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School of Agricultural Science, University of Tasmania, Private Bag 54, Hobart, Tasmania 7001, Australia
Correspondence
John P. Bowman
john.bowman{at}utas.edu.au
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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A figure showing the cellular morphologies of Subsaximicrobium wynnwilliamsii, Subsaxibacter broadyi and Lacinutrix copepodicola is available as supplementary material in IJSEM Online.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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, 2002) is one of the major branches of the Cytophaga–Flavobacterium–Bacteroides phylum, which was renamed (so far unofficially) ‘Bacteroidetes’ in volume 1 of the second edition of Bergey's Manual of Systematic Bacteriology (Garrity & Holt, 2001). The family Flavobacteriaceae includes many marine species that cluster together in 16S rRNA gene sequence phylogenetic trees as a well-defined ‘marine clade’ (Bowman, 2004). Flavobacterium, the type genus of the family, is positioned at the periphery of this marine clade. This genus includes species that are more adapted to freshwater and brackish ecosystems. Genera belonging to the family Flavobacteriaceae positioned beyond the genus Flavobacterium are generally adapted to non-marine ecosystems, including soil, water and animal habitats. Functionally, the members of this family are chemo-organotrophs and seem to play important roles in natural carbon cycles (Kirchman, 2002).
The marine clade of the family Flavobacteriaceae is particularly important in marine and marine-derived surface waters, where its members contribute strongly to the mineralization of primary-produced organic matter. They are major inhabitants of marine aggregates, conglomerations of organic detritus, which sink in the pelagic water column and which represent active biological zones in the oceans (Kirchman, 2002). Members of the marine clade also concentrate around or inhabit the surfaces of marine biota, where they seem to act primarily as commensals. Some are opportunistic pathogens, such as some Tenacibaculum species (Suzuki et al., 2001), or can potentially exacerbate existing disease (Bowman & Nowak, 2004). From molecular surveys, members of this family also make up a large community component in surface benthic sediments (Bowman & McCuaig, 2003), and are very abundant in sea ice (Brinkmeyer et al., 2003). In the cold, comparatively more nutrient-replete, surface waters of the Southern Ocean south of the Polar Front (located at about latitudes 55–60° S), many so far uncultured members of the family Flavobacteriaceae proliferate. Molecular analyses suggest that the abundance and diversity of this group markedly increases in Antarctic Zone waters, compared with the warmer, highly nutrient-limited waters north of the Polar Front (Abell & Bowman, 2005).
In Antarctic marine and coastal zones, members of the marine clade of the family Flavobacteriaceae have been isolated and described recently, from a wide range of ecosystems, including sea ice, quartz stone subliths, marine sediment, lake mats and sea water (Bowman, 2000; Bowman et al., 1997a, 1998, 2003; Bowman & Nichols, 2002; Gosink et al., 1998; Van Trappen et al., 2002, 2004). At this stage, molecular surveys provide a good appraisal of the diversity present in these habitats, but well-characterized isolates still only represent a small proportion of this diversity. However, because they can be readily cultured, many new genera of the family Flavobacteriaceae from non-polar marine ecosystems have been described recently (Bruns et al., 2001; Cho & Giovannoni, 2003, 2004; Ivanova et al., 2001, 2004; Nedashkovskaya et al., 2003a, b, 2004a, b, c, 2005a, b, c; Sohn et al., 2004). This expansion of knowledge of cultured members of the family Flavobacteriaceae continues in this study, with the description of several novel taxa from various Antarctic maritime samples, including sea ice, crustaceans and quartz stone cyanobacterial communities.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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; Bowman et al., 2003). Sea-ice brine drained from sea-ice cores was collected in glass bottles and then plated onto either marine agar (MA) or MA containing 10 % NaCl (w/v), and incubated at 4 or 12 °C. Melted sea-ice samples were also filtered through 0·8 µm polycarbonate filters, and the particulate material caught on the filter, mostly algal biomass, was diluted in sterile brine filtrate and plated onto MA and MA+10 % NaCl, and incubated at 4 or 12 °C. MA consisted of 0·5 % (w/v) peptone, 0·2 % (w/v) yeast extract and 1·5 % (w/v) agar, in 1000 ml artificial sea water. The artificial sea water comprised 35 g l–1 of a synthetic sea salts preparation (Aquasonic, Wauchope, NSW, Australia). Strains were also obtained from the surfaces of various crustaceans from various areas of the Vestfold Hills, East Antarctica, collected by using traps and plankton nets. Isolations were made from the calanoid copepod Paralabidocera antarctica, collected from Ace Lake, and from sea-ice algae-feeding amphipods collected at the sea ice–sea water interface in Long Fjord. The crustacean samples were rinsed gently several times in small quantities of sterile sea water before being used for isolation. Copepod samples were suspended for 24 h at 4 °C in a dilute marine medium consisting of 0·025 % (w/v) peptone and 0·005 % (w/v) yeast extract in sea water, before being plated. Material from the surface of the carapaces of the amphipod samples, which was 2–5 mm in length, was plated directly onto MA by gently using forceps that had been flame-sterilized with ethanol. Isolates and reference strains used in this study (Table 1) were maintained and grown routinely on MA at 12 °C, and stored at –70 °C in marine broth containing 30 % (v/v) glycerol.
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, 1998, 2003). MA was used as the basal medium, unless specified otherwise. Additionally, tests were performed using API 20E, API 20NE, API AUX and API 32ID AN test strips (bioMérieux–Vitek). For these tests, the inoculating or suspension medium contained 35 g synthetic sea salts l–1. The API 32ID AN test strips were incubated for 24 h at 20 °C, whereas the other test strips were incubated for up to 7 days at 12 °C. Gliding was confirmed by growing cultures for 1–4 days at 12 °C on a dilute marine medium (described above), solidified with 1 % (w/v) agar noble, on clean microscope glass slides and viewed by using phase-contrast microscopy (Bowman, 2000).
High molecular mass DNA for DNA G+C content and DNA–DNA hybridization was extracted by using the Marmur technique (Marmur & Doty,1962). DNA G+C content was determined by using the thermal denaturation procedure involving spectrophotometry (Sly et al., 1986); DNA–DNA hybridization was performed as described previously, by using the spectrophotometric renaturation kinetics approach (Bowman et al., 1998), adapted from Huß et al. (1983).
