Now, a Budding Bacterium from Hypersaline and Heliothermal Ekho Lake

hternational Journal of Systematic Bacteriology (1998), 48, 1363-1 372 Printed in Great Britain

Antarctobacter heliothermus gem nov,, SP. now, a budding bacterium from hypersaline and heliothermal Ekho Lake

Matthias Labrenz,' M. David Collins,' Paul A. Lawson,' Brian J. Tindallf3 Gesche Braker' and Peter Hirsch'

Author for correspondence : Peter Hirsch. Tel: +49 43 1 880 4340. Fax : + 49 43 1 880 2 194. e-mail: phirsch(a ifam.uni-kiel.de

lnstitut fur Allgemeine Four Gram-negative, aerobic, pointed and budding bacteria were isolated from Mikrobiologie, Universitat various depths of the hypersaline, heliothermal and meromictic Ekho Lake Kiel, D-24118 Kiel, Germany (Vestfold Hills, East Antarctica). The cells contained storage granules and formed rosettes. Daughter cells may be motile. Growth required sodium ions. Department of Microbiology, BBSRC Nitrate was reduced to nitrite, and dissimilatory reduction of nitrite was Institute of Food Research, possible. DNase and gelatinase were produced. Glutamate was metabolized Reading Laboratory, with and without an additional source of combined nitrogen. The most Reading RG6 6B2, UK abundant fatty acid was c18:1; other fatty acids present in lower concentrations 3 DSMZ - Deutsche were ClZZ13-OH, C16:1,C16:o, c18:o and C19:ocyc. The main polar lipids were Sammlung von Mikroorqanismen und phosphatidylglycerol and phosphatidylcholine. The DNA base composition was Zellkulturen, D-38124 62-3-6208mol0/o G+C. 165 rDNA sequence comparisons showed the isolates to Braunschweig, Germany be phylogenetically related to the genera Sagittula and Roseobacter. Morphological, physiological and genotypic differences to these and distinct characteristics supported the description of a new genus and a new species, Antarctobacter heliothermus gen. nov., sp. nov. The type strain is EL-21gT (= DSM 11445').

Keywords : Aiztarctohacter gen. nov., Antarctobacter heliothermus sp. nov., a-Proteohacteria, Antarctica, hypersaline lakes

INTRODUCTION cover. In this process, much salt is excluded as brine, which sinks downwards towards lower strata along the The ice-free area of the Vestfold Hills (East Antarctica) shore line. Consequently, zones of stepwise increasing abounds M ith lakes and ponds of various sizes. Several salinity with depth were formed. In Ekho Lake, a of these lakes originated from fjords and, at one time, summer salinity of 2-10%, was measured between 0 were open to the ocean. Thus, seawater determined and 1 m depth, whereas the salinity was 180%, at the their initial salt composition and concentration and, bottom depth of 42 m. At interfaces between zones of most likely, also the composition of their microbial increasing depth, the salt concentration increased communities. As the land rose about 5000-6000 years suddenly to the next higher level. Close to, and below, ago the fjords lost their connections to the ocean and these interfaces temperature increases were measured. became lakes. One such lake is Ekho Lake, which has Thus, these interfaces appeared to act as one-way since lost approximately 10 m of its water column due mirrors, allowing sun radiation to penetrate into the to evaporation; this and a minor influx of snow melt lower layers but preventing its escape due to downward water have resulted in increased salinity. In addition, a deflection. The strata below such interfaces were vertical gradient of salinity exists ; this was explained significantly warmer than the upper ones: the lake is by Ferris ('t al. (9) as follows. The annual freezing of considered to be heliothermally heated. A similar the lake water surface forms several metres of ice situation has been described for Solar Lake (Sinai) (19,

...... 21). Another consequence of separated water bodies Abbreviations: ASW, artificial seawater; FAME, fatty acid methyl ester. with high salinity is meromixis. Wind energy does not The EMBL accession numbers for the 165 rRNA sequences of strain EL-21gT turn over the lower strata in the summer, and during and strain EL-' 72, another representativeout of Ekho Lake, are Y11552 and winter a turnover is prevented by a thick ice cover. The Y11551, respectively. temperature of the lower layers of Ekho Lake remains

