Valery V. Mikhailov

Phenotypic diversity of Pseudoalteromonas citrea from different marine habitats and emendation of the description

Four strains of marine, aerobic, agar-decomposing bacteria with one polar flagellum and with DNA G + C contents of 38.9-40.2 mol% were isolated from the Far-Eastern mussels Crenomytilus grayanus and Patinopecten yessoensis. These four strains were identified as Pseudoalteromonas; however, they were phenotypically different from species described previously according to carbon compound utilization tests and the BIOLOG identification system. High agar-decomposing activity was found in two strains, in one of which agarase, alpha-galactosidase, pustulanase and laminarinase had been detected. The level of DNA homology of three of the strains was 70-100%. The fourth isolate was genetically less related to the others (67% DNA relatedness) and phenotypically was more distant from other members of this group; however, all four strains were assigned to a single species genotypically. DNA from the strains isolated from mussels showed 40-45% genetic relatedness with the DNA of Alteromonas atlantica, 8-36% with DNA of Pseudoalteromonas haloplanktis subsp. haloplanktis, Pseudoalteromonas haloplanktis subsp. tetraodonis, Pseudoalteromonas undina, Pseudoalteromonas nigrifaciens and Pseudoalteromonasas carrageenovora, 53% with Pseudoalteromonas elyakovii, 32-48% with marine P. nigrifaciens from mussels and 14-16% with Alteromonas macleodii. The DNA-DNA hybridization data revealed that the levels of relatedness between the strains isolated and the type strains of Pseudoalteromonas citrea and Pseudoalteromonas fuliginea described recently were significant (95-85%). These results were confirmed by serological data employing polyclonal antibodies to cell surface antigens. The strains isolated from mussels were identified as P. citrea. The hybridization data showed that the name P. fuliginea Romanenko et al. 1994 should be recognized as a junior subjective synonym of P. citrea Gauthier 1977. A notable phenotypic diversity of P. citrea which might be a reflection of their ecological habitats is discussed.

Idiomarina gen. nov., comprising novel indigenous deep-sea bacteria from the Pacific Ocean, including descriptions of two species, Idiomarina abyssalis sp. nov. and Idiomarina zobellii sp. nov.

Two bacterial strains, KMM 227T and 231T, were isolated from seawater samples collected from the north-western Pacific Ocean at a depth of 4000-5000 m and were characterized using polyphasic taxonomy. Both were Gram-negative, psychrotolerant, heterotrophic, aerobic and required NaCl for growth (0.6-15.0%). The temperature for growth was 4-30 degrees C. Both strains were rod-shaped, with a single flagellum. However, strain KMM 231T revealed a single long fimbrium. Cellular fatty acids detected in the isolates were predominantly odd-numbered and iso-branched, with 15 and 17 carbons (ca. 70%). Also present were saturated and monounsaturated straight-chain fatty acids. Results of phylogenetic analyses, employing three tree-making methods, strongly indicated that the two strains formed a distinct lineage within a clade containing the genera Alteromonas, Colwellia and Pseudoalteromonas, in the gamma-Proteobacteria. The two strains shared 16S rDNA sequence similarity of 96.9% and genomic DNA relatedness of 27%; the latter was determined by dot-blot hybridization. The strains were differentiated by the presence of fimbria, production of chitinase, ability to grow on 15% NaCl and BIOLOG profiles. Given the polyphasic evidence accumulated in this study, it is proposed that the two deep-sea isolates be classified in the genus Idiomarina gen. nov., as Idiomarina abyssalis sp. nov. (type strain is KMM 227T) and Idiomarina zobellii sp. nov. (type strain is KMM 231T).

Shewanella japonica sp. nov.

Two strains of agar-digesting bacteria, KMM 3299T and KMM 3300, respectively isolated from sea water and the mussel Protothaca jedoensis, have been characterized. Based on sequencing of the 16S rRNA gene, KMM 3299T showed the highest similarity (93-95%) to members of the genus Shewanella. The G+C contents of the DNAs of these strains were 43-44 mol%. The level of DNA homology between the two strains was conspecific (95%), indicating that they represent a distinct genospecies. These organisms were non-pigmented, Gram-negative, polarly flagellated, facultatively anaerobic, mesophilic, neutrophilic and able to degrade a wide range of high molecular mass polymers, including alginate, carrageenan, laminaran and agar. The novel organisms were susceptible to gentamycin, carbenicillin, lincomycin and oleandomycin. The predominant cellular fatty acids were i-15:0, 16:0, 16:1(n-7), 18:1(n-7). Eicosapentaenoic acid, 20:5(n-3), was detected in the two isolates at levels of 1-8%, depending on the temperature of cultivation. Phylogenetic evidence, together with phenotypic characteristics, showed that the two isolates studied constitute a novel species of the genus Shewanella. The name Shewanella japonica is proposed; the type strain is KMM 3299T(= LMG 19691T = CIP 106860T).