Strains were cultivated on MA at 15 or 20 °C for 3–5 days until good growth was achieved, and harvested for whole-cell fatty acid extraction. Procedures used for fatty acid analysis were those described previously (Bowman et al., 1997a, 2003). Isoprenoid quinones from lyophilized cell extracts were analysed by using reversed-phase liquid chromatography (Moss & Guerrant, 1983). Cellulophaga lytica ACAM 74T was included as a control for menaquinone-6 (MK-6) content.
Genomic DNA was obtained from small amounts of growth by using a Ultraclean PCR purification kit (Mo-Bio). The 16S rRNA genes from the DNA samples were amplified by PCR as described previously (Bowman et al., 1997b, 2003). Sequences obtained were 1366–1442 nucleotides in length and were aligned manually with sequences obtained from GenBank following N-BLAST searches (http://www.ncbi.nlm.nih.gov). The sequence data set was then analysed using the PHYLIP program package, as described previously (Bowman et al., 2003). The phylogenetic tree (Fig. 1) included the 16S rRNA genes of Rhodothermus marinus and Chlorobium limicola as outgroup sequences. The 16S rRNA gene sequence of Gelidibacter gilvus IC158T was determined during a previous study (Bowman et al., 1997b).
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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. 16S rRNA gene sequence analysis indicated that the strains formed several lineages in the marine clade of the family Flavobacteriaceae (Fig. 1
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). However, differences were evident between certain taxa for specific lipid components (Table 5).
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), which is an arid, ice-free region on the coast of East Antarctica. The growth of these sublithic (or hypolithic) communities is promoted in this region because it is protected from climatic extremes and includes a relatively diverse set of cold-adapted chemoheterotrophic bacteria (Smith et al., 2000). Strains from this ecosystem included one group comprising relatively fast-growing, rod-shaped bacteria (G#7T, G#4, O1-5, O3-5, O5-2, Y4-5T and Y5-5) (see panel A of Supplementary Figure in IJSEM Online), whereas a second group included slow-growing coccobacilli, such as strains P7T and O6-2 (Table 1, see also panel C of Supplementary Figure in IJSEM Online). Genotypic analysis indicated that the group containing G#7T included two genospecies. The first consisted of six strains, represented by G#7T, while the second comprised only strain Y4-5T. DNA–DNA hybridization analysis indicated that the genomic DNA of strain Y4-5T showed 47–59 % relatedness with that of strains G#7T, O1-5 and O3-5. By comparison, the DNA–DNA relatedness of the latter three strains was 89–99 %. Strain Y4-5T also differed from the other strains in a variety of phenotypic traits (Table 2); however, the fatty acid profiles of the strains were almost identical (Table 5). Both genospecies formed spheroplast-like bodies in old cultures (see panel B of Supplementary Figure in IJSEM Online). Strains P7T and O6-2 were found to form a single genospecies, with a mean DNA–DNA hybridization value of 88 %. These data concurred with the phenotypic (Table 2) and fatty acid (Table 5) data.
Phylogenetically, the two genospecies represented by strains G#7T and Y4-5T were most closely related to the genus Gelidibacter (16S rRNA gene sequence similarity of 92–93 %) (Fig. 1). The genospecies differed in various phenotypic traits from Gelidibacter species (Tables 2 and 4) and lacked the fatty acid i16 : 0, which was present in all Gelidibacter species (Table 5). Similarly, the genospecies represented by strain P7T also differed in terms of morphology and various phenotypic characteristics (Table 2) from Gelidibacter species, which were the closest relatives (similarity 95 %) (Fig. 1). Strains P7T and O6-2 contained high levels of the fatty acid 18 : 1
9c, which was absent from Gelidibacter species. The overall taxonomic data suggested that the quartz stone strains were sufficiently different not to be included in the genus Gelidibacter, but represent novel taxa at the genus level.
It is thus proposed that strains G#7T and Y4-5T represent a novel genus, named Subsaximicrobium gen. nov., containing two novel species designated Subsaximicrobium wynnwilliamsii sp. nov. (G#7T) and Subsaximicrobium saxinquilinus sp. nov. (Y4-5T). Subsaximicrobium wynnwilliamsii is the type species. It is also proposed that strain P7T represents the type strain of a new genus and novel species, named Subsaxibacter broadyi gen. nov., sp. nov.
Sea-ice algal isolates
Isolates were obtained from two distinct sea-ice sources: the sea-ice algal assemblage and brine drained from ice cores at the time of sampling. Samples collected from sea-ice communities have been analysed previously by using molecular and culture techniques (Bowman et al., 1997b; Brown & Bowman, 2001; Brinkmeyer et al., 2003) and novel taxa are slowly being described. Isolates studied here were obtained either from algae directly concentrated from basal ice algal assemblages or, in the case of strain IC162T, from particulate material concentrated from the sea-ice brine. All the strains appeared rod-shaped, and those grouped in the genus Gelidibacter exhibited gliding motility. Gliding was only evident for strain IC162T on dilute media. Some strains also formed short filaments, whereas others contained coccoidal bodies, possibly spheroplasts, in old cultures, as noted in the taxa descriptions below (similar to panel B of Supplementary Figure in IJSEM Online).
Phylogenetic analysis indicated that the sea-ice algae strains could be grouped either within the genus Gelidibacter (strains IC158T, IC159, IC163 and IC162T) or the genus Gillisia (IC157T, IC144, IC146, IC148T and IC154T). The sequences of strains IC158T, IC163 and IC159, which formed pale, viscid yellow colonies, with spreading margins, were identical, and only IC158T is shown in the phylogenetic tree (Fig. 1). Sequence analysis revealed that strains IC158T and IC162T were clearly distinct from each other and from Gelidibacter algens and Gelidibacter mesophilus (similarities 
97 %) (Fig. 1). In the case of the strains related to the genus Gillisia, strains IC157T, IC144 and IC146 had nearly identical 16S rRNA gene sequences but were quite different from IC148T and IC154T, as well as from Gillisia limnaea and Gillisia mitskevichiae (similarities 96–97 %), which are species from Antarctic lake cyanobacterial mats (Van Trappen et al., 2004) and sea water (Nedashkovskaya et al., 2005b), respectively.