~~ ~- ~~ 00769 0 1998 IUMS 1363 M. Labrenz and others at about + 13 "C in winter. During the austral summer, malate, 1.0 g; Ekho Lake water (salinity 98 yo,,), 980 ml; final intermediate strata reached as high as + 19 "C (P. pH after autoclaving 8.6. (11) Ekho Lake water samples Hirsch & J. Siebert, unpublished). Because of these (50 ml) were amended with 1 ml of a filter-sterilized solution differences of environmental parameters at increasing of 12.5 mg Bacto yeast extract in lake water and incubated at 15 "C and at 4.1 ymol photons ms2 SS'. (111) Ekho Lake depths, communities this hypersaline, microbial of water samples (0.5 ml) were spread directly on agar plates of heliothermal and meromictic Ekho Lake were studied. medium PYGV (41) which had been prepared with Ekho To date, only a few bacterial species or genera have Lake water of lo%, salinity. Incubation was at 15 "C and in been isolated from similar Antarctic lakes; some of the dark. Pure cultures were isolated by several dilution these were new. Nearby Organic Lake contained transfers on the corresponding agar media; they were kept as serial transfers on agar slants, lyophilized or deep-frozen at (6, lo), the Halonionas subglaciescola 16 strains of -72 "C. Flavobncterium-C~~toph~iga-Fle,~ihactergroup (4), Flavobacteriurn gondwanense and Flavobacteriurn sale- Bacterial strains. Type strains used for comparisons were of gens (5). Deep Lake contained the new taxon Halo- the following species : Rose0bactc.r dc>nitri$cans (DSM 700 1T), Roseobacter litoralis (DSM 6996') and Roscobactc.r ruhrurn (b Halobacterium') Iacusprofundi (1 5). Other new bacteria came from Ace Lake : Carnobacterium algicola (DSM 10251"). funditurn and Carnohacterium alterfunditurn (12), a Media and culture conditions. Medium PYGV contained spiral-shaped, psychrophilic and fermentative bac- (1-l): 0.25 g each of Bacto peptone, Bacto yeast extract and terium (1 3), a methanogenic and methylotrophic glucose as well as 20 ml HBM and 10 ml Vitamin Solution archaeon, Methanococcoides burtonii (14), and a psy- no. 6 (45). Solid media contained 1.8 % (w/v) Bacteriological chrophilic spirochaete lacking a cell wall (1 1). agar (Gibco). Where needed, liquid and solid media were prepared with 25%, (v/v) artificial seawater (ASW; 28) The presence of two Halonzonas and two Flavo- (strains EL-219T, EL-185, EL-165, R. algicola, R. dmitri- bacterium species in various hypersaline lakes of the $cans and R. litoralis) or, in case of EL-54, with 40%, ASW. Vestfold Hills was studied by James et al. (24), who Before autoclaving, medium PY GV containing ASW was employed immunofluorescence of specific antibodies. adjusted to a pH of 8.5 ; the final pH after autoclaving was These four serogroups were found during summer in 7-2. R2A agar (Difco) was prepared with the corresponding concentration of ASW and was employed to culture the those lakes which exceeded 61 %,, of total salts. The two strains for BIOLOG tests. Bacterial cultures for identifi- Halonzonas spp. made up 40% of the total bacterial cation tests were incubated at 20 "C; the anaerobic cul- numbers, but F. gondwanense contributed only 10 YOof tivation of isolates (at 4-21.5 pmol photons m-2SS') occurred these numbers in Organic and Ekho Lake. The at 16 "C. Biomass for chemotaxonomic studies was grown in microbial communities of Ekho Lake remained largely liquid PYGV+ASW at 20 "C that was bubbled with filter- unknown. sterilized air and harvested in the late-exponential phase after 4-8 d. The morphological diversity, total bacterial numbers and c.f.u. in Ekho Lake were recently studied by P. Microscopy. Cellular morphology was examined by phase- Hirsch, J. Siebert & H. R. Burton (unpublished). These contrast light microscopy of exponential phase broth cul- authors observed 135 prokaryotic morphotypes as tures employing 2 % water-agar-coated slides (3 l). For well as 52 eukaryotic morphotypes. Such an un- transmission electron microscopy, cells were washed with 0.5 M ammonium acetate, negatively stained with 1 YO(w/v) expectedly high morphological diversity in Ekho Lake phosphotungstic acid and observed with a Philips EM 300 prompted us to isolate some 250 bacterial pure cultures electron microscope at 80 kV. from Ekho Lake, and a high taxonomic (genetic) diversity was expected. Additionally, the isolation of Physiological and biochemical characterization. All physio- the major photosynthetic primary producer of Ekho logical tests were performed at 20 "C. Gram-staining occurred with 24 h cultures (39). Catalase production was Lake resulted in the description of a new green detected with 5 % H,O,. Peroxidase and cytochrome oxidase praseophyte, ' Tetraselmis antarctica' (20). This pub- activity was tested as described previously (7, 26). DNA lication describes four Ekho Lake bacterial isolates hydrolysis was indicated by clear zones around colonies on which represent a new genus and species. Bacto DNase Test agar (Difco)+ASW. Amylase and the relation to oxygen were studied as described previously (40). METHODS Cellulase was tested with bacterial suspensions with 4- methylumbelliferyl P-D-glucoside (Sigma) under UV light Water sampling, enrichment conditions and isolations. (366 nm). Motility was examined in hanging-drop pre- Water samples were taken aseptically at each metre of the parations. Additionally, EL-21 9" was examined for the depth profile using a Kammerer sampler at the 42-m-deep presence of flagella by electron microscopy after 1, 2, 3, 4 centre of Ekho Lake (20). Samples were stored on ice until and 8 d incubation in broth. The ability to grow under used for the inoculation of enrichments or plates after return various physical and chemical conditions was investigated in to the Davis Station laboratory (Vestfold Hills). Charac- liquid media and evaluated by determining the protein teristics of these water samples are shown in Table 1. content (1) of triplicate suspensions : osmotolerance in Enrichments were set up in three different ways. (I) A 5 ml PYGV prepared with 0, 10, 20-100, 130 and l50%, ASW Ekho Lake water sample was inoculated into 10 ml sterile was determined after 14 d incubation. The concentration of medium ' 398 ' and incubated for 4 weeks at 15 "C under dim l50%, ASW was achieved by adding NaCl to 1307&, ASW. light (4.1 ymol photons m-2 s-l). Medium '398' had the NaCl tolerance was determined in PYGV prepared with 0. following composition : Hutner's Basal Salts (HBM ; 2), 10-40, 60, 80, 100, 130 and 1500/,, NaCl after 14d in- 20 ml; Bacto yeast extract (Difco), 1.0 g; disodium D,L- cubation. The ability to grow at pH 5.3, 5.9, 6.2, 6-9, 7.8, 8.3