Characterization and identification of marine Alteromonas nigrifaciens strains and emendation of the description

Nine nonpigmented strains of gram-negative, aerobic, marine bacteria with polar flagella were isolated from the mussels Crenomytilus grayanus and Patinopecten jessoensis. These organisms were conspecific and exhibited relatively high levels of genetic relatedness (61 to 100%). The G+C contents of the DNAs of these strains were 38.5 to 40.2 mol%. The strains isolated from mussels were phenotypically distinct from previously described Alteromonas species that have similar DNA G+C contents (Alteromonas haloplanktis, Alteromonas tetraodonis, Alteromonas atlantica, and Alteromonas carrageenovora), and their DNAs exhibited only 12 to 41% similarity with the DNAs of the type strains of these species. DNA-DNA hybridization data revealed that the levels relatedness between the strains which we studied and the type strain of Alteromonas nigrifaciens were significant (66 to 70%). Production of a melanin-like pigment, which is characteristic of A. nigrifaciens, was observed only in tyrosine-containing media. The strains isolated from mussels were identified as A. nigrifaciens. We present an emended description of A. nigrifaciens that includes several phenotypic and chemotaxonomic characteristics.

Cellular fatty acids of Alteromonas species

Summary The fatty acid (FA) compositions of 7 type strains and 9 strains isolated from the marine environment, defined by general microbiological methods as members of the genus Alteromonas , were characterized by capillary gas-liquid chromatography (GLC) on polar and non-polar columns. Thirty fatty acids with concentrations exceeding 0.1% were identified, with 16:1(n-7), 16:0, 17:1, 15:0, 18:1(n-7) and 17:0 acids as main components. Principal coordinate analysis of fatty acid data have shown that the majority of the type strains and marine species, identified as Alteromonas sp., formed one group, without species separation. These findings, as well as low amounts of branched chain and hydroxy fatty acids, and an absence of cyclopropane fatty acids in Alteromonas , provides characteristic features for conveniently distinguishing members of this genus from Pseudomonas and other morphologically similar species.

Evaluation of Phospholipid and Fatty Acid Compositions as Chemotaxonomic Markers of Alteromonas-Like Proteobacteria

The cellular phospholipids (PLs) and fatty acids (FAs) were investigated in type and environmental strains of Pseudoalteromonas, Alteromonas macleodii, A. infernus, and in three type strains of Marinomonas, M. communis, M. vaga, M. mediterranea. A total of 40 strains (19 strains in this study and 21 reported in previous papers), including Idiomarina abyssalis, I. zobellii, and Glaciecola punicea, G. pallidula, aerobic Alteromonas-like proteobacteria showed genus-characteristic patterns of phospholipids and fatty acids useful for genera discrimination. The PL patterns of surface cultures of alteromonads, pseudoalteromonads, and marinomonads consisted almost entirely of phosphatidyl ethanolamine and phosphatidyl glycerol presented in different proportions. Neither diphosphatidyl glycerol nor glycophospholipids were found in bacteria studied. In addition, the minor amount of a glycolipid was found in all strains studied. Bacteria of the genera Marinomonas, Idiomarina, and Glaciecola were clearly distinguished by presence of one of the major FAs: 18:1 (n-7), i15:0, and 16:1 (n-7), respectively. The amounts of these FAs reached up to 40–60% of total FAs. Members of Alteromonas and Pseudoalteromonas were characterized by different ratio of the following major FAs:16:1(n-7), 16:0, 17:1 (n-8), and 18:1 (n-7).

Psychrobacter submarinus sp. nov. and Psychrobacter marincola sp. nov., psychrophilic halophiles from marine environments