The isolates belonging to the genus Gelidibacter were compared by DNA–DNA hybridization with Gelidibacter algens, Gelidibacter mesophilus, and each other. It was found that IC158T represented a distinct genospecies with strains IC163 and IC159 (hybridization values 93–100 %) and were genotypically (hybridization values 12–23 %) and phenotypically (Table 2) distinct from other type strains of the genus Gelidibacter. Strain IC162T, like IC158T, was also genotypically and phenotypically distinct from other Gelidibacter strains (hybridization values of 15–32 %). Fatty acids produced by the sea-ice strains were similar, and the profiles were qualitatively similar to those of Gelidibacter algens and Gelidibacter mesophilus (Table 5). The DNA G+C contents of the genospecies represented by strains IC158T and IC162T were 39 and 42 mol%, respectively, comparable with data from Gelidibacter algens and Gelidibacter mesophilus (Table 2). Phenotypically, the isolates were similar to members of the genus Gelidibacter, in particular the ability to glide, yellow pigmentation, non-exacting nutritional requirements, the ability to use carbohydrates and growth at low temperatures. However, with the expansion of the number of ecosystems in which the genus Gelidibacter has been found, the genus now exhibits an increased degree of phenotypic heterogeneity (Table 4). Overall, Gelidibacter species still possess a number of common traits, which differ from those of closely related taxa, including the novel quartz stone isolates discussed above.
From the DNA–DNA hybridization analysis, it was found that the strains belonging to the genus Gillisia clearly formed three genospecies. The bright-yellow-pigmented strains IC157T, IC144 and IC146 were grouped in one genospecies, whereas the orange-pigmented isolates IC148T and IC154T each formed the other two. DNA–DNA hybridization values between the genospecies ranged from 18 to 37 %. The DNA–DNA hybridization data also revealed that there was no significant similarity with Gillisia limnaea LMG 21470T, with hybridization values ranging from 11 to 31 %. These data were supported by the phenotypic results, which showed that the sea-ice strain groups were distinct from each other and from Gillisia limnaea (Table 2). The sea-ice isolates also differed phenotypically (Table 2) from the recently recognized species Gillisia mitskevichiae, which is much more halotolerant. The DNA G+C contents of the sea-ice strains were in the same general range as those of Gillisia limnaea and Gillisia mitskevichiae, with the value for the genospecies represented by strain IC157T being somewhat lower, with a mean value of 32 mol%. The fatty acid data indicated that the sea-ice strains of the genus Gillisia were similar to each other and to Gillisia mitskevichiae (Table 5) and Gillisia limnaea (data not shown).
Based on the polyphasic taxonomic data, it is proposed that the novel sea-ice algae-derived genospecies constitute novel species. Two of the novel species belong to the genus Gelidibacter, for which the names Gelidibacter gilvus sp. nov. (type strain, IC158T) and Gelidibacter salicanalis sp. nov. (type strain, IC162T) are proposed. Three novel species are also proposed for the genus Gillisia, with the names Gillisia illustrilutea sp. nov. (type strain, IC157T), Gillisia sandarakina sp. nov. (type strain, IC148T) and Gillisia hiemivivida sp. nov. (type strain, IC154T).
Sea-ice brine and crustacean isolates
As it freezes, sea ice traps and concentrates sea water within the ice matrix. The brine reaches temperatures as low as –20 °C in winter, although it does not freeze due to its high salinity, which can be as high as 150 
. Although an extreme environment, sea-ice brine channels still support psychroactive bacterial and algal communities, which have been found to undergo cell division at –10 °C (Junge et al., 2004). The brine samples examined here were directly plated on MA and on MA containing 10 % NaCl, in the hope of finding novel, highly salt-tolerant, but still highly cold-adapted, species. Primary isolation plates of different brine samples included various golden-yellow colonies and, for this study, yielded the strains HFDT, IC134 and IC164T. The strains were non-motile, with rod-shaped or sometimes spiral-shaped or curved cells, similar to the morphology found for the crustacean isolates discussed further below.
Strains HFDT and IC134 had almost identical 16S rRNA gene sequences, with a similarity of 97·2 % with the sequence of strain IC164T. The brine strains were most closely related to isolates from amphipods (HFDT, IC134 and IC164T; similarity 96 %) and to Bizionia paragorgiae (Nedashkovskaya et al., 2005c) (similarity 96–98 %). DNA–DNA hybridization analysis indicated that HFDT and IC134 belonged to the same genospecies (hybridization value, 83 %). However, strain IC164T appeared to represent a distinct genospecies, sharing only 36 % DNA hybridization with the genomic DNAs of the other brine isolates. No significant similarity was found between the brine isolates and the most closely related recognized species, B. paragorgiae, or with the epiphytic species Formosa algae (values of 4–22 %). Phenotypic data for the brine strains are shown in Table 3. The strains required Na+ ions and were distinctly salt tolerant, growing best in 1·0 M NaCl and tolerating up to 3·0 M NaCl. Several phenotypic differences were evident between the genospecies represented by strains HFDT and IC164T (Table 3).
Sea-salts-containing media onto which crustacean-derived material and bacterial cells were plated also revealed several golden-yellow-coloured colonies that were non-motile, with rod-shaped or sometimes spiral-shaped or curved cells. Four isolates were studied, including DJ3T, a rod-shaped strain (see panel D of Supplementary Figure in IJSEM Online) that was derived from the copepod sample, and APA-1T, APA-3 and ADA-4T, which were each derived from exoskeletal material of individual amphipod samples (Table 1). 16S rRNA gene sequence analysis indicated that strain DJ3T was phylogenetically quite separate from strains APA-1T, APA-3 and APA-4T (similarity 92–93 %), whereas the 16S rRNA gene sequences of APA-1T and APA-3 were identical, and quite similar to that of ADA-4T (similarity 97 %). DJ3T was most closely related to species of the genera Psychroserpens and Winogradskyella (similarity 94 %). Strains APA-1T and ADA-4T were related to the sea-ice brine isolates HFDT, IC134 and IC164T discussed above, and like these were most closely related to B. paragorgiae. Phenotypically and genotypically, the crustacean strains formed distinct genospecies. DJ3T did not show significant DNA–DNA hybridization (values 8–28 %) with any related strains, including APA-1T, ADA-4T, HFDT and IC164T, or Psychroserpens burtonensis ACAM 188T. In addition, the mean hybridization value for the genomic DNAs of strains APA-1T and ADA-4T was only 38 % and there was no genomic similarity with the sea-ice brine strains and F. algae (hybridization values 10–17 %). Apparent differences in phenotype between the different genospecies, represented by DJ3T (Table 2), APA-1T and APA-4T, supported the phylogenetic and genotypic data (Table 3). The differences between the ecosystems from which the isolates were obtained were also evident, with the sea-ice brine strains being much more salt tolerant than the crustacean isolates and B. paragorgiae (isolated from a coral species), which could not tolerate more than 2·0 M NaCl. However, they had many traits in common, including a requirement for yeast extract and a preference for the metabolism of proteins (such as casein and gelatin) and amino acids (e.g. L-histidine and L-tyrosine), but not carbohydrates.