1364 International Journal of Systematic Bacteriology 48 Antarctobacter gen. nov.

Table 1. Characteristics of the original 1989/90 Ekho D-glucose, methanol and methanesulfonic acid. Degradation Lake samples from which the isolates were obtained, of the following substrates was tested with the basal medium and enrichment conditions PYV + ASW (i.e. lacking glucose) : 0.2 YO(w/v) starch, 0.4 % gelatin, 1 YOTween 80 or 0.75% alginate. Production of Depth Salinity Temp. pH Isolate Enrichment bacteriochlorophyll a was tested with suspensions of cells grown in PYGV+ASW and analysed as described pre- (m) (040) ("C) method* viously (38). Methanolic extracts were examined with a ____ ~~ Beckman DU-600 spectrophotometer and by TLC (27). 5 60 + 12.9 8.3 EL-185 I 7 62 + 12.9 8.2 EL-219'" 11 Chemotaxonomy. Analysis of fatty acid methyl esters (FAMEs) was performed on 20 mg freeze-dried cells em- 14 75 + 16.0 8.0 EL-54 111 ploying methods which allowed selective hydrolysis of ester- 16 76 + 16.4 7.8 EL-I65 and amide-linked fatty acids. FAMEs were analysed by GC ~ ~~ ~ TI See Methods. (GC- 14A; Shimadzu) using a 0.2 mm x 25 m non-polar capillary column and flame-ionization detector. The chro- matography conditions were : injector and detector port temperature 300 "C, inlet pressure 80 kPa, split ratio 50: 1, and 9.0 and at temperatures of 3.0, 8-5, 16.0, 20.0, 26.0, 33.5 injection volume 1 pl and a temperature program of 130- and 43.5 "C was also tested. Requirement(s) for the following 3 10 "C at a rate of 4 "C min-l. Hydrogen was the carrier gas. vitamins was tested in six combinations, each lacking one The fatty acids were identified by converting retention times vitamin of Vitamin Solution no. 6: biotin, thiamin hy- to equivalent chain-length data (35). Saturated Clo-C:30 drochloride, nicotinic acid, sodium pantothenate or vitamin FAMEs were used as standards. Respiratory lipoquinones B, L. For these experiments casein hydrolysate (vitamin-free ; and polar lipids were extracted and analysed from 100 mg Merckj was used instead of Bacto peptone and Bacto yeast freeze-dried cells using the two-stage method as previously extract. Triplicate results were recorded after three serial described (43, 44). Purified cell wall preparations were transfers in the corresponding test media. Requirements for obtained by the methods of Schleifer & Kandler (36). Amino Na', K-, M$', Ca", C1- or SO:- were studied in PYGV+ acids and peptides of cell wall hydrolysates were analysed by ASW, where Na' was exchanged with K+,Mg2+ with Ca", two-dimensional ascending TLC on cellulose plates using C1- with SO:- and vice versa. Identical growth response solvent systems described previously (36). Diamino acids indicated independence of these cations or anions. Sus- were recognized by one-dimensional TLC on cellulose using ceptibility 10 antibiotics was determined with bioDiscs a solvent system of Rhuland rt a/. (32). (bioMerieu1) and the results were recorded after 4d. The Determination of DNA base composition. DNA G+C aerobic reduction of nitrate was tested after 14 d; the contents were analysed using HPLC (29). The HPLC anaerobic reduction of nitrate to nitrite or molecular apparatus (Pharmacia-LKB) was equipped with a Spheri- nitrogen was examined after 3 weeks incubation. In both sorb ODS I1 c,, column (5 pm; 4 x 250 mm; Bischoff, cases, PYG V + ASW which contained 5 mM NaNO, was Leonberg). Escherichia coli Strain B Type VIII-DNA employed. Inaerobic growth was examined in modified (Sigma) and lambda phage DNA from E. coli host strain PYGV+ASW, which contained (1-l) 0.1 g glucose, 0.1 g GM 119 (Sigma) were used as standards. acetate, 0.1 g glutamate and 0-5 g Tris. The medium was saturated with N, for 20 min, and 20 ml was distributed to DNA-DNA hybridization. Dot-blot hybridization experi- 100 ml type-111 serum bottles. For photolithoautotrophic or ments were carried out with the DIG DNA Labeling and photoorganGtrophic growth tests, the cultures were incu- Detection kit from Boehringer Mannheim following the bated under an atmosphere of H,/CO, (80: 20; 0.9 bar) and manufacturer's instructions. Gene probes were made from at light intensities of 4-21.5 pmol photons mP2 s-l. Pro- EL-219T and hybridized against chromosomal DNA of EL- duction of poly-p-hydroxybutyrate was determined in 185, EL-165, EL-54 and R. n'enitr8cans. The stringency of PYGV+ASW which was modified to contain (1-l) 20 ml 70 and 75% was calculated according to Sambrook et al. HBM, 10 ml Vitamin Solution no. 6, 0.1 g Bacto yeast (34). extract and 2.0 g succinate as previously described (40). 165 rRNA sequence determination and analysis of sequence Methyl red and Voges-Proskauer tests were performed in data. 16s rRNA gene fragments were generated by PCR as PYGV + ASW containing 0.2 '/o (w/v) Bacto peptone and previously described (23). A large fragment of the 16s rRNA 0.2 Yo (w/v) glucose. H,S and indole production was tested gene was amplified from DNA using universal primers pA with the sulfide indole motility medium (Merck) + ASW. (positions 8-28, E. coli numbering) and pH* (1542-1 522). Indole was detected with 1 ml Kovacs' reagent. The amplified product was purified by using a QIAquick To study the aerobic dissimilation of 95 carbon sources with PCR Purification kit (Qiagen) and sequenced directly using the BIOLOG system, the Microlog software, a Tandon primers to conserved regions of the rRNA. Sequencing was computer and a microplate photometer (Molecular Devices) performed using a PRISM Tay DyeDeoxy Terminator Cycle were used (Biolog). Suspensions were obtained from growth Sequencing kit (Applied Biosystems) and an automatic on R2A agar prepared with the individually required DNA sequencer (model 373A; Applied Biosystems). To concentration of ASW after 3-4 d at 20 "C. The cells were establish the closest relatives of the four Ekho Lake strains, suspended in the necessary ASW to a transmission of preliminary searches in the EMBL database were performed 53-59 '/o and inoculated into microplate wells. Test evalu- with the program FASTA (30). Sequences closely related to ation was performed after 4, 24 and 48 h. Additionally, those of the Ekho Lake strains were retrieved from the carbon source utilization was studied in a minimal medium EMBL database and aligned with the newly determined containing (1 I): HBM, 20 ml; Vitamin Solution no. 6, sequences using the program PILEUP (3). The rRNA align- 10 ml; Bacto yeast extract, 0.02 g; and NH,NO,, 0-25 g. The ment was corrected manually and approximately 100 bases following carbon sources were tested at 0-2YO (w/v) : acetate, at the 5' end of the molecule were omitted from further pyruvate, malate, citrate, succinate, butyrate, glutamate, a- analysis because of alignment uncertainties due to the highly