Two novel psychrophilic, halophilic, Psychrobacter-like bacteria, strains KMM 225T and KMM 277T, were isolated from sea water and the internal tissues of an ascidian Polysyncraton sp. specimen, respectively, and characterized using a polyphasic approach, which included phenotypic, genotypic, chemotaxonomic and phylogenetic analyses. The novel marine isolates were Gram-negative, aerobic, coccoid, oxidase- and catalase-positive, non-pigmented, non-motile, psychrophilic and halophilic and they utilized a restricted spectrum of carbon sources. Strains KMM 225T and KMM 277T required sea water or sodium ions for growth and were tolerant of up to 12-15% (w/v) NaCl. Growth of strains KMM 225T and KMM 277T was observed at 4-35 and 7-35 degrees C, respectively. The DNA G+C contents of KMM 225T and KMM 277T were respectively 46-8 and 50.7 mol %. Comparison of almost complete 16S rDNA sequences of strains KMM 225T and KMM 277T revealed that both strains were phylogenetically most closely related to each other (99.9% sequence similarity) and slightly less related to Psychrobacter glacincola, with 97.2 and 97.8% similarity, respectively. DNA-DNA reassociation between KMM 225T and KMM 277T revealed 15% similarity, whereas similarity to other Psychrobacter species was 14-25%. Strains KMM 225T and KMM 277T differed from one another in their growth temperature, organic substrate utilization, antibiotic sensitivity and DNA G+C content. Both strains examined could be distinguished from all previously described Psychrobacter species by their physiological, genotypic and phylogenetic characteristics. On the basis of the physiological and molecular properties of the novel isolates, the names Psychrobacter submarinus sp. nov. (type strain KMM 225T = DSM 14161T) and Psychrobacter marincola sp. nov. (type strain KMM 277T = DSM 14160T) are proposed.

Arenibacter gen. nov., new genus of the family Flavobacteriaceae and description of a new species, Arenibacter latericius sp. nov

Five dark-orange-pigmented, Gram-negative, rod-shaped, non-motile, aerobic bacterial strains were isolated from sandy sediment samples collected in the South China Sea in the Indian Ocean, from a holothurian, Apostichopus japonicus, in the Sea of Japan and from a brown alga, Chorda filum, from the Sea of Okhotsk in the Pacific Ocean. Phenotypic data were collected, demonstrating that the bacteria are chemo-organotrophic and require seawater-based media for growth. Polar lipids were analysed and 27% of the total extract comprised phosphatidylethanolamine as the major component. The predominant cellular fatty acids were branched-chain saturated and unsaturated [i-C15:0, i-C15:1, a-C15:0, C15:0, C16:1(n-7)]. The DNA base composition was 37.5-38.2 mol % G+C. The level of DNA homology of the five isolates was 83-94%, indicating that these isolates belong to the same species. A 16S rDNA sequence of the type strain KMM 426T was determined and phylogenetic analysis, based on neighbour-joining and Fitch-Margoliash methods, revealed that the type strain formed a distinct phyletic line in a clade corresponding to the family Flavobacteriaceae and represented a new genus. From the results of this polyphasic taxonomic analysis, it is proposed that the bacterial strains be classified in a new genus, Arenibacter gen. nov., and species, Arenibacter latericius sp. nov. The type strain is KMM 426T (VKM B 2137DT = LMG 19694T = CIP 106861T).

Degradation of fucoidan by the marine proteobacterium Pseudoalteromonas citrea

It was found that Pseudoalteromonas citrea strains KMM 3296 and KMM 3298 isolated from the brown algae Fucus evanescens and Chorda filum, respectively, and strain 3297 isolated from the sea cucumber Apostichopus japonicus are able to degrade fucoidans. The fucoidanases of these strains efficiently degraded the fucoidan of brown algae at pH 6.5–7.0 and remained active at 40–50°C. The endo-type hydrolysis of fucoidan resulted in the formation of sulfated α-L-fucooligosaccharides. The other nine strains of P. citrea studied (including the type strain of this species), which were isolated from other habitats, were not able to degrade fucoidan.

Lysobacter spongiicola sp. nov., isolated from a deep-sea sponge

An aerobic, Gram-negative bacterium, strain KMM 329(T), was isolated from a deep-sea sponge specimen from the Philippine Sea and subjected to a polyphasic taxonomic investigation. Comparative 16S rRNA gene sequence analysis showed that strain KMM 329(T) clustered with the species of the genus Lysobacter. The highest level of 16S rRNA gene sequence similarity (97.0 %) was found with respect to Lysobacter concretionis KCTC 12205(T); lower values (96.4-95.2 %) were obtained with respect to the other recognized Lysobacter species. The value for DNA-DNA relatedness between strain KMM 329(T) and L. concretionis KCTC 12205(T) was 47 %. Branched fatty acids 16 : 0 iso, 15 : 0 iso, 11 : 0 iso 3-OH and 17 : 1 iso were found to be predominant. Strain KMM 329(T) had a DNA G+C content of 69.0 mol%. On the basis of the phenotypic, chemotaxonomic, DNA-DNA hybridization and phylogenetic data, strain KMM 329(T) represents a novel species of the genus Lysobacter, for which the name Lysobacter spongiicola sp. nov. is proposed. The type strain is KMM 329(T) (=NRIC 0728(T) =JCM 14760(T)).