The fatty acid profiles of HFDT, IC164T APA-1T and APA-4T were practically indistinguishable, and were very similar to that of B. paragorgiae (Table 5), which was consistent with their phylogenetic relatedness. However, the fatty acid profile of DJ3T differed from those of the other strains, in the lack of both i17 : 1 and a17 : 1. These fatty acids were present in all the other isolates and related species examined in this study. Although strain DJ3T was in some ways phenotypically similar to the sea-ice brine and amphipod strains (Table 3), there were also a number of key differences (Table 4), including non-exacting nutritional requirements and the ability to utilize carbohydrates. Together with the inability to form branched-chain C17 mono-unsaturated fatty acids (Table 5), various phenotypic characteristics and the DNA G+C content also separated it from the phylogenetically related genera Psychroserpens and Winogradskyella (Table 4). Thus, based on taxonomic data, strain DJ3T appears to represent a new genus and a novel species, for which the name Lacinutrix copepodicola gen. nov., sp. nov. is proposed.
The sea-ice brine strains and amphipod-derived strains all formed a common phylogenetic cluster with B. paragorgiae (Fig. 1). The four genospecies represented by these strains also have many characteristics in common with B. paragorgiae, and make up a distinct genus that differs phenotypically from related members of the family Flavobacteriaceae, including their closest relatives F. algae and Algibacter lectus (Table 4). The DNA G+C content values of members of the novel genospecies (40–45 mol%) were also distinctive, and somewhat higher than is typical for many members of the family Flavobacteriaceae, but much less than the 55 mol% value recorded for Robiginitalea biformata (Cho & Giovannoni, 2004). On the basis of the combined taxonomic data, it is proposed that the sea-ice brine- and amphipod-derived strains should be included within the genus Bizionia, comprising four distinct species, Bizionia saleffrena sp. nov. (type strain, HFDT), Bizionia gelidisalsuginis sp. nov. (type strain, IC164T), Bizionia algoritergicola sp. nov. (type strain, APA-1T) and Bizionia myxarmorum sp. nov. (type strain, ADA-4T).
Description of Subsaximicrobium gen. nov.
Subsaximicrobium (Sub.sa.xi.mi.cro'.bi.um. L. pref. sub below; L. neut. n. saxum stone; N.L. neut. n. microbium microbe; N.L. neut. n. Subsaximicrobium microbe living below stone).
Gram-negative, rod-shaped cells, approximately 0·3–0·5 µm in width and 1–6 µm in length. Older cultures contain many spherical cells (diameter 1–3 µm). Exhibit gliding motility when grown on dilute agar media. Cell mass is either yellow or orange. Flexirubin pigments are not formed. Do not form resting cells or spores. Strictly aerobic and chemoheterotrophic. Produce catalase. Major fatty acids include i15 : 110c, a15 : 110c, i15 : 0, a15 : 0, 16 : 17c, 3-OH a15 : 0 and 3-OH i16 : 0. DNA G+C content is 39–40 mol%. A member of the family Flavobacteriaceae, class Flavobacteria, phylum ‘Bacteroidetes’.
The type species is Subsaximicrobium wynnwilliamsii.
Description of Subsaximicrobium wynnwilliamsii sp. nov.
Subsaximicrobium wynnwilliamsii (wynn.wil.li.ams'i.i. N.L. gen. n. wynnwilliams of Wynn-Williams, named in honour of the late D. D. Wynn-Williams, British Antarctic microbiologist).
Description is as for the genus plus the following. Cell mass is either a bright-orange or golden-yellow colour. Colonies are circular, convex, with an entire edge and a butyrous consistency on MA. Colonies exhibit spreading margins on dilute media. Growth occurs at –2 °C in marine broth. Good growth occurs on MA at 1–20 °C. Growth at 25 °C is weak or negative. Requires Na+ ions for growth. Grows in 0·25–2·0 M NaCl, with optimal growth occurring in approximately 0·3–0·4 M NaCl. Grows poorly in media prepared with 2x strength (70 g l–1) sea salts; no growth occurs with 4x strength (140 g l–1) sea salts. Grows well on trypticase soya agar (TSA) containing 1 % (w/v) NaCl, but grows poorly on TSA and nutrient agar (NA). Does not require yeast extract or vitamins for growth, and can use inorganic nitrogen sources such as sodium nitrate and ammonium chloride. Grows in mineral salts media with various sole carbon and energy sources, including D-glucose, glycogen, maltose, sucrose and L-proline. Acid production from carbohydrates on Leifson's O–F medium is undetectable. Hydrolyses gelatin, Tween 80, L-tyrosine and aesculin, but not DNA or urea. Most strains also decompose starch and weakly degrade casein. Produces 
-glucosidase and alkaline phosphatase. Does not produce arginine dihydrolase or lecithinase, or reduce nitrate. Other phenotypic data are shown in Table 2. Mean DNA G+C content is 40 mol%.
The type strain is G#7T (=ACAM 1070T=CIP 108525T), which was isolated from cyanobacterial biofilms attached to the undersides of partially buried quartz stones, found in the Vestfold Hills, an ice-free region of East Antarctica.
Description of Subsaximicrobium saxinquilinus sp. nov.