~_ ~- International Journal of Systematic Bacteriology 48 1365 M. Labrenz and others variable region V1. Percentage sequence similarities were hybridization group) was also almost completely calculated and corrected for substitution rates by using sequenced. Sequence searches of the EMBL database Jukes & Cantor parameters (25). A phylogenetic tree was revealed the newly determined sequences were related constructed according to the neighbour-joining method (33) to the a-subclass of Proteobacteria (data not shown). with the program NEIGHBOR. For this analysis, the following Strain EL-219T displayed the highest 16s rRNA additional taxa, listed by genera and accession number, were sequence relatedness (approximately 92-94 %) with included : Rhodomicrobium vannielii (M34127), ‘ Methylo- sulfonomonas methylovora’ (U62893), Rhodobium marinum species of the genus Roseobacter (viz. R. algicola, R. (D30790), Rhodo b ium or ien t is (D3 0792), Rhodospir illum litoralis and R. denitrificans), the latter was previously salexigens (M59070), Rhodovulum euryhalinum (D13479), named Erythrobacter sp. Och 114 (37), Sagittula Rhodovulum sulJidophilum (U5 5277), Rkodovulunz adri- stellata (1 6), Szilfitobacter pontiacus (42), Octadeca- aticum (D16418), Rhodobacter blasticus (D16429), Rhodo- bacter antarcticus (17), a Sargasso sea isolate and bacter capsulatus (D 16428), Rhodobacter sphaeroides the Ekho Lake strain EL-172. Other proteobacterial (D16425), Rhodobacter veldkampii (D16421), Rhodospi- species examined showed significantly lower levels of r illum rub rum (D307 7 8), Rhodospir illum p hotome t r icum relatedness. An unrooted tree depicting the phylo- (D30777), Rhodospirillum fulvum (D 14433), Rhodospirillum genetic relationships of the Ekho Lake strains and rnolisch ianum (M 59067), Rhodosp ir illum salina r um their close relatives is shown in Fig. 3. The results of (M59069), Rhodospirillum sodomense (M59072), Rhodopila globformis (M59066), Rhodoplanes roseus (D253 13), Blasto- treeing analyses confirmed that the Ekho Lake strain chloris viridis (D253 14), Rhodopseudomonas palustris EL-2 19T was phylogenetically most closely related to (D253 12) and Rhodopseudomonas acidophila (M34128). Roseobacter species and their close relatives. Strain The stability of the groupings was estimated by bootstrap EL-2 19Tformed a distinct subline within this grouping analysis (500 replications) using the programs SEQBOOT, and clustered with another strain of Ekho Lake DNADIST, NEIGHBOR and CONSENSE of the PHYLIP package (8). (EL- 172). However, bootstrap resampling showed the relationship between Ekho Lake strains EL-219T and RESULTS EL-172 was supported in only 63 % of trees. Further- more, although bootstrap resampling showed that the Isolation of bacteria, morphology and motility bacterium from Ekho Lake was associated with the Three different enrichment procedures yielded four above-mentioned Roseobacter cluster (Fig. 3 ; boot- pure cultures with similar morphology ; they were strap value = loo), the unknown bacterium did not obtained from 5, 7, 14 and 16 m Ekho Lake samples possess a particularly significant phylogenetic affinity (Table 1). These isolates are referred to as EL-219T, with any species within this clade. EL-54, EL-165 and EL-185. They all were Gram- negative rods with one or both cell poles narrower, and Culture and growth characteristics of variable length (Fig. la, b). Cells had a tendency to form rosettes (Table 2; Fig. lc). Cell growth appeared Visible aerobic growth appeared after 3-5 d on me- to be monopolar since one cell end was usually dium PYGV+25%, (or 40%,) ASW and at 20°C. narrower and shorter, possibly indicating a budding Colonies were circular, smooth, convex, 14mm in process (18). In such cases, the ‘daughter cell’ was diameter and brownish-yellow. On R2A agar + ASW, often positioned at an angle to the ‘mother cell’ (Fig. the colonies were brownish-red. Optimal growth oc- lc). All four isolates contained electron-transparent curred between 16 and 26 “C and pH values of 6.9-7.8. poly-/3-hydroxybutyrate granules. Their cell sizes were The four isolates had an absolute requirement for very similar ; a peculiar characteristic of laboratory- Na+; the pairwise replacement of the other cations and cultured cells, as well as those from Ekho Lake anions (see Methods) did not influence normal growth samples, was the frequent formation of elongated cells yields. Likewise, R. denitriJicans did not need C1- or of up to 33 pm in length (Table 2). Motility was SO:- under our growth conditions. The EL isolates observed in all strains except EL-219T. Electron had a requirement for thiamin, nicotinic acid and microscopy revealed one to three subpolar flagella in possibly biotin, but neither pantothenate nor vitamin strains EL-54 and EL-185 (Fig. 2). B,, were required, as was also the case with R. denitrzficans. Their osmotolerance ranged from < 10 165 rRNA sequence determinations and phylogenetic to > l50%, ASW with an optimum between 10 and analyses 40%,; however, strain EL-54 had an optimum between 40 and 70%, ASW. The NaCl tolerance of EL-219T Partial 16s rRNA gene sequences of the four Ekho and EL-54 ranged from < 10 to loo%, with an Lake isolates were determined by direct sequence optimum of 20-60%, NaCl, and that of EL-165 and analysis of PCR-amplified products. Based on a EL-185 from < 10-20 to 40-60%, NaCl with an comparative analysis of a continuous stretch of optimum at 20%,. approximately 800 bases, the isolates formed a distinct group, exhibiting 100 YO16s rRNA sequence similarity Physiological and biochemical characteristics among each other. The almost complete 16s rRNA gene sequence (> 1400 bases) of strain EL-219T as a All four strains exhibited cytochrome oxidase, per- representative was determined. For comparison, Ekho oxidase and catalase activity. They did not produce Lake strain EL- 172 (a representative of another acetoin or acids from glucose. They were susceptible to

