Biogeographic and quantitative analyses of abundant uncultivated gamma-proteobacterial clades from marine sediment.

16S rRNA gene-based molecular analyses revealed the presence of several large and so far uncultivated clades within class gamma-Proteobacteria, designated gamma-proteobacterial marine sediment (GMS) clades 1 to 4, in marine sediment. The GMS clades appear only indigenous to marine sediment and so far have an unknown functionality. SYBR Green-based real-time PCR analyses using GMS clade-specific primers indicated GMS clades were a significant part of the bacterial community (0.3-8.7% of total 16S rRNA genes) in both polar and temperate marine sediment samples. Univariate statistical analyses indicated that GMS clade communities were indistinguishable in two temperate coastal sediment samples even though these possessed very different mean grain sizes, organic contents, and organic loading rates. GMS clade communities were slightly different (p < 0.05) between polar and temperate sites, suggesting that psychrophilic adaptation among GMS clade taxa corresponds only to subtle phylogenetic differences. Similar levels of difference were also observed through a sediment core reflecting that through the sediment core history, which spanned approximately 3000 years, GMS clonal diversity shifted only marginally.

Prokaryotic metabolic activity and community structure in Antarctic continental shelf sediments.

The prokaryote community activity and structural characteristics within marine sediment sampled across a continental shelf area located off eastern Antarctica (66 degrees S, 143 degrees E; depth range, 709 to 964 m) were studied. Correlations were found between microbial biomass and aminopeptidase and chitinase rates, which were used as proxies for microbial activity. Biomass and activity were maximal within the 0- to 3-cm depth range and declined rapidly with sediment depths below 5 cm. Most-probable-number counting using a dilute carbohydrate-containing medium recovered 1.7 to 3.8% of the sediment total bacterial count, with mostly facultatively anaerobic psychrophiles cultured. The median optimal growth temperature for the sediment isolates was 15 degrees C. Many of the isolates identified belonged to genera characteristic of deep-sea habitats, although most appear to be novel species. Phospholipid fatty acid (PLFA) and isoprenoid glycerol dialkyl glycerol tetraether analyses indicated that the samples contained lipid components typical of marine sediments, with profiles varying little between samples at the same depth; however, significant differences in PLFA profiles were found between depths of 0 to 1 cm and 13 to 15 cm, reflecting the presence of a different microbial community. Denaturing gradient gel electrophoresis (DGGE) analysis of amplified bacterial 16S rRNA genes revealed that between samples and across sediment core depths of 1 to 4 cm, the community structure appeared homogenous; however, principal-component analysis of DGGE patterns revealed that at greater sediment depths, successional shifts in community structure were evident. Sequencing of DGGE bands and rRNA probe hybridization analysis revealed that the major community members belonged to delta proteobacteria, putative sulfide oxidizers of the gamma proteobacteria, Flavobacteria, Planctomycetales, and Archaea. rRNA hybridization analyses also indicated that these groups were present at similar levels in the top layer across the shelf region.

Taxonomy of Antarctic Flavobacterium species: description of Flavobacterium gillisiae sp. nov., Flavobacterium tegetincola sp. nov., and Flavobacterium xanthum sp. nov., nom. rev. and reclassification of [Flavobacterium] salegens as Salegentibacter salegens gen. nov., comb. nov.

16S rRNA phylogenetic analysis of a number of yellow- and orange-pigmented strains isolated from a variety of Antarctic habitats including sea ice, lakewater and cyanobacterial mats indicated a close relationship to the genus Flavobacterium but distinct from known Flavobacterium species. Phenotypic properties, DNA G+C content and whole-cell fatty acid profiles of the Antarctic strains were consistent with those of the genus Flavobacterium. DNA-DNA hybridization analysis indicated the presence of two distinct and novel genospecies each isolated from a different Antarctic habitat. From polyphasic taxonomic data it is proposed that the two groups represent new species with the following proposed names: Flavobacterium gillisiae (ACAM 601T) and Flavobacterium tegetincola (ACAM 602T). In addition polyphasic analysis of the species '[Cytophaga] xantha' (Inoue and Komagata 1976), isolated from Antarctic mud, indicated it was a distinct member of the genus Flavobacterium and was thus revived as Flavobacterium xanthum. Phylogenetic and fatty acid analyses also indicate that the species [Flavobacterium] salegens (Dobson et al. 1993), from Organic Lake, Antarctica, is misclassified at the genus level. It is proposed that this species belongs to a new genus, Salegentibacter salegens gen. nov., comb. nov.

The microbial composition of three limnologically disparate hypersaline Antarctic lakes.

