 1 w4c 16:1w9c 16:lw7c 16:lw5c 17: 1 w8c 17: lw6c 18:lw5c

2-OH i15:O 3-OH i15:O 3-OH i16:O

3-OH i17:O 3-OH a17:O

2-OH 15:O

3-OH 1610

0.2 (0.1) 10.1 (2.8) 0.4 (0.1)

tr 0.4 (0.1)

10.0 (1.3) 10.4 (1.0) 14.1 (3.4) 8.4 (2.3) 0.6 (0.5) 9.1 (2.7)

-

- -

1.5 (0.5) t r

6.4 (0.6) 3.5 (0.8) 7.6 (0.8) 0.7 (0.6) 0.4 (0.3) 6.9 (1.9) 1.2 (0.4) 1.5 (0.4)

-

- - - - - - - -

0.3 (0.1) 8.8 (2.5) 1.4 (0.1) 0.2 (0.1)

7.9 (4.3)

- 0.5 (0.2)

13.2 (2.8) 11.3 (3.8) 8.8 (2.9) 8.0 (2.0)

7.7 (2.6) 1.5 (0.1) 4.9 (1.6) 5.2 (0.5)

-

-

3.5 (0.1) -

1.3 (0.1)

5.9 (0.8) 1.5 (0.8)

3.7 (0.8)

-

-

- -

tr 0.5 (0.3) 0.8 (0.6) 0.3 (0.1) 0.4 (0.4) 0.4 (0.4)

1 .o 3.4 1.9 0.4 2.0 4.3

48.7 0.7

14.0 - - - 4.8 - - - - -

5.4 - -

2.9 0.6 tr t r - - - 6.6

0.4

1.2

-

-

- 0.9 (0.2) -

2.0 (0.2) 1.2 (0.2) -

13.0 (1.1) 1.4 (0.2)

11.7 (0.9)d - - -

1.7 (0.2)d - - -

2.5 (0.2)'

4.2 (0.4) -

- - - - - -

1.3 (0.1)

18.9 (1.5) 20.6 (1.5) 7.5 (0.9) 0.9 (0.1) 6.9 (0.8)

1.1 (0.2)

-

" Fatty acids which account for less than 1% of the total fatty acids in all species are not shown. tr, trace (less than 0.1%); -, not detected. The values are averages; the values in parentheses are standard deviations. ' Data for [Flectobacillus] glomeratus ACAM 171T are from reference 38.

Data for [Flexibacter] maritimus are from reference 3. The double bond position was not confirmed. The double bond and branching position were not determined.

Based on polyphasic taxonomic data (51) from phenotypic, chemotaxonomic, genotypic, and phylogenetic comparisons, the Antarctic strains form two taxa which represent novel genera within the family Flavobacteriaceae. A variety of phenotypic traits and DNA G + C contents can be used to differentiate the Antarctic taxa from other marine members of the Flavobacte- n'aceae (Table 5) . Thus, we propose that the Burton Lake isolates should be placed in Psychroserpens burtonensis gen. nov., sp. nov., while the sea ice isolates should be placed in Gelidibacter algens gen. nov., sp. nov.

Description of Psychroserpens gen. nov. Psychroserpens. (Psy. chro.ser'pens. Gr. adj. psychros, cold; L. n. serpens, serpent; M.L. n. Psychroserpens, cold serpent). Gram negative. Mor- phology varies with the age of the culture. Young cultures contain rodlike or vibrioid cells that are 2 to 6 pm long and 0.5 to 0.6 pm wide, and coiled and helical filamentous cells up to 20 pm long also frequently occur. Cells in older cultures eventually transform into nonrefractile, coccoid cells that are 0.5 to 2.0 pm in diameter. Nonmotile. Requires seawater, yeast ex-