1366 lnternational Journal of Systematic Bacteriology 48 Antnrctobacter gen. nov.

Fig. 1. Phase-contrast light micrographs of strains EL-21gT (a, b) and EL-165 (c) on agar-coated slides. Note differences in morphology and size of EL-21gT cells due to either growth for 12 d on PYGV+67xo agar (a), or growth for 56 d on PYGV+25%, NaCl agar (b). Cells of EL-I65 (c) were cultivated for 29 d on PYGV+67%, ASW. Bar, 10 pm.

Table 2. Morphological characteristics of the EL strains, Sagittula stellata, R. algicola, R. denitrificans, R. litoralis and Ma rinosu lfono monas m ethy lo trop ha '

Organism ' C ell bhape Cell huddingt Storage granules: Rosettes Cell size (mean length) (pm) Flagella ; Colony colour on motility$ PYGVI

~~~ ~ ~- Rods. I 2 poles pointed + PHBA + 0.8 1.1x 3.5-10.9 (5.5) - Brownish-yellow Rods. I 2 pole\ pointed + PH BA + 0.8 I.?x 2.0-1 1.0 (3%) 1-3; sp Brownish-yellow Long rodi, I 2 pole.; pointed + PH BA + 0.8-1.8 x 2.5-336 (7.7) v Brownish-yellow Rotla. I 2 poles pointed + PHBA + 0.8-1.5 x 3.5-13.8 (6.8) 1-2: sp Brownish-yellow Rod\. I pole narro\s . UI) I'HRA + 0.9 x 2.3 +/- Cream-coloured" holdf~>ti,:fibrils

Ovoid rods lu 1) 1-2: sp Brownish-beige" Ovoid or rocis N 1) 1-3; sp Red" Ovoid or rod.; NI) 1-3; sp Red" Rod\ NI) - Semi-translucent'

~ ~~ ~___~__~

N D , N o t d t' t c rni i n ed . "Data from reference: N, (16); h, (27); c', (37); (I, (22). ? Daughte! cells substantially smaller than mother cells and often positioned at an angle. PH B A. yol y -p-h yd rox yb u t y ra t e . #sp. subp'olar to lateral; v, variable I/ a, On M,irine agar; 6, present data supplement published diagnoses; c', on methanesulfonic acid/Phytagel (1 %, w/v) medium. a- Present ciala supplement published diagnoses. chloraniphenicol (30 pg), penicillin G (10 U), strep- but not Tween 80 or alginate. Other hydrolysis data tomycin 10 pg), tetracycline (30 pg) and polymyxin B and differences in the metabolism of carbon com- (300 U). All strains reduced nitrate to nitrite. Nitrite pounds as revealed with the BIOLOG system are was not reduced aerobically, and anaerobic reduction shown in Table 3. to was variable. H,S or indole was not produced. TheN, following carbon sources were utilized in the In the BIOLOG system, the isolates metabolized presence of available nitrogen : succinate, butyrate, N-acetyl-D-galactosamine, N-acetylglucosamine, ado- glutamate, acetate, pyruvate, malate, citrate and a-~- nitol, D-arabitol, i-erythritol, D-fructose, L-fucose, glucose. Not utilized were methanol and methane- ~-galactose,gentiobiose, a-D-glucose, a-lactose, a-D- sulfonic xid. In the absence of other nitrogen sources, lactose-lactulose, maltose, D-mannitol, D-mannose, glutamate was utilized as a carbon and nitrogen source. D-melibiose, u-raffinose, L-rhamnose, D-sorbitol, Without any added nitrogen source there was slight sucrose, D-trehalose, turanose, acetic acid, citric acid, growth with acetate, butyrate, pyruvate, malate or itaconic acid, propionic acid, glucuronamide, L-alanyl- succinatc. The strains hydrolysed gelatin and DNA, glycine and uridine. They did not metabolize a-

__~_ __ ~--~ ~ ~~ ~~ ~ ______International Journal of Systematic Bacteriology 48 1367 M. Labrenz and others

~~ ~