16S rRNA clone library analysis was used to examine the biodiversity and community structure within the sediments of three hypersaline Antarctic lakes. Compared to sediment of low to moderate salinity Antarctic lakes the species richness of the hypersaline lake sediments was 2-20 times lower. The community of Deep Lake (32% salinity, average sediment temperature -15 degrees C) was made up almost entirely of halophilic Archaea. The sediment communities of two meromictic hypersaline lakes, Organic Lake (20% salinity, -7 degrees C) and Ekho Lake (15% salinity, 15 degrees C) were more complex, containing phylotypes clustering within the Proteobacteria and Cytophagales divisions and with algal chloroplasts. Many phylotypes of these lakes were related to taxa more adapted to marine-like salinity and perhaps derive from bacteria exported into the sediment from the lower salinity surface waters. The Ekho Lake clone library contained several major phylotypes related to the Haloanaerobiales, the growth of which appears to be promoted by the comparatively high in situ temperature of this lake.

Diversity and community structure within anoxic sediment from marine salinity meromictic lakes and a coastal meromictic marine basin, Vestfold Hilds, Eastern Antarctica.

16S rDNA clone library analysis was used to examine the biodiversity and community structure within anoxic sediments of several marine-type salinity meromictic lakes and a coastal marine basin located in the Vestfolds Hills area of Eastern Antarctica. From 69 to 130 (555 total) 16S rDNA clones were analysed from each sediment sample, and restriction fragment length polymorphism (RFLP) and sequence analysis grouped the clones into 202 distinct phylotypes (a clone group with sequence similarity of >0.98). A number of phylotypes and phylotype groups predominated in all libraries, with a group of 10 phylotypes (31% of clones) forming a novel deep branch within the low G+C Gram-positive division. Other abundant phylotypes detected in several different clone libraries grouped with Prochlorococcus cyanobacteria, diatom chloroplasts, delta proteobacteria (Desulfosarcina group, Syntrophus and Geobacterl Pelobacter/Desulphuromonas group), order Chlamydiales (Parachlamydiaceae) and Spirochaetales (wall-less Antarctic spirochaetes). Most archaeal clones detected (3.1% of clones) belonged to a highly diverged group of Euryarchaeota clustering with clones previously detected in rice soil, aquifer sediments and hydrothermal vent material. Little similarity existed between the phylotypes detected in this study and other clone libraries based on marine sediment, suggesting that an enormous prokaryotic diversity occurs within marine and marine-derived sediments.

Flavobacterium hibernum sp. nov., a lactose-utilizing bacterium from a freshwater Antarctic lake.

Four freshwater Antarctic lakes were examined for the presence of beta-galactosidase-producing bacteria using mineral medium enrichments and lactose. Enrichments from only one of the lakes produced growth and two strains were isolated that were very similar in phenotype and fatty acid profile, and shared considerable homology in their DNA (DNA-DNA hybridization = 93 +/- 7%). The strains were psychrotrophic with theoretical Tmax, Tmin and Topt of 30-31, -7 degrees and 26 degrees C, respectively. The beta-galactosidase in cell extracts had an optimal activity at 39 degrees C. The strains were Gram-negative rods, showed gliding motility, contained branched and hydroxy fatty acids, and menaquinone 6 as the major respiratory quinone. The strains did not form microcysts and utilized lactose while using ammonium ions as a source of nitrogen, and a range of other sugars. The G + C content of the DNA was 34 mol%. Phylogenetic analysis of one of the strains, by comparison of 16S rDNA sequences, showed that it was most similar, but not identical to, Flavobacterium columnare and '[Sporocytophaga] cauliformis'. Both species could be differentiated phenotypically from the Antarctic isolates. DNA-DNA hybridization of the Antarctic isolate with six different members of the Flavobacterium 16S rDNA cluster showed no strain with greater than 18% relatedness. The nearest type species to the Antarctic isolate in the phylogenetic analysis was Flavobacterium aquatile. The name Flavobacterium hibernum is proposed for the Antarctic strains, and the type strain is ATCC 51468T (= ACAM 376T).

Shewanella gelidimarina sp. nov. and Shewanella frigidimarina sp. nov., novel Antarctic species with the ability to produce eicosapentaenoic acid (20:5 omega 3) and grow anaerobically by dissimilatory Fe(III) reduction.

A polyphasic taxonomic study was performed to characterize dissimilatory iron-reducing strains mostly isolated from Antarctic sea ice. The strains were isolated from samples of congelated (land-fast) sea ice, grease ice, and ice algal biomass collected from the coastal areas of the Vestfold Hills in eastern Antarctica (68 degrees S 78 degrees E). The strains were facultatively anaerobic, motile, and rod shaped, were capable of anaerobic growth either by fermentation of carbohydrates or by anaerobic respiration, and utilized a variety of electron acceptors, including nitrate, ferric compounds, and trimethylamine N-oxide. A phylogenetic analysis performed with 16S rRNA sequences showed that the isolates formed two groups representing novel lineages in the genus Shewanella. The first novel group included seawater-requiring, psychrophilic, chitinolytic strains which had DNA G + C contents of 48 mol%. The members of the second strain group were psychrotrophic and did not require seawater but could tolerate up to 9% NaCl. The strains of this group were also unable to degrade polysaccharides but could utilize a number of monosaccharides and disaccharides and had G + C contents of 40 to 43 mol%. The whole-cell-derived fatty acid profiles of the sea ice isolates were found to be similar to the profiles obtained for other Shewanella species. The omega-3 polyunsaturated fatty acid eicosapentaenoic acid (EPA) (20:5 omega 3) was detected in all of the sea ice isolates at levels ranging from 2 to 16% of the total fatty acids. EPA was also found at high levels in Shewanella hanedai (19 to 22%) and Shewanella benthica (16 to 18%) but was absent in Shewanella alga and Shewanella putrefaciens. On the basis of polyphasic taxonomic data, the Antarctic iron-reducing strains are placed in two new species, Shewanella frigidimarina sp. nov. (type strain, ACAM 591) and Shewanella gelidimarina sp. nov. (type strain, ACAM 456).