[ Cytophaga] marinoflava

[ Cytophaga] lytica

[Flexibactefj maritimus

gas vacuolated strain 23-P

gas vacuolated strain 301

[ F/ectobaci//us] glomeratus 70

r ACAM 188

ACAM 167

r ACAM 550

1

n II & ACAM 537

Burton Lake isolates

sea ice isolates

LACAM 536

[ Cytophaga] lafercula

[Flavobacterium] salegens 1 -i=- [Flavobacferium] gondwanense

[Cytophaga] uliginosa

5%

FIG. 3. Phylogenetic tree based on almost complete 16s rDNA sequences comparing the Antarctic isolates with members of the [Flenibacter] maritimus rRNA branch. The numbers at the branch nodes are bootstrap values expressed as percentages.

tract, and/or vitamins for growth. Psychrophilic. Yellow carotenoid pigments are formed. Flexirubin pigments are absent. Strictly aerobic chemoheterotroph. The major cellular lipids are il5:lwlOc, a15:1~10c, i15:0, a15:0, 15:1011c, 15:lw4c, 15:0, and i16:lwl lc. Member of the family Flavobacteriaceae (located on the [Flexibacter] maritimus 16s rRNA branch). The genus contains one species, Psychroselpens burtonensis, which is the type species.

Description of Psychroserpens burtonensis. Psychroserpens burtonensis (bur.ton.en'sis. M.L. adj. burtonensis, of Burton Lake, Antarctica, the body of water from which the organism was isolated). The characteristics of Psychroserpens burtonensis are the same as those of the genus. In addition, growth occurs

in 0.4X to 1.5X seawater. Psychrophilic, with growth occurring at 0°C and optimal growth occurring at 10 to 12°C. No growth occurs at 20°C. No gas vesicles are formed. Weakly proteolytic. Tweens are attacked, and associated growth stimulation occurs. Phosphatase and catalase are produced. Starch, chitin, agar, dextran, esculin, DNA, L-tyrosine, urate, and xanthine are not hydrolyzed. Negative for oxidase activity, urease activity, nitrate reduction, and hydrogen sulfide production (from thiosulfate), and acid is not formed from carbohydrates. Able to utilize a variety of organic acids, but unable to utilize carbohydrates as sole sources of carbon and energy (see Table 2 for additional phenotypic information). The DNA G+ C content is 27 to 29 mol% (as determined by the thermal denaturation procedure). Colonies on seawater media are circular, raised convex with entire edges and a viscid consistency, 4 to 5 rnm in diameter after 7 to 10 days of incubation, and yellow- orange to tan. No diffusible pigment is formed. The type strain is ACAM 188, which was isolated by McGuire (32) from the pycnocline of Burton Lake, Vestfold Hills, Antarctica.

Description of Gelidibacter gen. nov. Gelidibacter (Ge.li.di. bac'ter. L. adj. gelidus, ice-cold or icy; Gr. masc. n. bacter, rod; M.L. masc. n. Gelidibacter, ice-cold or icy rod). Gram negative. The cells are generally rodlike, 1 to 4 pm long, and 0.5 pm wide. May form filaments up to 50 pm long, which fragment into shorter cells. As the cultures age, cells become progressively shorter, with nonrefractile, coccoid cells that are 0.5 to 2.0 km in diameter eventually predominating. Ring-shaped, coiling, and helical cells are not formed. Gliding motility. Re- quires seawater and an organic nitrogen source (i.e., L-glutamate) for growth. Yellow carotenoid pigments are formed. Flexirubin pigments are absent. Strictly aerobic chemoheterotroph. The major cellular lipids are il5:lwlOc, a15:1wlOc, i15:0, a15:0, 15:0, i16:106c, i16:0, 16:lw7c, and a17:1w7c. A member of the family Flavobacteriaceae (located in the [Flexi- bacter] maritimus 16s rRNA branch). The genus contains one species, Gelidibacter algens, which is the type species.