Fig. 2. Electron micrograph of phosphotungstic acid negatively stained cells and flagella of strain EL-54 after cultivation for 3 d in liquid medium PYGV+750;, ASW. The cell without a flagellum shows monopolar growth. Bar, 2 pm.

I00 Paracoccus kociirii JCM 7684 (D3224 1) Pavacoccus denilrificans LMG 421 8 (X69159) ‘Silicibacter lacuscuerulensis’ IT1 1 157 (U77644) Antarctic strain EL-1 72 (Y 1 155 1 ) I00 Antnrctobncter heliotliermus gen. nov. sp. nov. EL-2 19‘ (Y 1 1552) Roseobacter algicola ATCC 5 1440 (X783 15) Fig. 3. Unrooted tree showing phylogenetic relationships of strain EL-21gT with EL-172 Octadecabactcr arcticus 238 (1173725) and closely related proteobacteria. The tree ‘Marinosulfonomonusmethylotropha ’ PSCH4 (U62894) was constructed using the neighbour-joining Sargasso sea isolate LFR (L 15345) method and K,,, values; it was based Prionitis lunceolata gall symbiont (U37762) on a comparison of approximately 1400 nucleotides. Bootstrap values, expressed as a cter denitrificans ATCC 33942 (M96746) percentage of 500 replications, are given at Sulfitobucter pontiacus CHLG-I 0 (Y 13 155) branching points; only those above 90% are Sagittulu stellatu E-37 (U58356) -0.0 I shown. -~

cyclodextrin, dextrin, glycogen, Tween 80, p-methyl-D- a was never found in cell suspensions or in methanolic glucoside, methyl pyruvate, cis-aconitic acid, mono- extracts. Absorption spectra of methanolic extracts methylsuccinate, formic acid, D-galactonic acid were very similar to those of R. ulgicola (27) grown on lactone, D-galacturonic acid, a-hydroxybutyric acid, PYGV+25%, ASW. The G+C content ranged from P-hydroxybutyric acid, y-hydroxybutyric acid, p- 62-3to 62.8 mol %. Gene probes from EL-2 19’ hybrid- hydroxyphenylacetic acid, a-ketobutyric acid, a-keto- ized with DN.4 from the other three Ekho Lake strains glutaric acid, a-ketovaleric acid, DL-lactic acid, sebacic but not with DNA of R. denitr$cans. acid, succinic acid, bromosuccinic acid, succinamic acid, D-alanine, L-aspartic acid, glycyl-L-glutamic acid, glycyl-L-aspartic acid, L-histidine, hydroxy-L-proline, Chemotaxonomic parameters L-leucine, L-ornithine, L-phenylalanine, L-proline, D-serine, L-pyroglutamic acid, DL-carnitine, y-amino- The peptidoglycan of all four isolates contained MI- butyric acid, urocanic acid, phenylethylamine, diaminopimelic acid, and the peptidoglycan of strain putrescine, 2-aminoethanol, glycerol, glucose 6-phos- EL- 185 was of the directly cross-linked type A1 y (36). phate or DL-a-glycerophosphate. The ubiquinone of the four strains was Q-10. Polar lipid patterns and fatty acid compositions of the strains Strains EL-54 and EL-I 65 grew microaerobically, but grown on PYGV+ASW are listed in Table 4 and they did not grow photolithoautotrophically under compared to some other members of the a-Protoo- anaerobic conditions with HJCO, (80:20) in the gas bacteria. Their fatty acid profile was characterized by phase or photoorganotrophically . Bacteriochlorophyll the predominance of C,,:

~~~ ~ ~____-~~~_____ 1368 In termtio na I I ou maI of System a tic 5a c teriology 48 Antarctohacter gen. nov.

