Polaribacter Gen. Nov., with Three New Species, P

lnternational Journal of Systematic Bacteriology (1 998), 48, 223-235 Printed in Great Britain

Polaribacter gen. nov., with three new species, P. irgensii sp. nov., P. franzmannii sp. nov. and P. filamentus sp. nov., gas vacuolate polar marine bacteria of the C'ophaga- Flavobacterium-Bacteroides group and reclassification of 'Flectobacillus glomeratus as Polaribacter glomeratus comb. nov.

John J. Gosink," Carl R. Woese' and James T. Staley'

Author for correspondence: James T. Staley. Fax: + 1 206 543 8297 e-mail: [email protected]

Department of Several psychrophilic, gas vacuolate strains of the Cytophage-Flavobacterium- Microbiology, Box 357242, Bacteroides (CFB) phylogenetic group were isolated from sea ice and water University of Washington, Seattle, WA 98195-7242, from the Arctic and the Antarctic. The closest taxonomically defined species by USA 16s rRNA sequence analysis is 'Flectobacillus glomeratus '. However, 'Flc. Department of glomeratus ' is phylogenetically distant from the Flectobacillus type species, Microbiology, University of Flc. major. On the basis of phenotypic, genotypic and 165 rRNA sequence Illinois, 131 Burrill Hall, analyses we propose a new genus, Polaribacter, with three new species, Urbana, IL 61801, USA Polaribacter irgensii strain 23-P (ATCC 700398), Polaribacter franzmannii strain 301 (ATCC 700399) and Polaribacter filamentus strain 215 (ATCC 700397). P. filamentus is the type species of the genus. None of these species exhibits a cosmopolitan or bipolar distribution. This is the first taxonomic description of gas vacuolate bacteria in the CFB group. Additionally, we propose that 'Flc. glomeratus ' be reclassified to the genus Polaribacter as P. glomeratus, comb. nov.

Keywords: Polaribacter gen. nov., Polaribacter irgensii sp. nov., Polaribacter franzmannii sp. nov., Polaribacter Jilamentus sp. nov., Polaribacter glomeratus comb. nov.

INTRODUCTION gas vesicles. In spite of this diversity, only about two percent of taxonomically defined species are described Gas vesicles are small, hollow, proteinaceous struc- as gas vacuolate (producing gas vesicles) (20, 34). tures found in some species of prokaryotes (for a Our laboratory has concentrated on studying gas review, see 35). Gas vesicles provide these organisms vacuolate bacteria isolated from arctic and antarctic with buoyancy in shallow, stratified aqueous environ- sea ice and sea water (1 1, 17, 32). These strains are the ments. Species from seven of the twelve major phylo- first known examples of heterotrophic gas vacuolate genetic groups of Bacteria are known to produce gas marine bacteria. Polar marine bacteria are among the vesicles. These include the alpha, beta and gamma most psychrophilic bacteria known, with some strains Proteobacteria, cyanobacteria, Gram-positive bac- not exhibiting growth above 10 "C (1 7). Gas vacuolate teria, green sulfur bacteria, and Planctomycetes. polar marine species might be expected to be endemic Archaea of the group Euryarchaeota including species to one pole or the other because of their physiological of extreme halophiles and methanogens also produce requirements (shallow, stratified, cold, marine water) and the great distance between the poles (10). Studying these bacteria may provide insight into the biogeo- Abbreviations :CFB, Cytophaga-Flavobacterium-Bacteroides; RDP, Ri bo- soma1 Database Project. graphic distribution of prokaryotes. The GenBank accession number for the 165 rDNA sequence of strain 215 is Our gas vacuolate polar strains were initially grouped U73726. on the basis of fatty acid composition. Some of these