Subsaximicrobium saxinquilinus (sax.in.qui.li'nus. L. n. saxum stone; L. masc. n. inquilinus the denizen; N.L. masc. n. saxinquilinus the denizen of stone).
Description is as for the genus plus the following. Cell mass is an orange colour. Colonies are circular, convex, with an entire edge and a butyrous consistency on MA. Colonies exhibit spreading margins on dilute media. Growth occurs at –2 °C in marine broth. Good growth occurs on MA at 1–20 °C. No growth occurs at 25 °C or higher. Requires Na+ ions for growth. Grows in 0·1–2·0 M NaCl, with optimal growth occurring in approximately 0·3–0·4 M NaCl. Grows well in media prepared with 2x strength sea salts. No growth occurs with 4x strength sea salts. Grows well on TSA containing 1 % (w/v) NaCl, but grows poorly on TSA and NA. Does not require yeast extract or vitamins for growth, and can use inorganic nitrogen sources such as sodium nitrate and ammonium chloride. Grows in mineral salts media with various sole carbon and energy sources, including D-glucose, glycogen, D-mannose, maltose and L-proline. Acid production from carbohydrates on Leifson's O–F medium is undetectable. Hydrolyses gelatin, casein, Tween 80, starch, aesculin and DNA, but not elastin, L-tyrosine or urea. Produces -glucosidase, 
-glucosidase and alkaline phosphatase. Produces arginine dihydrolase. Lecithinase production and nitrate reduction are negative. Other phenotypic data are shown in Table 2. Mean DNA G+C content is 39 mol%.
The type strain is Y4-5T (=ACAM 1063T=CIP 108526T), which was isolated from cyanobacterial biofilms attached to the undersides of partially buried quartz stones, found in the Vestfold Hills, an ice-free region of East Antarctica.
Description of Subsaxibacter gen. nov.
Subsaxibacter (Sub.sa.xi.bac'ter. L. pref. sub below; L. neut. n. saxum stone; N.L. masc. n. bacter rod; N.L. masc. n. Subsaxibacter bacterial rod living below stone).
Gram-negative, coccobacilli, approximately 0·3–0·5 µm in width and 0·4–1·0 µm in length. Exhibit gliding motility. Cell mass is orange. Flexirubin pigments are not formed. Do not form resting cells or spores. Strictly aerobic and chemoheterotrophic. Produce catalase. Major fatty acids include i15 : 110c, a15 : 110c, i15 : 0, a15 : 0, 16 : 17c, a17 : 1, 18 : 19c and 3-OH i16 : 0. A member of the family Flavobacteriaceae, class Flavobacteria, phylum ‘Bacteroidetes’.
The type species is Subsaxibacter broadyi.
Description of Subsaxibacter broadyi sp. nov.
Subsaxibacter broadyi (broa.dy'i. N.L. gen. n. broadyi of Broady, named in honour of P. A. Broady, Antarctic microbiologist from New Zealand).
Description is as for the genus plus the following. Colonies are small (<1 mm), circular and convex, with a spreading or entire edge and a butyrous to viscid consistency on MA. Colonies exhibit spreading margins on dilute media. Growth occurs slowly at –2 °C in marine broth. Psychrophilic. Moderate to good growth occurs on MA at 1–15 °C. Weak growth occurs at 20 °C. No growth occurs at 25 °C or higher. Requires Na+ ions for growth. Grows in 0·1–1·25 M NaCl, with optimal growth occurring in approximately 0·3–0·4 M NaCl. Grows well in media prepared with 2x strength sea salts. No growth occurs with 4x strength sea salts. Grows on TSA containing 1 % (w/v) NaCl. No growth occurs on TSA or NA. Requires yeast extract for good growth. Peptone also supports poor to moderate growth. Cannot use sodium nitrate or ammonium chloride as sole nitrogen sources. Does not utilize any of the common substrates tested as sole carbon and energy sources (see Table 2 for substrates tested). Acid production from carbohydrates on Leifson's O–F medium is undetectable. Hydrolyses gelatin and Tween 80, but not casein, elastin, starch, DNA or urea. May weakly decompose L-tyrosine or aesculin. Produces alkaline phosphatase. Does not produce arginine dihydrolase or lecithinase, or reduce nitrate. Other phenotypic data are given in Table 2. Mean DNA G+C content is 35 mol%.
The type strain is P7T (=ACAM 1064T=CIP 108527T), which was isolated from cyanobacterial biofilms attached to the undersides of partially buried quartz stones, found in the Vestfold Hills, an ice-free region of East Antarctica.
Description of Lacinutrix gen. nov.
Lacinutrix [La.ci.nu'trix. L. n. lacus lake; L. fem. n. nutrix feeder; N.L. fem. n. Lacinutrix lake feeder (in the sense of being basically important for the food chain)].
Gram-negative, straight or slightly curved rods, approximately 0·4–0·5 µm in width and 1–2 µm in length. Non-motile. Cell mass is golden-yellow. Flexirubin pigments are not formed. Do not form resting cells or spores. Strictly aerobic and chemoheterotrophic. Produce catalase. Major fatty acids include i15 : 110c, a15 : 110c, i15 : 0, a15 : 0, br16 : 1 and i16 : 0. A member of the family Flavobacteriaceae, class Flavobacteria, phylum ‘Bacteroidetes’.
The type species is Lacinutrix copepodicola.
Description of Lacinutrix copepodicola sp. nov.
Lacinutrix copepodicola [co.pe.pod.i'col.a. N.L. neut. pl. n. copepoda copepods (small types of crustacea); L. fem. or masc. suff. -cola the dweller, inhabitant; N.L. masc. or fem. n. copepodicola the inhabitant of copepods].