Table 3. Differences in physiological properties of the EL the major respiratory quinone only in members of the strains a-subclass of the Proteobacteria, although some ...... , . . , ...... organisms, such as Legionella spp. in the y-subclass of +, Positive. -. negative; v, variable; w, weak; ND, not Proteobacteria, also produce Q- 10, which is accom- determined. panied by significant quantities of other, long-chain homologues. The fatty acid pattern of the EL strains Character EL-219T EL-54 EL-165 EL-185 was dominated by the presence of a C18:1fatty acid. _.______~~~~ ______- This predominance, in combination with the presence BIOLOG data of Q- 10, is again typical of members of the a-subclass Tween 40 of Proteobacteria. The only indication of the presence L-Arabinosc - + - V of non-ester-linked fatty acids (potentially amide- Cellobiose linked fatty acids) was found among the 3-hydroxy m-Inosi to1 fatty acids. The polar lipid composition of all strains Psicose indicated that the organisms contained phospholipids Xylitol as the major polar lipids. Taken together, the sum of D-Gluconiz acid respiratory lipoquinones, fatty acids and polar lipid D-Gluc~satninic acid data indicated that the strains examined belonged to a D-Glucuronic acid group of organisms (at the genus or family rank) Malonic acid within the a-subclass of the Proteobacteria. Quinic acid D-Saccharic acid Comparative 16s rDNA gene sequencing showed also Alaninamide that the unknown bacteria from Ekho Lake, as L- Alani n e exemplified by EL-219’, were members of the a- L-Asparagine subclass of Proteobacteria and were specifically asso- L-Glutamic acid ciated with the Roseobacter supercluster of organisms, L-Serine which included ‘ Marinosulfonomonas’ (22), Prionitis Inosine Ianceolata gall symbiont, Octadecabacter, Roseobacter, Thymidinc Sagittula, SulJltobacter and an unknown Sargasso sea 2,3-Butancdiol isolate. This cluster was supported by a bootstrap Glucose 1 -phosphate value of 100 YO.Sequence divergence values of > 5 YO showed that the unknown bacteria were genetically Hydrolysis of: distinct from all currently recognized members of the Starch Proteobacteria. Furthermore, bootstrap resampling B-D-Glucohide showed that EL-219’ from Ekho Lake did not possess a particularly significant phylogenetic affinity with any individual species within the above-mentioned genera (Fig. 3). The association between Ekho Lake strains DISCUSSION EL-219‘ and EL-1 72 was recovered in only 63 Y of The three very different enrichment procedures use( trees (500 replicates). It is evident from the treeing for Ekho Lake samples yielded four nearly identical analyses that the genus Roseobacter as currently bacterial isolates, although these came from 5-16 m recognized is not monophyletic, with several taxa (e.g. depth. Apart from the salinity (60-76 %J, the original Prionitis lanceolata gall symbiont, Su@tobacter) inter- samples were fairly similar, and thus it may be possible dispersed with species of this genus. Based on sequence that the isolates were widely distributed in the upper divergence values combined with tree topology con- layers of the lake. DNA of all four isolates hybridized siderations, it is evident that the Ekho Lake strain EL- with gene probes of strain EL-219T (75 Yo stringency), 219T merits a similar taxonomic rank (i.e. genus) as and sequencing of 16s rDNA confirmed the isolates that of Sagittula, Prionitis lanceolata gall symbiont, were genealogically homogeneous and members of a SulJitobacter and Octadecabacter. In view of the distinct tason within the a-3-subclass of the Proteo- phylogenetic separation of the unknown bacteria and their phenotypic distinctiveness (Table we conclude bacteria. The four isolates displayed very similar mor- 5), phologies (Table 2). Strain EL-219’ did not exhibit that the four Ekho Lake isolates are representatives of directed motility but rather a tumbling and twitching a new and distinct taxon for which we formally propose of daughter cells. There were only minor differences in the name Antarctobacter Izeliothermus gen. nov., sp. biochemical and physiological activities such as in the nov. hydrolysis of starch or in the metabolism of carbon compounds as revealed by the BIOLOG system (Table Description of Antarctobacter gen. nov. 3). The strains were also identical in the battery of chemotaxonomic tests employed : they all contained Antarctobacter [Ant.arc.to.bac’ter. Antarctica M.L. ubiquinones as the sole respiratory quinones, indi- name of the South Pole continent, derived from Gr. cating that the organisms were members of the a-, p- or pref. anti against (on the other side), Gr. adj. arcticos y-subclasses of the Proteobacteria. Generally, Q- 10 is northern; Gr. n. baktron a rod or staff; M.L. masc. n.

______~ ~~ In terna tionaI Journal of Systematic Bacteriology 48 1369 M. Labrenz and others

Table 4. Chemotaxonomic characteristics of the EL strains, Sagittula stellata, R. algicola, R. denitrificans, R. litoralis, 0. arcticus and 0. antarcticus

For the FAME composition data, values less than 1 % and unidentified FAMEs are not shown. All polar lipid data are from the present study; strains were cultivated on PYGV +ASW. ND, Not determined.

Characteristic EL- EL- EL- EL- S. R. R. R. 0. 0. 219T 54 165 185 stellata* algicolal- f: denitrijicanss litoralistrj arcticus 11 antarcticus /I

~ FAME composition (YO) ClO 0 3-OH 4.2 1.9 4.0 2.0 C,, 3-OH 3.1 3.1 3.0 3-1 3.6 C,, 2-OH 3.9 0.8 1.1 0.8 0.6 1.4 8.0 12.0 Cl6 1 Cl, 0 2-5 2.7 2.5 2.3 8.6 1.6 1.6 1.1 6.0 6.0 C,, L 1.6 7.1 1.4 1 83.2 81.9 81.4 84.0 11 9 1.5 84.6 88.8 75.0 77.0 Cl, Cl, 0 1.0 0.7 1.1 1.4 6.8 2.2 1.2 1.3 Cl9 0 CYC 2.4 2.5 2.7 1.9 1.8 Polar lipids Phosphatidylglycerol + + + + ND + + ND ND Phosphatidylcholine + + + + ND + - ND ND Diphosphatidylglycerol - - - - ND + + ND ND Phospha tidylethanolamine - - - - ND - ~ ND ND Unknown aminolipid + + + + ND + + ND ND Unknown phospholipid(s) + + + + ND + + ND ND * Data from reference (16). ?Grown on Marine broth. f: Data from the present study. rj Present data supplement published diagnoses. /I Data from reference (17). 7 Major fatty acid, but not quantified.