00611 0 1998 IUMS 223 J. J. Gosink, C. R. Woese and J. T. Staley strains were found to belong to the Cytophaga- Vitamin requirements were assayed in a similar manner. In Flavobacterium-Bacteroides (CFB) group by 16s this case the basal medium consisted of SWCm without rRNA phylogenetic analysis (12). This was the first tryptone, beef extract, yeast extract, vitamins or trace report of gas vacuolate members of the CFB group. elements solution, but to which vitamin-free Casamino acids The present study examines the phenotypic, genotypic, (Difco) had been added to a final concentration of 2 g 1-l. Four different preparations of this basal medium were made, and phylogenetic identity of three members of this each of which had all but one of the following vitamins: fatty acid group, two strains from Antarctica (23-P 0.01 pg biotin ml-', 1 pg nicotinic acid ml-', 1 pg thia- and 301) and one strain from the Arctic (215). Other mine. HCl ml-l, or 1 pg pantothenic acid ml-l. Another workers have also reported isolating members of the preparation was made without any vitamins and a final CFB group from polar marine environments (4, 5,26, preparation contained all of the vitamins. Cells were scraped 30). One of these strains, ' Flectobacillus glomeratus', is from Petri plates, washed twice and resuspended to faint included in this study because of its close phylogenetic turbidity in cold SWCm without any vitamins, trace ele- relationship with the gas vacuolate polar strains. ments or nutrients. As in the carbon source experiments, 50 pl of these cell suspensions were inoculated in quadruplicate into the wells of the microtitre plates. Aliquots METHODS (10 pl) of cells were passed through two subsequent serial transfers to fresh wells of the corresponding vitamin type Bacterial strains. Strains were collected from the sea ice and after 34 and 19 d growth, respectively. Growth was scored in water of the Arctic and Antarctica as previously described. the final set of wells 16 d after the last transfer using a Strain 23-P was isolated between 10 and 25 m depth in microtitre plate reader as described above. Penola Strait, Antarctica, in November 1986 (17). Strain 215 was isolated in April 1992 from surface sea water in a small Strains were tested for the ability to grow on various solid lead, 350 km offshore from Deadhorse, Alaska (73" 01' N, and liquid media. Solid media included Nutrient agar (NA) 148" 31' W). Strain 301 was isolated at 25-50 cm from the (Difco), NA + 2.5 % (w/v) NaC1, Trypticase soy agar (TSA) bottom of an ice core at site 5 in McMurdo Sound, (BBL), TSA + 3 % (w/v) sucrose (TSAS), TSA + 3 Oh (w/v) Antarctica, in November 1992 (12). Strain 84-W(gv)l was D-glucose (TSAG), TSA + 2.5 % (w/v) NaC1, Marine agar isolated from water 18 m beneath the ice at site 7 in 2216 (Difco), CLED agar (Difco), MacConkey agar (Difco), Antarctica in December 1987 (32). Strains 215,23-P and 301 Levine EMB agar (Difco), SWCm agar, Mueller-Hinton have been deposited in ATCC under numbers 700397- medium (Difco), peptonized milk agar (PMA) (Difco), MS 700399, respectively. agar and MS agar+3.0% (w/v) NaCl. Liquid media included MSB (26), MSB + 1.5 % (w/v) NaC1, MSB + 3.0 YO Microscopy. Confirmation of gas vesicles in presumptive gas (w/v) NaC1, Marine medium 2216 (Difco) and SWCm. vacuolate strains was made by electron microscopy. Electron micrographs were obtained of unstained whole cells using a Oxidation and fermentation of sugars, alcohols, and related JEOL- 1OOB transmission electron microscope at 60 kV. carbohydrates were tested using SWCm to which had been Colony colour, colony shape, cell morphology, coccoid added 0.5 g Tris base l-l, 0.01 g Phenol red l-l, and 2.5 g agar body formation and coil formation were all examined in cells 1-1 (Difco). The medium was adjusted to pH 7.5 with HC1, grown on SWCm (17) or half-strength MS (22) (also called steamed for 15 min and aliquoted into flasks. The medium Microcyclus Medium 956 by ATCC) plates (for Flecto- was then autoclaved and 0.1 vol. 10% filter-sterilized test bacillus major). carbon source solution was added to each flask. Aliquots (2-5-3.0 ml) were dispensed into 75 x 100 mm tubes. The Fatty acid methyl ester analysis. Whole-cell fatty-acid com- tubes were chilled overnight to 4 "C. Turbid suspensions of positions were determined using the MIDI system as each strain were stab-inoculated into each of the tubes in described previously (12). groups of six. One half of the tubes were then overlaid with Nutritional requirements and physiology. The ability of mineral oil. Tubes were incubated at 10 "C (23-P, 215, 301 strains to grow on a variety of carbon sources was tested and ' Flc. glomeratus'), or 22 "C (Flectobacillus marinus). using SWCm without tryptone or beef extract, and only Turbidity and pH changes were scored 3, 7, 14, 21 and 28 d 0.05 g yeast extract 1-l. In the case of Flc. major, the basal post-inoculation. salts solution (SWCm salts) was diluted 1: 10 as this strain Biochemical tests including urease, lysine and ornithine would not grow in full-strength SWCm. Aliquots (200 pl) of decarboxylase, oxidase, catalase, indole formation, Voges- this base broth along with specific carbon sources were Proskauer reaction, NO, reduction and H,S formation were pipetted into each well of Falcon Microtest I11 96-well tissue conducted by the methods of Smibert & Krieg (31). In these culture plates. The plates were chilled to 4 "C. Cells were tests, distilled water was replaced with the basal salt solution scraped from Petri plates and suspended to slight turbidity in of SWCm. In the case of the NO, reduction assay, SWCm cold SWCm without added nutrients. Aliquots (50 pl) of basal salts were used with the omission of NH,Cl. p- each cell suspension were inoculated in sets of four into the Galactosidase activity was determined by streaking cultures wells of the microtitre plates so that the final concentration onto SWCm agar plates amended with 0.1 mM IPTG and of the test carbon sources was 1.0 g 1-l. Plates were incubated 20 pg X-Gal ml-l. Cultures were incubated at 10 "C (23-P, at 10 "C (1 5 "C for ' Flc. glomeratus') and growth was scored 215, 301 and 'Flc. glomeratus') or 22 "C (Flc. marinus and 7, 14, 21 and 28 d post-inoculation with an EL31 lsx Flc. major). microtitre plate reader (BIO-TEK) at 600 nm. Growth was scored as negative if the OD,,, did not increase twofold Macromolecule hydrolysis by the strains was similarly tested above the initial level, it was scored weak if turbidity using SWCm base (Flc. marinus, ' Flc. glomeratus', 23-P, 21 5 increased to between two- and fourfold greater than the and 301), or on a half-strength MS medium base (Flc major). initial level, and it was scored as positive if the turbidity Aesculin, starch, casein and gelatin hydrolysis were tested by increased fourfold or more over the initial level. the methods of Smibert & Kreig (31). Chitin hydrolysis was

224 International Journal of Systematic Bacteriology 48 Polaribacter gen. nov.

Table 1. Organisms and environmental clone 16s rDNA sequences used in phylogenetic analyses

Species or environmental clone GenBank Source no.