Description of Gelidibacter algens. Gelidibacter algens (al- 'gens. L. part. adj. algens, feeling cold, referring to the sea ice habitat). The characteristics of Gelzdibacter algens are the same as those of the genus. In addition, growth occurs in 0.4X to 1.5X seawater. Psychrophilic, with growth occurring at 0°C and optimal growth occurring at 15 to 18°C. No growth occurs at temperatures of 25°C or higher. Gas vesicles are not formed. Phosphatase and catalase are produced. Starch, dextran, esculin, DNA, various Tweens, and L-tyrosine (no diffusible pig-

TABLE 5. Differentiating characteristics of P. burtonensis, G. ulgens, and other marine species of the family Flavobacteriaceae"

Gliding Flexirubin Growth Requires Requires Growth Carbohydrate Starch Nitrate G+C

motility pigments at 25°C seawater amino acids On seawater peptone- utilization hydrolysis reduction (mol%)b 'Ontent Species

Psychroserpens burtonensis Gelidibacter algens [Flectobacillus] glomeratus [Flexibacter] maritimus [Flexibacter] ovolyticus (Cytophaga] latercula [Flavobacterium] gondwanense [Flavobacterium] salegens ICytophaga] lytica [Cytophaga] marinoflava [Cytophaga] uliginosa