Table 5. Comparison of isolate EL-21gTand the other related strains with Sagittula stellata (1 6), R. algicola (27), R. denitrificans (37), R. litoralis (37), Sulfitobacter pontiacus (42), 0. arcticus (17), 0. antarcticus (1 7) and Marinosulfonomonas methylotropha (22)

+ , Positive; -, negative; ND, not determined; w, weak reaction.

Characteristic EL-219"' S. stellata R. algicola R. denitrifcanv R. litoralis S. pontiacus 0. arcticus 0. antarcticus ' M. meth~ylotropha'

Cell shape Rods Rods Ovoid Ovoid Ovoid Rod5 Rods Rods Rods Rosettes formed + + - - + - - + Colony colour Brown-yellow Cream Brown-beige Pink Pink Colourless White White Translucent Gas vesicles - - - - - + + - Motility +/- +* + + + + - - - Maximum length (pm) 10'9-33.6 2.3 23 7 3 2-5 4.4 48 2.5 Minimum temp. ("C) 3-8.5 <4 10 <2 7 4 <4 <4 20 Maximum temp. ("C) 33,543.5 > 41 > 31 > 30 > 30 35 > 15 ia 15 31 Nicotinic acid requirement - - + + + ND + ND N 11 Biotin requirement W - + + + ND - ND NII Dissimilatory NO; reduction + - - + - ND - - h'D - - - Assimilatory NO; reduction + +t ti I1 ND ND + Carbon source: methanol - + - - - + Carbon source: butyrate + + -4 - - + - - ND Cellulase activity - + NII N D ND ND ND ND ND Gelatine hydrolysis + - + + + - - - ND Bacteriochlorophyll o - - + + - - - - G+C content (mol%) 62.3-62.8 65 64t 59.6 56.3-58.1 62.1 57 56 57 * Motility was assumed since flagella were found in suspension, but motile cells were not seen. l- Data from the present study. f: Positive in the present study.

1370 International Journal of Systematic Bacteriology 48 Antarctobacter gen. nov. bacter; M.L. masc. n. Antarctohncter a rod-shaped EL-2 19T was deposited in the Deutsche Sammlung von bacterium from Antarctica]. Mikroorganismen und Zellkulturen (DSM 1 1445T). Gram-negative rods, multiplying by monopolar Additional isolates are EL-165 (DSM 11440), EL-54 growth, i.e. by a budding process. Daughter cells may and EL-185. be motile. Cell sizes vary due to age and growth conditions: 0.8-1-2 x 2.0-33.6 pm. The cells may con- ACKNOWLEDGEMENTS tain poly-/%hydroxybutyrate;rosettes are formed. Do Skilful technical assistance of B. Hoffniann, M. Beese and R. not form spores. Colonies smooth, convex and brown- Erncke is gratcfully ackiiowledged: J. Siebert assistcd with ish to ycllowish on medium PYGV with ASW, or the Antarctic field work and H. Volker (Kicl) helped with brownish-red on medium R2A with ASW. The tem- clectron microscopy. P. Schumann (DSMZ, Jena) has helped perature range for growth is < 3-43.5 "C. The cells with the identification of futty acids and N. Weiss (DSMZ have an absolute requirement for Na+, and grow in the Braunschweig) with cell wall analyses. We wish especially to range of < 10 to < loo%, NaCl. They grow between express our gratitudc to thc Australian Antarctic Di\.ision < 10 and > 150%, ASW. The pH tolerance range is (Kingston, Tasmania) for supporting tn'o eventful visits to 5.3- > 9. Strictly aerobic, non-fermentative hetero- the Vestfold Hills (Antarctica), and especially to H. R. trophs. The cells exhibit cytochrome oxidase, per- Burton and T. A. McMeekiii (Hobart, Tasmania) for their oxidase and catalase activities. No growth on glucose prLicticii1 help and hospitality. We are grateful to the Deutsche ~orschun~sgemeiiiscliaft(grants Hi 68/ 16-3, Hi anaerobically in the absence of nitrate. They do not 68/ 19-3 and Hi 68/25 1 /2) and thc European Union (CT 93- grow photoautotrophically with H,/CO, (80 : 20) or 0 194 and CT 93-0 1 19) for support. photoorganotrophically with acetate or glutamate ; they do not contain bacteriochlorophyll a. The peptidoglycan contains m-diaminopimelic acid and is of REFERENCES the directly cross-linked type A 1y. The respiratory 1. Bradford, M. M. (1976). A rapid and sensitive method for the quinone is Q- 10. The following polar lipids are present : quantitation of microgram quantities of protein utilizing phosphatidylglycerol, phosphatidylcholine as well as the principle of protein-dye binding. Anal Biochenz 72, unknown phospholipids and an aminolipid. The domi- 248-254. nant fattv acid is CIS:; another characteristic fatty 2. Cohen-Bazire, G., Sistrom, W. R. & Stanier, R. Y. (1957). acid is ('.12:1 3-OH. The G+C content is 62.3- Kinetic studies of pigment synthesis by non-sulfur purple 62.8 mol"6. 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~ International Journal of Systematic Bacteriology 48 1371 M Labrenz and others

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1372 International Journal of Systematic Bacteriology 48