Strain 215 U73726 ATCC 700397 Strain 301 U14586 ATCC 700399 Strain 23-P M61002 ATCC 700398 Strain 84-W(gv)1 NA ' Flectobacillus glomeratus' M58775 ATCC 43844; UQM 3055 Flexibacter maritimus M64629 ATCC 43398; NCIMB 2145 Marine snow associated clone agg50 L 10947 NA Cytophaga marina D12667 JCM 8137 Cytophaga johnsonae strain MYX. 1.1.1 M59051 ATCC 17061 ; DSM 2064; NCIMB 11054 Flavobacterium aquatile M28236 ATCC 11947; DSM 1132 Capnocytophaga canimorsus strain 2340-2-61 L14637 ATCC 35979; CDC 7120 Flavobac ter ium odora tumT M58777 ATCC 4651 ; NCTC 11036 Cytophaga lytica strain LIM-21 M28058 ATCC 23 178 ; DSM 2039 Flavobacterium gondwanense M92278 DSM 5423 ; ATCC 5 1278 Cyt ophaga mar inoflava M58770 NCIMB 397 Weeksella virosa M93 152 ATCC 43766; NCTC 11634 Bergeyella zoohelcum M93 153 ATCC 43767; NCTC 1160 Microscilla aggregans subsp. catalatica M58791 ATCC 23 190 strain HI-3 Ornithobacterium rhinotracheale L19156 LMG 9086 Cytophaga fermentans M58766 ATCC 19072; NCIMB 2218 Cytophaga salminocolor subsp. agarovorans D12654 ATCC 19043; NClMB 2217 Bacteroides splanchnicus L 16496 NCTC 10825 Cytophaga mar in ofla va M58770 NCIMB 397 Flexibacter flexilis strain Lewin M62794, ATCC 23079; IF0 15060 M28056 Flectobacillus major M62787 ATCC 29496; DSM 103 Flectobacillus marinus strain WH-A M62788, ATCC 43824 M2780 1

NA, Not available.

tested using chitin overlay plates (16). Cellulose hydrolysis tation using the spooling technique were employed until was tested both by cellulose overlay plates (31) and by high quality, pure DNA was obtained. Thermal de- examining strips of filter paper in liquid cell cultures for naturation (3 1) was used to determine the mol% G + C from dissolution (1 5). the DNA of the gas vacuolate polar strains. Ethanol extracts of the cells were examined by spectroscopy. DNA hybridization. A thermal renaturation method (8) was One-litre cultures of each strain were grown in SWCm. Cells used to determine the percentage DNA-DNA reassociation were harvested by centrifugation, resuspended in 10 ml cold between the strains and type species. The initial denaturation 95 YO ethanol, and sonicated for 10 s. The sonicated cell temperature was set at 97 "C and the reannealing tem- material was then filtered through a 0.22 pm nitrocellulose perature was set at 63 "C. All pairwise reassociations were GSWP (Millipore) filter and the absorption spectrum be- run six to ten times. tween 250 and 700 nm was determined with an Hitachi U- 165 rDNA cloning, sequencing and analysis. The 16s rDNA 2000 scanning spectrophotometer. The samples were then nucleotide sequence of strain 215 was obtained in the same alkalinized with 0-1 vol. 0.1 M NaOH. The absorption manner as that of strain 301 (12). The sequences of strains spectra were then reobtained and the peaks were examined 23-P and 84-W(gv)l were determined in the laboratory of for a bathychromatic shift, which is a characteristic of Carl Woese (38). The sequence of strain 23-P had been flexirubins (37). previously deposited in GenBank under the name ' Vesicu- G+C content. DNA for mol% G+C determination and latum antarcticum ' or ' Antarcticum vesiculatum ' (Gen- DNA-DNA hybridization was obtained by a hexadecyltri- Bank accession no. M61002). The 16s rRNA sequences methylammonium bromide miniprep method described listed in Table 1 were also included in the analysis. previously (2). Multiple rounds of phenol/chloroform ex- Sequences were submitted to the Ribosomal Database tractions and chloroform extractions with ethanol precipi- Project (RDP; version 4.0 or 5.0) (23) for alignment by the

International Journal of Systematic Bacteriology 48 225 J. J. Gosink, C. R. Woese and J. T. Staley