-

+ + +

+ + + + + + - -

V -

+

-

+ + - -

+ + + + + +

27-29 36-38 33 31-32 29-30 32 36 37-38 32-33 37 42

~~~

a Data from references 1, 13, 22, 29, and 43 and this study. Determined by the thermal denaturation method. -, negative; +, positive; (+), weakly positive; v, variable; +, previously reported results differ. Also has a vitamin requirement.

676 BOWMAN ET AL. INT. J. SYST. BACTERIOL.

ment is formed on tyrosine agar) are degraded. Acid is produced from a limited number of carbohydrates, including L-arabinose, D-glucose, D-mannose, dextran, and starch. Strains may degrade casein and/or gelatin. Chitin, agar, urate, and xanthine are not degraded. Negative for oxidase activity, urease activity, P-galactosidase activity (0-nitrophenyl-P-D-galactopyranoside test), nitrate reduction, and hydrogen sulfide production (from thiosulfate). Able to utilize a variety of organic acids and some carbohydrates, including D-glucose, as sole sources of carbon and energy (see Table 2 for additional phenotypic information). The DNA G+C content is 36 to 38 mol% (as determined by the thermal denaturation procedure). Colonies have spreading edges and a mucoid and sticky consistency, are 2 to 3 mm in diameter after 7 to 10 days of incubation, and are yellow pigmented. No diffusible pigments are formed. The type strain is ACAM 536, which was isolated from a sea ice core collected from Ellis Fjord, Vestfold Hills, Antarctica.

ACKNOWLEDGMENTS

This work was supported by the Australian Research Council and the Antarctic Science Advisory Committee.

We thank Bronwyn Innes and Peter Grewe (CSIRO Marine Labo- ratories) for performing automated DNA sequencing of dye M13 primer reaction mixtures and Jenny Skerratt (Antarctic CRC) and David Nichols for providing source plates for some of the sea ice strains and for assistance in fatty acid analyses.

REFERENCES

1. Bernardet, J.-F., and P. A. D. Grimont. 1989. Deoxyribonucleic acid relatedness and phenotypic characterization of Flexibacter columnaris sp. nov., nom. rev,, Flexibacter psychrophilus sp. nov., nom. rev., and Flexibacter maritimus. Wakabayashi, Hikida, and Masumura 1986. Int. J. Syst. Bacteriol. 3 9

2. Bernardet, J.-F., E. Keroualt, and C. Michel. 1994. Comparative study on Flexibacter maritimus strains isolated from farmed sea bass (Dicentrarchus labrax) in France. Fish Pathol. 29:105-111.

3. Bernardet, J.-F., P. Segers, M. Vancanneyt, F. Berthe, K. Kersters, and P. Vandamme. 1996. Cutting a gordian knot: emended classification and description of the genus Flavobactenum, emended description of the family Flavobacteriaceae, and proposal of Flavobacterium hydatis nom. nov. (ba- sonym, Cytophaga aquatilis Strohl and Tait 1978). h t . J. Syst. Bacteriol. 4 6

346-354.

128-148. 4. Bowman, J. P. Unpublished data. 5. Bowman, J. P., J. J. Austin, J. Cavanagh, and K. Sanderson. 1996. Novel

Psychrobacter species from Antarctic ornithogenic soil. Int. J. Syst. Bacteriol.

6. Bowman, J. P., and D. S. Nichols. Biodiversity and ecophysiology of bacteria associated with sea ice. Antarct. Sci., in press.

7. Burke, C. M., and H. R. Burton. 1988. The ecology of photosynthetic bacteria in Burton Lake, Vestfold Hills, Antarctica. Hydrobiologica 1651-11.

8. Damelin, L. H., G. A. Dykes, and A. Vonholy. 1995. Biodiversity of lactic acid bacteria from food-related ecosystems. Microbios 83:13-22.

9. Dees, S. B., C. W. Moss, D. G. Hollis, and R. E. Weaver. 1986. Chemical characterization of Flavobactenum odoratum, Flavobacterium breve, and Fla- vobacterium-like groups IIe, IIh, and IIF. J. Clin. Microbiol. 23:267-273.

10. Delille, D. 1992. Marine bacterioplankton at the Weddell Sea ice edge, distribution of psychrophilic and psychrotrophic populations. Polar Biol. 12:

11. Delille, D. 1993. Seasonal changes in the abundance and composition of marine heterotrophic bacterial communities in an Antarctic coastal area. Polar Biol. 13:463-470.

12. Dobson, S. J., and P. D. Franzmann. 1996. Unification of the genera Deleya (Baumann et al. 1983), Halomonas (Vreeland et al. 1980), and Halovibrio (Fendrich 1988) and the species Paracoccus halodenitrijicans (Robinson and Gibbons 1952) into a single genus, Halomonas, and placement of the genus Zymobucter in the family HuIomonaduceae. Int. J. Syst. Bacteriol. 46:550- 558.