Diversity and association of psychrophilic bacteria in Antarctic sea ice.

The bacterial populations associated with sea ice sampled from Antarctic coastal areas were investigated by use of a phenotypic approach and a phylogenetic approach based on genes encoding 16S rRNA (16S rDNA). The diversity of bacteria associated with sea ice was also compared with the bacterial diversity of seawater underlying sea ice. Psychrophilic (optimal growth temperature, < or = 15 degrees C; no growth occurring at 20 degrees C) bacterial diversity was found to be significantly enriched in sea ice samples possessing platelet and bottom ice diatom assemblages, with 2 to 9 distinct (average, 5.6 +/- 1.8) psychrophilic taxa isolated per sample. Substantially fewer psychrophilic isolates were recovered from ice cores with a low or negligible population of ice diatoms or from under-ice seawater samples (less than one distinct taxon isolated per sample). In addition, psychrophilic taxa that were isolated from under-ice seawater samples were in general phylogenetically distinct from psychrophilic taxa isolated from sea ice cores. The taxonomic distributions of psychrotrophic bacterial isolates (optimal growth temperature, > 20 degrees C; growth can occur at approximately 4 degrees C) isolated from sea ice cores and under-ice seawater were quite similar. Overall, bacterial isolates from Antarctic sea ice were found to belong to four phylogenetic groups, the alpha and gamma subdivisions of the Proteobacteria, the gram-positive branch, and the Flexibacter-Bacteroides-Cytophaga phylum. Most of the sea ice strains examined appeared to be novel taxa based on phylogenetic comparisons, with 45% of the strains being psychrophilic. 16S rDNA sequence analysis revealed that psychrophilic strains belonged to the genera Colwellia, Shewanella, Marinobacter, Planococcus, and novel phylogenetic lineages adjacent to Colwellia and Alteromonas and within the Flexibacter-Bacteroides-Cytophaga phylum. Psychrotrophic strains were found to be members of the genera Pseudoalteromonas, Psychrobacter, Halomonas, Pseudomonas, Hyphomonas, Sphingomonas, Arthrobacter, Planococcus, and Halobacillus. From this survey, it is proposed that ice diatom assemblages provide niches conducive to the proliferation of a diverse array of psychrophilic bacterial species.

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

Psychrophilic, yellow-pigmented, seawater-requiring bacteria isolated from the pycnocline of meromictic Burton Lake and from sea ice cores obtained in the Vestfold Hills (68 degrees S, 78 degrees E) in eastern Antarctica were characterized. Phenotypic analysis showed that the strains isolated formed two distinct taxa. The first taxon included nonmotile, nutritionally fastidious strains that were isolated from the pycnocline of Burton Lake. The cells of these strains were morphologically variant, ranging from vibrioid to ring shaped to coiled and filamentous; in addition, the strains were unable to metabolise carbohydrates or polysaccharides and had DNA G + C contents of 27 to 29 mol%. The strains of the second taxon, which were isolated from sea ice cores and from ice aigal biomass, were saccharolytic, exhibited rapid gliding motility, were rodlike to filamentous, and had DNA G + C contents of 36 to 38 mol%. A 16S ribosomal DNA (rDNA) sequence analysis revealed that the two Antarctic taxa formed related but distinct lineages within the [Flexibacter] maritimus rRNA branch of the family Flavobacteriacrae. The levels of 16S rDNA sequence similarity between the taxa were 90.5 to 91.3%, while the levels of similarity to other members of the [F.] maritimus rRNA branch were 85 to 90%. The whole-cell lipid profiles of the Antarctic strains were mainly comprised of branched and unbranched monounsaturated C15 to C17 fatty acids. The presence of significant levels of the lipids a 15:1 omega 10c and a17:1 omega 7c appeared to be useful biomarkers for the new Antarctic taxa and for differentiating these organisms from other members of the family Flavobacteriaceae. On the basis of polyphasic taxonomic data we propose that the new taxa are novel bacterial species designated Psychroserpens burtonensis gen. nov., sp. nov. (type strain, ACAM 188) and Gelidibacter algens gen. nov., sp. nov. (type strain, ACAM 536).