ALIGN-SEQUENCE program (23). Small adjustments were sequences of strains 301 (12), 23-P (4), and 84-W(gv)l made by manual comparison to secondary structues pro- (9) had been determined earlier. The approximate vided by the RDP (13). Additional, prealigned, 16s rRNA phylogenetic identity of these strains was determined sequences were also obtained from the RDP. BLAST (1) through the RDP’s SIMILARITY-RANK program (27). searches were additionally performed against GenBank to Closely related 16s rRNA sequences were retrieved determine if other closely related sequences existed in this database. The aligned datasets were trimmed to leave only from the RDP and aligned with the gas vacuolate sequence positions between, but not including, the regions polar bacterial sequences. defined by the 8 FPL and the 1492 RPL primers (29). An initial phylogenetic analysis was performed using Phylogenetic trees were generated using PAUP 3.0s (33) for DNADIST NEIGHBOR parsimony analysis, SEQBOOT, DNADIST,NEIGHBOR and FITCH and (7) under a Kimura (7) for distance analysis, fastDNAml (6, 28) for likelihood 2-parameter model (1 8) with the transition to trans- analysis, and DNAML (7) for Kishino-Hasegawa tests. version ratio (T) set at 1.0. The Tvalue of the resulting MacClade 3.05 (24) was used to determine transition and tree, 1-17, with ‘Fk. glomeratus’, Flc. marinus and transversion frequencies. Flexibacter JEexilis as the outgroup, did not change upon subsequent iterations of the analysis. Taxa not closely related to the ‘Flc. glomeratus’ group, and taxa RESULTS not necessary to define the major groups of closely Strains of gas vacuolate polar bacteria were initially related CFB, were removed from the aligned dataset grouped on the basis of whole-cell fatty-acid com- leaving only the 25 sequences shown in Table 1. The position. The principal fatty acids of one group of gas V1 loop (14, 39) of the aligned dataset was also vacuolate polar strains included high levels of several removed because these positions could not be unam- branched hexadecanoic (palmitic) acid derivatives. biguously aligned for all of these taxa. These fatty acids were often found in Flavobacterium and Cytophaga (20). Two of these strains, 215 and 301, ‘ Final ’ phylogenetic analyses were then performed on were investigated in further detail. Two other isolates, this reduced dataset. First, another round of iterative 23-P and 84-W(gv)l, had also been partially charac- neighbour-joining analyses were performed under a terized previously (9, 17). The mean fatty acid com- Kimura 2-parameter model until a stable T value of positions of three different preparations of each of the 1.05 (using the same outgroup as above) was reached. four strains are shown in Table 2. One hundred bootstrap datasets using jumbled sequence addition order and the same parameters as The phylogenetic relationship of these strains to each above were similarly analysed. Likelihood analysis other, and to other previously described species was using fastDNAml(6, 28) was also performed with the determined by 16s rDNA sequence analysis. The T value set to 1.05 and using the ‘empirical base frequencies’ option (Fig. 1). One hundred bootstrap analyses were likewise performed using fastDNA- ml-boot (6, 28) with the jumbled sequence addition. Table 2. Predominant fatty-acid composition of Generalized, unweighted (Fitch) parsimony analysis Polaribacter and related strains was also conducted on the reduced dataset and 100 bootstrap resampled datasets using a heuristic search Percentage of total cellular method (33). The reduced dataset gave rise to four fatty acids” equally parsimonius trees. These trees were analysed with MacClade 3.05 to produce a ‘ typeset’ to correct 23-P 301 215 84-W(gv)l for uneven rates of the 12 types of transitions and transversions. This typeset was calculated using the 13:O is0 255 2 negative natural logarithm of the fraction of each type 15: 1 is0-F-f ND ND ND 22 of nucleotide substitution out of the total tree length. 15: 1 is0-G-f 6 11 12 ND Because of computational expense, however, it was 15:l iso-I/H/13:O 3-OHS ND ND 6 0.4 only possible to analyse the topology of taxa that were 15:O is0 12 9 22 20 closely related to the gas vacuolate polar strains. 15 : 0 anteiso 646 16 MacClade 3.05 was used to collapse these clades to 15: lo6cis 399 ND polytomies. The typeset described above was then 16: lo7cis 294 1 applied to the dataset and a tree with the partially 15 :0 iso-3-OH 38 17 22 18 collapsed clades was analysed using PAUP 3.0s. 15:O 3-OH 10 7 2 ND Each of these tree-building methods produced slightly ND,Not detected. different topologies. The KishineHasegawa test (1 9) *Only fatty acids representing at least 5% of the total fatty was employed to evaluate the relative strength of these acids of at least one of the strains are shown. topologies under a likelihood framework with a T t 15: 1 is0 F, G were resolved but the position of the double value of 1.05. The results show that the topology of the bond could not be specifically identified by the MIDI system. tree produced under a maximum likelihood framework $ This fatty acid was identified as either a congener of 15 : 1 or with T set to 1.05, and the tree produced under a 13:0 3-OH. parsimony framework with the applied typeset were