Description is as for the genus plus the following. Colonies are golden-yellow, circular, convex, with an entire edge and a butyrous consistency on MA. Growth occurs at –2 °C in marine broth. Good growth occurs on MA at 1–25 °C. No growth occurs at 30 °C or higher. Requires Na+ ions for growth. Grows in 0·1–2·0 M NaCl, with optimal growth occurring in approximately 0·3–0·4 M NaCl. Grows well in media prepared with 2x strength sea salts. No growth occurs with 4x strength sea salts. Grows poorly on TSA containing 1 % (w/v) NaCl. Does not grow on either TSA or NA. Does not require yeast extract or vitamins for growth, and can utilize inorganic nitrogen sources such as sodium nitrate and ammonium chloride. Grows in mineral salts media with various sole carbon and energy sources, including D-glucose, glycogen, D-mannose, maltose, sucrose and L-proline. Acid production from carbohydrates on Leifson's O–F medium is undetectable. Hydrolyses gelatin, Tween 80 and L-tyrosine, but not casein, elastin, starch, aesculin, DNA or urea. Produces alkaline phosphatase and weakly produces N-acetyl--D-glucosaminidase. Does not produce arginine dihydrolase and lecithinase, or reduce nitrate. Other phenotypic data are shown in Table 2. Mean DNA G+C content is 37 mol%.
The type strain is DJ3T (=ACAM 1055T=CIP 108538T), which was isolated directly from the calanoid copepod species Paralabidocera antarctica dwelling in Ace Lake in the Vestfold Hills, an ice-free region of East Antarctica.
Description of Bizionia saleffrena sp. nov.
Bizionia saleffrena [sal.ef.fre'na. L. masc. n. sal salt; L. fem. adj. effrena unbridled; N.L. fem. adj. Saleffrena unbridled by salt (referring to the species' good growth on salt-containing media)].
Cells are rod-like, 0·4–0·5x1·5–5 µm. Colonies are golden-yellow, circular and convex, with an entire edge and a butyrous consistency on MA. Flexirubin pigments are not produced. Growth occurs at –2 °C in marine broth. Good growth occurs on MA at 1–20 °C. Growth is poor at 25 °C. No growth occurs at 30 °C or higher. Requires Na+ ions for growth. Grows in 0·2–3·0 M NaCl, with optimal growth occurring in approximately 1·0 M NaCl. Grows well in media prepared with 4x strength sea salts. Does not grow on TSA containing 1 % (w/v) NaCl, TSA or NA. Requires yeast extract or peptone for growth, and cannot use inorganic nitrogen sources such as sodium nitrate or ammonium chloride. Does not utilize any of the common substrates tested as sole carbon and energy sources (see Table 3 for substrates tested). Acid production from carbohydrates on Leifson's O–F medium is undetectable. Hydrolyses gelatin, casein, elastin, Tween 80, L-tyrosine and DNA, but not starch, aesculin or DNA. Produces lecithinase. Produces alkaline phosphatase. Does not produce arginine dihydrolase or reduce nitrate. Other phenotypic data are shown in Table 3. Mean DNA G+C content is 40 mol%.
The type strain is HFDT (=ACAM 1059T=CIP 108534T), which was isolated from sea-ice brine, drained and collected from fast sea-ice cores obtained from the coastal areas of the Vestfold Hills, an ice-free region of East Antarctica.
Description of Bizionia gelidisalsuginis sp. nov.
Bizionia gelidisalsuginis (gel.id.i'sal.su.gin.is. L. adj. gelidus icy; L. fem. n. salsugo -inis the brine; N.L. gen. n. gelidisalsuginis of icy brine).
Cells are rod-like and 0·4–0·5x1·5–3·5 µm. Colonies are golden-yellow, circular and convex, with an entire edge and a butyrous consistency on MA. Flexirubin pigments are not produced. Growth occurs at –2 °C in marine broth. Good growth occurs on MA at 1–25 °C. No growth occurs at 30 °C or higher. Requires Na+ ions for growth. Grows in 0·2–3·0 M NaCl, with optimal growth occurring in approximately 1·0 M NaCl. Grows well in media prepared with 4x strength sea salts. Does not grow on TSA containing 1 % (w/v) NaCl, TSA or NA. Requires yeast extract or peptone for growth, and cannot use inorganic nitrogen sources such as sodium nitrate or ammonium chloride. In minimal growth media can utilize sodium acetate, sodium propionate, L-alanine and L-histidine as sole carbon and energy sources. Acid production from carbohydrates on Leifson's O–F medium is undetectable. Hydrolyses gelatin, casein and L-tyrosine, but not elastin, Tween 80, starch, aesculin, DNA or urea. Produces alkaline phosphatase. Produces arginine dihydrolase. Does not produce lecithinase or reduce nitrate. Other phenotypic data are shown in Table 3. Mean DNA G+C content is 39 mol%.
The type strain is IC164T (=ACAM 1057T=CIP 108536T), which was isolated from sea-ice brine, drained and collected from fast sea-ice cores obtained from the coastal areas of the Vestfold Hills, an ice-free region of East Antarctica.
Description of Bizionia algoritergicola sp. nov.
Bizionia algoritergicola (al.go.ri.ter.gi'col.a. L. n. algor the cold; L. n. tergum outer covering or surface; L. fem. or masc. suff. -cola the dweller, inhabitant; N.L. fem. or masc. n. algoritergicola the inhabitant of a cold surface/covering).
Cells are rod-like, 0·3–0·5x1–3 µm. Colonies are golden-yellow, circular and convex, with an entire edge and a butyrous consistency on MA. Flexirubin pigments are not produced. Growth occurs at –2 °C in marine broth. Good growth occurs on MA at 1–20 °C. Poor growth occurs at 25 °C. No growth occurs at 28 °C or higher. Requires Na+ ions for growth. Grows in 0·2–2·0 M NaCl, with optimal growth occurring in approximately 0·3–0·4 M NaCl. Grows well in media prepared with 2x strength sea salts, whereas growth in 4x strength sea salts is very poor. Does not grow on TSA containing 1 % (w/v) NaCl, TSA or NA. Requires yeast extract or peptone for growth, and cannot use inorganic nitrogen sources such as sodium nitrate or ammonium chloride. In minimal growth media utilizes L-histidine as a sole carbon and energy source. Acid production from carbohydrates on Leifson's O–F medium is not detectable. Hydrolyses gelatin, casein, Tween 80, L-tyrosine, DNA and urea, but not elastin, starch or aesculin. Produces alkaline phosphatase. Produces arginine dihydrolase. Does not produce lecithinase or reduce nitrate. Other phenotypic data are shown in Table 3. Mean DNA G+C content is 45 mol%.
The type strain is APA-1T (=ACAM 1056T=CIP 108533T), which was isolated from the exoskeletal slime on an unidentified sea-ice, algae-feeding amphipod, collected from the sea ice–sea water interface in coastal areas of the Vestfold Hills, an ice-free region of East Antarctica.