13. Dobson, S. J., R. R. Colwell, T. A. McMeekin, and P. D. Franzmann. 1993. Direct sequencing of the polymerase chain reaction-amplified 16s rRNA gene of Flavobacterium gondwanense sp. nov. and Flavobacterium salegens sp. nov., two new species from a hypersaline Antarctic lake. Int. J. Syst. Bacte- riol. 43:77-83.

14. Fautz, E., and H. Reichenbach. 1980. A simple test for flexirubin-type pigments. FEMS Microbiol. Lett. 8237-91.

46:841-848.

205-210.

15.

16.

17.

18.

19.

20.

21.

22.

23.

24.

25.

26.

27.

28.

Fautz, E., L. Grotjahn, and H. Reichenbach. 1981. Hydroxy fatty acids as valuable chemosystematic markers in gliding bacteria and flavobacteria, p. 127-134. In H. Reichenbach and 0. B. Weeks (ed.), The Flavobacterium- Cytophaga group. Verlag-Chemie, Weinheim, Germany. Felsenstein, J. 1993. PHYLIP (phylogeny inference package), version 3.57~. University of Washington, Seattle. Franzmann, P. D., P. P. Deprez, A. J. McGuire, T. A. McMeekin, and H. R. Burton. 1990. The heterotrophic bacterial microbiota of Burton Lake, Ant- arctica. Polar Biol. 10261-264. Gosink, J. J., R. L. Irgens, and J. T. Staley. 1993. Vertical distribution of bacteria in arctic sea ice. FEMS Microbiol. Ecol. 102:85-90. Gosink, J. J., and J. T. Staley. 1995. Biodiversity of gas vacuolate bacteria from Antarctic sea ice and water. Appl. Environ. Microbiol. 61:3486-3489. Grossmann, S., and G. S. Dieckmann. 1994. Bacterial standing stock, activity, and carbon production during formation, and growth of sea ice in the Weddell Sea, Antarctica. Appl. Environ. Microbiol. 60:2746-2753. Gutell, R. R. 1994. Collection of small subunit (16s- and 16s-like) ribosomal RNA structures: 1994. Nucleic Acids Res. 22:3502-3507. Hansen, G. H., 0. Bergh, J. Michaelsen, and D. Knappskog. 1992. Flexibacter ovolyticus sp. nov., a pathogen of eggs and larvae of Atlantic halibut, Hip- pogfossus hippoglossus L. Int. J. Syst. Bacteriol. 42:451-458. Helmke, E., and H. Weyland. 1995. Bacteria in sea ice and underlying water of the eastern Weddell Sea in midwinter. Mar. Ecol. Prog. Ser. 11:269-287. HUSS, V. A. R., H. Festl, and K.-H. Schleifer. 1983. Studies on the spectrophotometric determination of DNA hybridization from renaturation rates. Syst. Appl. Microbiol. 4184-192. Irgens, R. L., I. Suzuki, and J. T. Staley. 1989. Gas vacuolate bacteria obtained from marine waters of Antarctica. Curr. Microbiol. 18261-265. Kottmeier, S. T., and C. W. Sullivan. 1990. Bacterial biomass and production in pack ice of Antarctic marginal ice edge zones. Deep Sea Res. 3T1311- 1330. Kottmeier, S. T., S. M. Grossi, and C. W. Sullivan. 1987. Sea ice microbial communities. VIII. Bacterial production in annual sea ice of McMurdo Sound, Antarctica. Mar. Ecol. Prog. Ser. 35175-186. Leifson, E. 1963. Determination of carbohydrate metabolism of marine bacteria. J. Bacteriol. 851183-1184.

29. Lewin, R. A., and D. M. Lounsbery. 1969. Isolation, cultivation, and characterization of flexibacteria. J. Gen. Microbiol. 58:145-170.

30. Marmur, J., and P. Doty. 1962. Determination of the base composition of deoxyribonucleic acid from its thermal denaturation temperature. J. Mol. Biol. 5109-118.

31. McConville, M. J., and R. Weatherbee. 1983. The bottom-ice microalgal community from annual ice in the inshore waters of East Antarctica. J. Phycol. 19:431-439.

32. McGuire, A. J. 1985. The microbiota of Burton Lake, Vestfold Hills, Ant- arctica. Honors thesis. Department of Agricultural Science, University of Tasmania, Hobart, Tasmania, Australia.

33. McGuire, A. J., P. D. Franzmann, and T. A. McMeekin. 1987. Flectobacillus glomeratus sp. nov., a curved, non-motile, pigmented bacterium isolated from Antarctic marine environments. Syst. Appl. Microbiol. 9265-272.

34. Mencier, F. 1972. Methode simple et rapide de mise en evidence des micro- organismes producteurs de dextranase. Ann. Inst. Pasteur (Paris) 122:153- 157.

35. Naganuma, T., and K. Horikoshi. 1994. Cellular fatty acids of marine, aga- rolytic bacteria, gliding bacteria. Syst. Appl. Microbiol. 17:125-127.