226 International Journal of Systematic Bacteriology 48 Polaribacter gen. nov.

Growth of these strains was difficult, often requiring 1-3 weeks of incubation on SWCm at 10 "C. Strain 23- - B. splanchnicus P had been previously shown to be capable of growth from - 1.5 to 12 "C (17). Strain 301 was capable of Berg. zoohelcum growing at 4 "C and 10 "C but not at 15 "C. Strain 2 15 0. rhinotracheale was capable of growing at 4-19 "C but growth was weaker at 15 "C and especially so at 19 "C. Other than for strain 23-P, growth at temperatures lower than 4 "C was not tested. All gas vacuolate polar strains grew as long rods or filaments. Unlike the genus, Flectobacillus, however, these rods were not coiled or spiral-shaped (Fig. 2). Older cultures of the polar strains often gave rise to coccoid cells. Polar flagella were noted in strain 23-P (Fig. 2c), but motility was never observed in wet mounts. Strains 21 5 and 301 were never observed to be motile or to possess flagella. Strains 23-P, 215 and 301 all produced gas vesicles whereas ' Flc. glomeratus' did not. Strains 23-P, 21 5 and 301 grew as orange- or salmon- coloured colonies on SWCm agar plates. Pigments extracted from these cells showed absorption spectra similar to those of carotenoids (Fig. 3). Alkalinization 0.1 of these extracts did not lead to a bathychromatic shift, indicating that these pigments are not flexirubins, a feature which is typical of Flavobacteriurn species (37). Fig- 1. Phylogenetic relationship of Polaribacter species and related species. This tree was produced using fastDNAml (6, 28) The ability of these strains to grow on various carbon with a transition to transversion ratio (7)of 1.05. The scale bar sources was tested using a basal medium with 0.05 g represents 0.1 changes per average nucleotide position. yeast extract to stimulate growth. All strains grew Percentage bootstrap support is indicated next to the branches. 1-I The same tree topology was produced using weighted well on yeast extract and Casamino acids. The only parsimony as described in the text. The full names of the taxa other carbon source on which strain 23-P grew was DL- used in this analysis are listed in Table 1. malate. Strain 2 15 grew on L-glutamate and glycerol. Strain 301 grew on glycerol and N-acetyl-p-glucosamine. In contrast to the results shown by others (21, 26), Flc. marinus was not capable of growth on L- both the same (Fig. 1). This tree is both the shortest tartartic acid, DL-malate, citrate, succinate, pyruvate (parsimony) tree and the most likely tree of all those or acetate. This could be the result of differences in produced by the above methods. The Kishine growth conditions between this study and previous Hasegawa test rules out the neighbour-joining and the work. It should be noted, however, that Flc. marinus bootstrap neighbour-joining trees as significantly was capable of excellent growth on other carbon worse than the best tree. The Fitch parsimony, sources. Also, the carbon source tests in this study bootstrap parsimony and bootstrap likelihood trees were conducted in quadruplicate and the results were were all longer and less likely (but not significantly so) scored quantitatively with a sensitive automated than the best tree. microtitre plate reading system. The results of these analyses clearly show that strains Macromolecule hydrolysis tests showed that all of the 23-P, 215 and 301 are most closely related to 'Flc. strains could weakly hydrolyse starch. Strains 215 and glomeratus'. These results also show that ' Flc. glomer- 301 could hydrolyse gelatin, albeit only weakly for atus' is only distantly related to the type strain and strain 21 5. Strain 301 was also capable of hydrolysing species of the genus Flectobacillus, Flc. major'. It was aesculin. None of the strains was capable of growing also revealed that Flexibacter maritimus and Cyto- on the casein medium provided. Although all of the phaga marina are similarly distant from the type strains strains were capable of growing on the chitin and of their respective genera, but these relationships were cellulose overlay plates, none of them induced degra- not investigated in further detail in this study. Strain- dation of these macromolecules. 84-W(gv)l was not closely related to any of the other All of the strains demonstrated the ability to produce gas vacuolate polar strains and it was not analysed acid from a number of sugars, alcohols and other further in this study. The high degree of similarity carbon sources in oxidation/fermentation agar deeps. between 'Flc. glomeratus' and strains 23-P, 215 and Low-level acid production on the basal medium (no 301 as shown in Fig. 1 warranted further investigation carbon source added) was often seen under both by phenotypic and genotypic methods. aerobic and anaerobic conditions. Unfortunately, true

~ International Journal of Systematic Bacteriology 48 227 J. J. Gosink, C. R. Woese and J. T. Staley

Figrn2. Phase micrographs (left) and electron micrographs (right) of (a) strain 215, (b) strain 301 and (c) strain 23-P. Scale bars on the phase and electron micrographs are 10 pm and 1 pm, respectively.

glomeratus’ and Flc. marinus showed distinct acid production on a number of carbon sources which were in contrast to previous studies (21, 26). ‘Flc. glomeratus’ also grew on xylose in contrast to the results of McGuire et al. (26). Again, this may be the result of different growth conditions between our study and those of previous studies. The gas vacuolate polar strains were tested for the ability to grow on various types of media. Both Marine 300 400 500 600 700 medium 2216 (Difco) and SWCm as agar plates and Wavelength (nm) liquid broth allowed good growth of these strains. In addition, strain 215 was capable of limited growth on NA 2.5 NaCl plates. Again, the results for ‘Flc. Fig. 3. In vitro absorption spectrum of an ethanol extract of + YO strain 215 showing the characteristic absorption peaks of glomeratus’, Flc. major and Flc. marinus were slightly carotenoids at approximately 450, 475 and 505 nm with a small different from those listed in the literature (21, 26). As shoulder peak at approximately 425 nm. Alkalinization with shown in Table 3, these differences included the 0.1 vol. 0-1 M NaOH did not lead to any shift in peak positions. inability to grow on some types of media, and the presence of growth on other types of media which has not been reported previously. anaerobic fermentation was difficult to discern in these tests as the low temperatures and long incubation None of the strains showed activity for any of the times may have permitted some diffusion of oxygen biochemical tests employed, with the exception of into the media. However, none of the strains grew on strain 301 for P-galactosidase. This result was not SWCm agar plates at 10 “C in an anaerobic Gas-Pak unexpected as the oxidation/fermentation tests re- jar (BBL). All of the gas vacuolate polar strains were vealed oxidation of lactose by this strain. Unfortu- able to oxidize or produce acid from D-fructose, D- nately, neither of the decarboxylase test media allowed glucose and mannose. None of the strains were able to the growth of these gas vacuolate polar strains. oxidize or produce acid from cellobiose, lactose, Likewise, the H,S medium did not permit the growth dextrin, dulcitol, sorbitol, adonitol, raffinose, methyl of strains 23-P or 301. Again, in contrast to the results a-D-glucopryanoside, melibiose or myoinositol. ‘ Flc. of McGuire et al. (26), ‘Flc.glomeratus’ demonstrated