Description of Bizionia myxarmorum sp. nov.
Bizionia myxarmorum [myx.ar.mor'um. Gr. n. myxa slime; L. gen. pl. n. armorum defensive armour; N.L. pl. gen. n. myxarmorum of armour slime (of the slime on the carapace of crustacean host)].
Cells are rod-like, 0·3–0·5x1·5–3·5 µm. Colonies are golden-yellow, circular and convex, with an entire edge and a butyrous consistency on MA. Flexirubin pigments are not produced. Growth occurs at –2 °C in marine broth. Good growth occurs on MA at 1–25 °C. Poor growth occurs at 30 °C. No growth occurs above 32 °C. Requires Na+ ions for growth. Grows in 0·2–2·0 M NaCl, with optimal growth occurring in approximately 0·3–0·4 M NaCl. Grows well in media prepared with 2x strength sea salts, whereas no growth occurs in 4x strength sea salts. Does not grow on TSA or NA. Requires yeast extract or peptone for growth, and cannot use inorganic nitrogen sources such as sodium nitrate or ammonium chloride. In minimal growth media can utilize sodium acetate, sodium valerate, L-alanine, L-histidine, L-proline and L-serine as sole carbon and energy sources. Acid production from carbohydrates on Leifson's O–F medium is undetectable. Hydrolyses gelatin, casein, Tween 80, L-tyrosine, DNA and urea, but not elastin, starch or aesculin. Produces alkaline phosphatase. Produces arginine dihydrolase and lecithinase, but does not reduce nitrate. Other phenotypic data are shown in Table 3. Mean DNA G+C content is 43 mol%.
The type strain is ADA-4T (=ACAM 1058T=CIP 108535T), which was isolated from the exoskeletal slime on an unidentified sea-ice, algae-feeding amphipod, collected from the sea ice–sea water interface in coastal areas of the Vestfold Hills, an ice-free region of East Antarctica.
Description of Gelidibacter gilvus sp. nov.
Gelidibacter gilvus (gil'vus. L. masc. adj. gilvus pale yellow).
Gram-negative. Cells are rod-shaped, approximately 0·4–0·6x2–8 µm. Large coccoid bodies (2–3 µm in diameter) form in older cultures. Motile by gliding. Cell mass is yellow. Flexirubin pigments are not formed. Colonies are circular and convex, with a thin spreading margin and a viscid consistency on MA. Growth occurs at –2 °C in marine broth. Good growth occurs on MA at 1–25 °C. No growth occurs at 30 °C or higher. Halophilic. Grows in 0·1–1·5 M NaCl, with optimal growth occurring in approximately 0·3–0·4 M NaCl. Grows in media prepared with 2x strength sea salts, whereas no growth occurs in 4x strength sea salts. Grows well on TSA containing 1 % (w/v) NaCl, and weakly on TSA and NA. Requires yeast extract or peptone for growth, and cannot use inorganic nitrogen sources such as sodium nitrate or ammonium chloride. In minimal growth media can utilize D-glucose, glycogen, N-acetyl-D-glucosamine, DL-arabinose, maltose, sucrose, sodium acetate, sodium propionate and L-proline as sole carbon and energy sources. Acid production from carbohydrates on Leifson's O–F medium is positive for D-glucose, L-arabinose, D-mannose, D-galactose, D-fructose, D-rhamnose, D-xylose, D-mannitol, sucrose, N-acetyl-D-glucosamine, lactose, cellobiose, trehalose, maltose, D-melibiose, inositol and glycerol. Acid is not formed from melezitose, raffinose, dextran, adonitol or D-sorbitol. Hydrolyses Tween 80 and aesculin, but not gelatin, casein, elastin, L-tyrosine, starch, DNA or urea. Produces lecithinase. Produces 6-phospho--galactosidase, -galactosidase, -galactosidase, -glucosidase, -glucosidase, -fucosidase and N-acetyl--D-glucosaminidase, but not alkaline phosphatase. Does not produce arginine dihydrolase or reduce nitrate. Other phenotypic data are shown in Table 2. Mean DNA G+C content is 39 mol%.
The type strain is IC158T (=ACAM 1054T=CIP 108531T), which was isolated from sea-ice algae, collected from fast sea-ice cores obtained from the coastal areas of the Vestfold Hills, an ice-free region of East Antarctica.
Description of Gelidibacter salicanalis sp. nov.
Gelidibacter salicanalis (sal.i.can.a.l'is. L. n. sal salt; L. n. canalis channel; N.L. gen. n. salicanalis of the salt channel).
Gram-negative. Cells are rod-shaped or filamentous, approximately 0·4–0·6x2–15 µm. Small coccoid bodies (approximately 1–1·5 µm diameter) form in older cultures. Motile by gliding (visible on very dilute growth media only). Cell mass is golden yellow. Flexirubin pigments are not formed. Colonies are circular and convex, with an entire edge and a butyrous consistency on MA. Growth occurs at –2 °C in marine broth. Good growth occurs on MA at 1–25 °C. No growth occurs at 30 °C or higher. Non-halophilic. Grows in 0–2·5 M NaCl, with optimal growth occurring at approximately 0·3–0·4 M NaCl. Grows in media prepared with 2x strength sea salts, whereas poor growth occurs in 4x strength sea salts. Grows on TSA containing 1 % (w/v) NaCl, TSA and NA. Does not require yeast extract for growth, and can utilize inorganic nitrogen sources such as sodium nitrate and ammonium chloride. In mineral salts growth media can utilize D-glucose, glycogen, N-acetyl-D-glucosamine, maltose, L-proline and L-serine as sole carbon and energy sources. Acid production from carbohydrates on Leifson's O–F medium is positive for D-glucose, L-arabinose, D-mannose, D-galactose, N-acetyl-D-glucosamine, maltose, lactose, cellobiose, sucrose and trehalose. Hydrolyses gelatin, casein, L-tyrosine and aesculin, but not elastin, starch, Tween 80 or DNA. Urea is weakly decomposed. Produces 6-phospho--galactosidase, -galactosidase, -galactosidase, -fucosidase, N-acetyl--D-glucosaminidase and alkaline phosphatase. Produces arginine dihydrolase, but does not produce lecithinase or reduce nitrate. Other phenotypic data are shown in Table 2. Mean DNA G+C content is 42 mol%.