36. Nakagawa, Y., and K. Yamasoto. 1996. Emendation of the genus Cytophaga and transfer of Cytophaga agarovorans and Cytophaga salmonicolor to Mari- nolabilia gen. nov.: phylogenetic analysis of the Flavobacterium-Cytophaga complex. Int. J. Syst. Bacteriol. 46599-603.

37. Nichols, D. S., P. D. Nichols, and T. A. McMeekin. 1993. Polyunsaturated fatty acids in Antarctic bacteria. Antarct. Sci. 5149-160.

38. Nichols, D. S., P. D. Nichols, and T. A. McMeekin. 1995. Ecology and physiology of psychrophilic bacteria from Antarctic saline lakes and sea ice. Sci. Prog. 78:311-347.

39. Nichols, P. D., J. B. Guckert, and D. C. White. 1986. Determination of monounsaturated fatty acid double-bond position and geometry for microbial monocultures and complex consortia by capillary GC-MS of their di- methyldisulphide adducts. J. Microbiol. Methods 549-55.

40. Overmann, J., and N. Pfennig. 1989. Pelodictyonphaeoclathratifoime sp. nov., a new brown-colored member of the Chlorobiaceae forming net-like colonies. Arch. Microbiol. 152:401-406.

41. Oyaizu, H., and K. Komagata. 1981. Chemotaxonomic and phenotypic characterization of the strains of species in the Flavobacterium-Cytophaga complex. J. Gen. Appl. Microbiol. 2757-107.

42. Putt, M., G. Miceli, and D. Stoecker. 1994. Association of bacteria with Phaeocystis sp. in McMurdo Sound, Antarctica. Mar. Ecol. Prog. Ser. 105:

43. Shivja, S., M. K. Ray, N. Shyamda Rao, L. Saisree, M. V. Jagannadham, G. Seshu Kumar, G. S. N. Reddy, and P. M. Bhargawa. 1992. Sphingobacterium antarcticus sp. nov., a psychrotrophic bacterium from the soils of Schirma- cher Oasis, Antarctica. Int. J. Syst. Bacteriol. 42102-106.

44. Skerratt, J. H., P. D. Nichols, C. A. Mancuso, S. R. James, S. J. Dobson, T. A.

179-1 89.


45.

46.

47.

48.

49.

McMeekin, and H. Burton. 1991. The phospholipid ester-linked fatty acid composition of members of the family Halomonadaceae and genus Flavobac- terium. A chemotaxonomic guide. Syst. Appl. Microbiol. 148-13. Skerratt, J. H., P. D. Nichols, J. P. Bowman, and L. I. Sly. 1992. Occurrence and significance of long-chain (o-1)-hydroxy fatty acids in methane utilizing bacteria. Org. Geochem. 1892-99. Sly, L. I., L. L. Blackall, P. C. Kraat, T. Tian-Shen, and V. Sangkhobol. 1986. The use of second derivative plots for the determination of mol% guanine plus cytosine of DNA by the thermal denaturation method. J. Microbiol. Methods 5139-156. Staley, J. T., J. A. Fuerst, S. Giovannoni, and H. Schlesner. 1991. The order Planctomycetales and the genera Planctomyces, Pirellula, Gemmata, and Zsosphaera, p. 3710-3731. In A. Balows, H. G. Triiper, M. Dworkin, W. Harder, and K.-H. Schleifer (ed.), The prokaryotes, 2nd ed. Springer-Verlag, New York, N.Y. Sullivan, C. W., and A. C. Palmisano. 1984. Sea ice microbial communities: distribution, abundance, and diversity of ice bacteria in McMurdo Sound, Antarctica, in 1980. Appl. Environ. Microbiol. 47:788-795. Vandamme, P., M. Vancanneyt, K. Van Hove, R. Mutters, J. Hommez, F.

Dewhirst, B. Paster, K. Kersters, E. Falsen, L. A. Devriese, M. Bisgaard, K.-H. Him, and W. Mannheim. 1994. Omithobacterium rhinotracheale gen. nov., sp. nov., isolated from the avian respiratory tract. Int. J. Syst. Bacteriol.

50. Wayne, L. G., D. J. Brenner, R. R. Colwell, P. A. D. Grimont, 0. Kandler, M. I. Krichevsky, L. H. Moore, W. E. C. Moore, R. G. E. Murray, E. Stackebrandt, M. P. Starr, and H. G. Triiper. 1987. Report of the Ad Hoc Committee on Reconciliation of Approaches to Bacterial Systematics. Int. J. Syst. Bacteriol. 37:463464.

51. White, D. C., W. M. Davis, J. S. Nickels, J. D. King, and R. J. Bobbie. 1979. Determination of the sedimentary microbial biomass by extractable lipid phosphate. Oecologica 40:51-62.

52. Yabuuchi, E., and C. W. Moss. 1982. Cellular fatty acid composition of strains of three species of Sphingobacteriurn gen. nov. and Cytophaga johnso- nae. FEMS Microbiol. Lett. 13537-91.

53. Zdanowsky, M. K., and S. P. Donachie. 1993. Bacteria in the sea ice zone between Elephant Island and the South Orkneys during the Polish sea-ice zone expedition, (December 1988 to January 1989). Polar Biol. 13:245-254.

4 4 24-3 7.

Documents

Psychroserpens burtonensis gen. nov., sp. nov., and Gelidibacter algens gen. nov., sp. nov., psychrophilic bacteria isolated from Antarctic lacustrine and sea ice habitats