228 International Journal of Systematic Bacteriology 48 Polaribacter gen. nov.

Table 3. Nutritional, physiological and genotypic properties of Polaribacter and Flectobacillus strains

Data are from this study and others (17, 21, 26). F, Fermentation/acid production in anaerobic tubes; 0, oxidation/acid production in aerobic tubes but not in anaerobic tubes; w, weak result; NG, no growth; NA, results not available; *, difference between the results of this study and previously reported results (21, 26); t, difference between the results reported by McGuire et al. (26) and Larkin & Borrall (21).

23-P 215 301 'Flc. Flc. major Flc. marinus glomeratus '

Cell morphology Filamentous Filamentous Irregular rod Curved rods Curved rods Coils and coils Cell size : Length (pm) 0.848 16-32 416 1.6-3-2 5-240 1.62-4 Diameter (pm) 0.25-0-5 0.5-1.2 0.8-1.6 0.6-0.9 0.8-1 *6 06-0.8 Coil formation - - - + + + Coccoid cells + + + - - + Motility ------Gas vesicles + + + - - - Grain strain ------Colony colour Orange Salmon Orange Orange Orange Pink/orange Colony morphology : Form Circular Circular Circular Circular Circular Circular Elevation Convex Flat /convex Flat/convex Convex Convex Convex Margin Entire Entire Entire Entire Entire Entire Appearance Smooth Smooth Smooth Smooth Smooth Smooth Opacity Translucent Opaque Opaque Opaque Translucent Opaque Texture Butyrous Butyrous Viscous Butyrous Butyrous Butyrous Phase variation ------Utilization as a carbon source: Yeast extract + + + + + + L-Tartartic acid ------* Ethanol - - - - Casamino acids + + + + + - DL-Malate + - - - + - Sucrose - - - + + + L-Leucine - - - - L-Proline - - - - L-Glutamate - + - + D- Ri bose - - - - DL-Asparta te - - - - 2-Oxoglutarate - - - - Fumarate - - - - Citrate - - - - Succinate - - - - Pyruva te - - - - Propionate - - - - Acetate - - - - Benzoate - - - - Glycolic acid - - - - Glycerol - + + - Methanol - - - - N-Acetyl-P-glucosamine - - + - Continued on following page

International Journal of Systematic Bacteriology 48 229 J. J. Gosink, C. R.Woese and J. T. Staley

Table 3 (cont.)

23-P 215 301 'FIc. FIc. major FIc. marinus glomeratus '

Growth on various liquid media: MSB ++ - - MSB + 1.5 Oh NaCl + MSB + 3 Yo NaCl - - + 2216 ++ ++ ++ ++ - ++ SWCm + ++ ++ ++ - ++ Growth on various solid media: NA ++ - NA + 2.5 % NaCl - - ++ TSA + + ++ TSAS + - + TSAG - - - TSA + 2.5 % NaCl - * - ++ 2216 ++ -* ++ CLED - ++* - MacConkey - - EMB +* - SWCm - ++ Mueller-hint on + ++ PMA - * - MS ++ - MS + 3.0 % NaCl - + Half-strength MS ++ - Oxidation/fermentation of various carbon sources : DL-Arabinose + F D- Ri bose + F Xylose + F Rhamnose + F D-Fructose + F D-Galactose + F D-Glucose + F Mannose + F Cellobiose t F Lactose + F Maltose + F Sucrose + F Trehalose + F Dextrin + F Inulin d F Glycerol - - Erythritol - - Dulcitol - - Mannitol - F* Sorbitol - - Adonitol NA - Salicin + F Raffinose + F Methyl a-D-glucopryanoside + F Melibiose + F Myoinositol NA -

230 International Journal of Systematic Bacteriology 48 Polaribacter gen. nov.

Table 3 (cont.)