The type strain is IC162T (=ACAM 1053T=CIP 108532T), which was isolated from sea-ice algae collected from pack-ice brine, Southern Ocean.
Description of Gillisia illustrilutea sp. nov.
Gillisia illustrilutea (il.lus.tri.lu.te'a. L. adj. illustris bright; L. fem. adj. lutea yellow; N.L. fem. adj. illustrilutea bright yellow).
Gram-negative. Cells are rod-shaped, approximately 0·3–0·5x1–10 µm. Non-motile. Cell mass is bright yellow. Flexirubin pigments are not formed. Colonies are circular and convex, with an entire edge and a butyrous consistency on MA. Growth occurs at –2 °C in marine broth. Good growth occurs on MA at 1–20 °C. No growth occurs at 25 °C or higher. Non-halophilic. Grows in 0–1·25 M NaCl, with optimal growth occurring in approximately 0·2–0·3 M NaCl. Grows in media prepared with 2x strength sea salts, whereas no growth occurs in 4x strength sea salts. Grows on TSA containing 1 % (w/v) NaCl, TSA and NA. Does not require yeast extract for growth, and can use inorganic nitrogen sources such as sodium nitrate and ammonium chloride. In mineral salts growth media can utilize D-glucose, D-mannose, maltose, sucrose, sodium propionate and L-proline as sole carbon and energy sources. Acid production from carbohydrates on Leifson's O–F medium is negative for D-glucose. Hydrolyses DNA, but not casein, elastin, L-tyrosine, aesculin, starch, Tween 80 or urea. Produces -galactosidase, -glucosidase and -glucosidase. Does not produce arginine dihydrolase or lecithinase, or reduce nitrate. Other phenotypic data are shown in Table 2. Mean DNA G+C content is 32 mol%.
The type strain is IC157T (=ACAM 1062T=CIP 108530T), which was isolated from sea-ice algae collected from fast sea-ice cores, obtained from the coastal areas of the Vestfold Hills, an ice-free region of East Antarctica.
Description of Gillisia sandarakina sp. nov.
Gillisia sandarakina [san.da.ra.kin'a. N.L. fem. adj. sandarakina (from Gr. fem. adj. sandarakinê) of orange colour)].
Gram-negative. Cells are rod-shaped, approximately 0·4–0·5x1–10 µm. Non-motile. Cell mass is light orange. Flexirubin pigments are not formed. Colonies are small (1 mm diameter), circular and convex, with an entire edge and a butyrous consistency on MA. Growth occurs at –2 °C in marine broth. Good growth occurs on MA at 1–20 °C. No growth occurs at 25 °C or higher. Requires sea salts for growth. Media supplemented only with Na+ ions do not support growth. Grows in 0·2–1·5 M NaCl, with optimal growth occurring in approximately 0·3–0·5 M NaCl. Grows in media prepared with 2x strength sea salts, but no growth occurs with 4x strength sea salts. Does not grow on TSA containing 1 % (w/v) NaCl, TSA or NA. Does not require yeast extract for growth, and can use inorganic nitrogen sources such as sodium nitrate and ammonium chloride. In mineral salts growth media can utilize D-glucose, DL-arabinose, D-mannose, sodium propionate (weak growth) and L-proline as sole carbon and energy sources. Acid production from carbohydrates on Leifson's O–F medium is negative for D-glucose. Hydrolyses gelatin, starch, Tween 80 and L-tyrosine, but not casein, elastin, aesculin, DNA or urea. Produces -glucosidase, -glucosidase and alkaline phosphatase. Does not produce arginine dihydrolase or lecithinase. Reduces nitrate to nitrite. Other phenotypic data are shown in Table 2. Mean DNA G+C content is 36 mol%.
The type strain is IC148T (=ACAM 1060T=CIP 108529T), which was isolated from sea-ice algae collected from fast sea-ice cores, obtained from the coastal areas of the Vestfold Hills, an ice-free region of East Antarctica.
Description of Gillisia hiemivivida sp. nov.
Gillisia hiemivivida [hi.em.i'vi.vi.da. L. fem. n. hiems -emis the cold (of winter); L. fem. adj. vivida lively; N.L. fem. adj. hiemivivida lively in the cold)].
Gram-negative. Cells are rod-shaped, approximately 0·4–0·6x1–7 µm. Non-motile. Cell mass is light orange. Flexirubin pigments are not formed. Colonies are circular and convex, with an entire edge and a butyrous consistency on MA. Growth occurs at –2 °C in marine broth. Good growth occurs on MA at 1–25 °C. No growth occurs at 30 °C or higher. Requires sea salts for growth. Media supplemented only with Na+ ions do not support growth. Grows in media containing 0·2–1·5 M NaCl, with optimal growth occurring in approximately 0·3–0·5 M NaCl. Grows in media prepared with 2x strength sea salts, but no growth occurs in 4x strength sea salts. Does not grow on TSA containing 1 % (w/v) NaCl, TSA or NA. Does not require yeast extract for growth, and can use inorganic nitrogen sources such as sodium nitrate and ammonium chloride. In mineral salts growth media can utilize D-glucose, glycogen, N-acetylglucosamine, DL-arabinose, D-mannose, maltose, sucrose, sodium propionate, L-alanine (weak growth) and L-proline as sole carbon and energy sources. Acid production from carbohydrates on Leifson's O–F medium is negative for D-glucose. Hydrolyses gelatin, starch, Tween 80, L-tyrosine and urea, but not casein, elastin, aesculin or DNA. Produces -glucosidase, -glucosidase and alkaline phosphatase. Does not produce arginine dihydrolase or lecithinase, or reduce nitrate. Other phenotypic data are shown in Table 2. Mean DNA G+C content is 34 mol%.
The type strain is IC154T (=ACAM 1061T=CIP 108528T), which was isolated from sea-ice algae collected from fast sea-ice cores, obtained from the coastal areas of the Vestfold Hills, an ice-free region of East Antarctica.
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, >90 %; 
, 60–90 %). Bar, evolutionary distance of 0·05.