23-P 215 301 'Flc. Flc. major Flc. marinus glomeratus '

Macromolecule hydrolysis : Aesculin - + + - t + Casein NG NG NG NG - NG Starch W W W + + - Gelatin - W + + + - Chitin ------Cellulose ------Biochemical tests : Urease - - - - + - Lysine decarboxylase NG NG NG - NA NG Ornithine decarboxylase NG NG NG - - W p-Galactosidase - - + - t + Oxidase W - + + + + Catalase + + + W W + Indole formation ------Voge s-Pr o skauer ------Nitrate reduction - - - +* - - H,S formation NG - NG - - - Temperature growth range : - 1.5 "C + NA NA NA NA NA 4 "C + + + + NA NA 10 "C + + + + NA NA 15 "C - + - + NA NA 19 "C - W - + NA NA 21 "C - - - + + + 25 "C - - - - + + Absorbance wavelength of 450 45 1 45 1 45 1 455 NA ethanolic extracts (nm) 475 475 476 48 1 506 506 506 505 505 G + C content (mol%) 31 32_+1 32f 1 33.2 39.5-40.3 34-38

a distinct, albeit low-level ability to reduce nitrate to Finally, strains 23-P, 215, 301, and 'Flc. glomeratus' nitrite . were tested to determine the degree to which their DNAs would hybridize with each other. As shown in In addition to the discrepancies noted above between Table 5, none of these strains shows greater that 34% the results of this study and previously reported work, DNA-DNA hybridization with any of the other there were also several differences between the reported strains. Pairs of bacteria that have 70% or greater results of McGuire et al. (26) and Larkin & Borrall(21) DNA-DNA hybridization are defined as being mem- for Flc. major including cellobiose use, aesculin hy- bers of the same species. However, members of the drolysis and P-galactosidase activity. As with the other same genus may exhibit much lower levels of hybrid- discrepancies noted above, the test conditions em- ization (36). Therefore all of these bacteria may be ployed in some of our studies may have differed members of the same genus, but are not the same slightly from the conditions used in other studies. species. The G + C content of strains 21 5 and 301 as determined by thermal denaturation (3 1) was 32 & 1 mol%, which Description of Polaribacter gen nov. is very similar to the value of 3 1 mol% reported earlier for strain 23-P (17). These values are typical for Polaribacter (Po.lar.i.bac'ter. Polaro M.L. adj. polaris members of the CFB phylogenetic group (3, 20), and pertaining to the geographic poles; bacter from the Gr. similar to the value obtained for ' Flc. glomeratus' (33-2 n. baktron for rod or staff; Polaribacter rod-shaped mol%) (26). bacterium from a polar region). A summary of the major distinguishing features of Psychrophilic or psychrotrophic Gram-negative non- strains 23-P, 21 5, 301 and ' Flc. glomeratus' is given in motile rods. Cell size varies between 2 and 48 pm in Table 4. length and 0.25-1-6 pm in diameter depending on

International Journal of Systematic Bacteriology 48 23 1 J. J. Gosink, C. R. Woese and J. T. Staley

Table 4. Distinguishing features of Polaribacter strains and 'Flectobacillus glorneratus'

23-P 215 301 'Flc. glomeratus'

Cell morphology Filamentous Irregular rod Curved rods Coil formation - - + Gas vesicles + + - Utilization as a carbon source : DL-Malate - - - L-Glutamate + - + Glycerol + + - N-Acetyl-P-glucosamine - + - Oxidation/ fermentation of: DL- Ara bino se F - - D-Ribose NG NG - Xylose, erythritol and - NG - salicin Rhamnose F NG F D-Fructose - F - Lactose - 0 - Maltose - 0 F Sucrose - F F Trehalose - F - Dextrin - - F Inulin - - - Glycerol F - - Hydrolysis of: Aesculin W + - Gelatin W + + P-Galactosidase - + - Growth at 21 "C - - + Absorbance wavelength 45 1 45 1 45 1 of ethanolic extracts (nm) 475 476 506 506 505 F, Fermentation; NG, no growth; 0, oxidation; w, weak result.

Table 5. Percentage DNA-DNA hybridization between these bacteria. Cells can grow on yeast extract and strains 23-PI 21 5, 301 and 'Nectobacillus glorneratus' Casamino acids. G + C content range is 3 1-33 mol%. All strains have been isolated from polar marine 23-P 215 301 environments and grow well at low temperatures. 16s rRNA sequence analysis clearly indicates that 23-P - this genus of bacteria forms a distinct clade of 215 26+ 15 - the Cyt ophaga-Fla vobac ter ium-Bac teroides phylum . 301 34f 12 13f5 - PolaribacterJilamentus is the type species of the genus. 'Flc. glomeratus' 14+6 31 f8 18+5 Description of Polaribacter filamentus sp. nov. Polaribacter Jilamen tus (fil.a .men? us. Jilamen t us from L. v. Jilare to spin; Polaribacter filamentus a fila- growth medium, temperature, and age of the culture. mentous bacterium of the genus Polaribacter). Coccoid bodies are often seen in ageing cultures. May form coils. Cells produce orange-, salmon- or pink- Long rods or filaments measuring 0-5-1.2 pm in width coloured pigments which are not flexirubins. Gas and 16-32 pm in length. Cells are orange- to salmon- vesicles are produced in some strains. Some strains coloured and may produce gas vesicles. This species may be flagellated, but motility has never been ob- can use L-glutamate and glycerol as carbon sources and served in wet mounts. Aerobic. Cells grow well in it can hydrolyse gelatin. It also produces acid from DL- media which contain NaCl. Cells produce acid from a arabinose, rhamnose, D-galactose, D-glucose, mannose number of carbohydrates. Starch is hydrolysed by and glycerol. These cells can grow from < 4-19 "C,

232 lnternational Journal ofSystematic Bacteriology 48 Polaribacter gen. nov. but not at 21 "C. G+C content is 32k 1 mol%. Reclassification of 'flectobacillus glomeratus' to Principal fatty acids include 22 YO15 :0 iso-3-OH, 22 YO Polaribacter glomeratus corn b. nov. 15:O is0 and 12 YO 15: 1 iso-G (position of double bond unknown) when grown on SWCm agar at 10 "C. In addition to the features listed previously (26), this Isolated from surface sea water, 350 km north of species can grow on sucrose and L-glutamate. Produces Deadhorse, Alaska. Type strain of the species is strain acid from rhamnose, D-galactose, D-glucose, mannose, 21 5 (= ATCC 700397). maltose, sucrose and dextrin. Hydrolyses gelatin. Reduces nitrate to nitrite. Oxidase and (weakly) catalase positive. Cells can grow at temperatures from Description of Polaribacter irgensii sp. nov. ,< 4 to 21 "C. Does not produce coccoid bodies. May Polaribacter irgensii (ir.gen'.si.i. irgensii in honour of produce curved rods or coils depending on culture Roar L. Irgens, American microbiologist and polar conditions. Not known to produce gas vesicles. Mol% researcher who first observed gas vacuolate polar G + C is 33-33.2 as determined by thermal denatu- marine bacteria). ration. Additionally, phylogenetic considerations based on 16s rRNA sequence analysis show that this Forms long, thin rods or filaments 0-25-0-5 pm in species is only distantly related to the type species of width and 0.8-48 pm long. Coccoid bodies are often Flectobacillus, Flc. major, but that it is very closely found in most cultures, particularly ageing cultures. related to the genus Polaribacter. Cells are orange coloured and may be gas vacuolate. Have polar flagella but motility has never been observed. Strain 23-P can grow on DL-malate. This species produces acid from a number of carbohydrates DISCUSSION including D-glactose, D-glucose, mannose and glycerol. It is capable of growth from - 1-5-12 "C (17). G+C One of the most notable differences between strains 23- content is 31 mol% (17). Principal fatty acids include P, 215, 301 and ' Flectobacillus glomeratus' is that the 38% 15:O iso-3-OH, 12% 15:O is0 and 10% 15:O former all produce gas vesicles, whereas the latter does 3-OH when grown on SWCm agar at 10 "C. Isolated not. This is the first taxonomic description of gas from sea water near the ice edge in Penola Strait on the vacuolate members of the CFB phylogenetic group of Antarctic penninsula. The type strain of this species is bacteria. Gas vesicles have been found in a number of strain 23-P (ATCC 700398). evolutionarily diverse bacteria including six of the 12 major radiations of Bacteria and both the Cren- Description of Polaribacter franzmannii sp. nov. archaeota and Euryarchaeota (35). The results in this study indicate that species of the CFB phylogenetic Polaribacter franzmannii (franz.man'.ni .i. franzmannii group also produce gas vesicles. in honour of Peter D. Franzmann, Australian microbiologist and polar researcher). The ecological role of gas vesicles in the polar strains Members of this species grow as large rods 0.8-1-6 pm described here is unknown. These bacteria were iso- in width and 4-16 pm in length. Gas vesicles are lated from polar marine waters and sea ice. Gas present in these light to dark orange coloured cells, but vacuolate bacteria are usually found in calm, vertically sometimes sparingly depending on the growth me- stratified aquatic environments (35). Sea ice forms a dium. Cells are capable of growth on glycerol and N- strong vertical stratification that reduces surface mix- acetyl-P-glucosamine. Acid is produced from growth ing (25) of underlying marine waters. In any event it is on D-fructose, D-galactose, D-glucose, mannose, lac- known that the undersurface of sea ice provides a tose, maltose, sucrose and trehalose. Aesculin and habitat for algae, diatoms and bacteria, especially in gelatin are hydrolysed by this species. Cells can grow the spring and autumn. This region of high biological from 4 to 10 OC, but not at 15 "C. Principal fatty activity may be easily reached by bacteria buoyed with < gas vesicles. acids include 17 YO 15 :0 iso-3-OH, 11 YO 15: 1 iso-G (position of double bond unknown) and 9% each of 15:O iso, 15:106cis and 16:107cis when grown on More work on the biogeographic distribution of free- living bacterial species in general is warranted. For SWCm agar at 10 "C. The G + C content as determined microbiologists to understand microbial diversity, they by thermal denaturation is 32 2 1 mol%. Isolated from will need to determine whether endemism occurs, as sea ice in McMurdo Sound, Antarctica. The type well as the range within ecosystems of cosmopolitan strain of this species is strain 301 (= ATCC 700399). and endemic species. Members of the same genus, Phylogenetic, phenotypic and genotypic similarities Polaribacter, occur associated with the sea-ice mi- between ' Flectobacillus glomeratus ' and members of crobial communities at opposite poles. None of the the genus Polaribacter suggest that this species should individual species was found at both poles. These be reclassified as Polaribacter glomeratus. In particu- results show that there is no clear evidence of cosmo- lar, DNA-DNA hybridization between this species politan species in this genus. Greater sampling is and Polaribacter species is 3 1-14 YO,within the range required, however, to conclude whether or not these of values for members of the same genus. species are endemic to their respective poles.

International Journal of Systematic Bacteriology 48 233 J. J. Gosink, C. R. Woese and J. T. Staley

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