Galina M. Frolova

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).

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).

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)).

Winogradskyella arenosi sp. nov., a member of the family Flavobacteriaceae isolated from marine sediments from the Sea of Japan.

An aerobic, Gram-negative, yellow-pigmented, gliding bacterial strain, designated R60(T), was isolated from a marine sediment sample obtained from the Sea of Japan and was characterized by using a polyphasic approach. 16S rRNA gene sequence analysis showed that strain R60(T) belonged to the genus Winogradskyella, sharing <97 % sequence similarity with the type strains of recognized Winogradskyella species. The main fatty acids of strain R60(T) were iso-C(15 : 0) 3-OH, iso-C(16 : 0) 3-OH, iso-C(17 : 0) 3-OH, anteiso-C(17 : 0) 3-OH and iso-C(15 : 0), consistent with its assignment to the genus Winogradskyella. On the basis of phenotypic distinctiveness and phylogenetic divergence, strain R60(T) is considered to represent a novel species of the genus Winogradskyella, for which the name Winogradskyella arenosi sp. nov. is proposed. The type strain is R60(T) (=KMM 3968(T) =NRIC 0748(T)= JCM 15527(T)).

Occurrence and antagonistic potential of Stenotrophomonas strains isolated from deep-sea invertebrates

Stenotrophomonas maltophilia is known to be of significance as opportunistic pathogen as well as a source of biocontrol and bioremediation activities. S. maltophilia strains have been isolated from rhizospheres, soil, clinical material, aquatic habitats, but little is known about Stenotrophomonas strains recovered from marine environments. During a survey of the biodiversity of Pseudomonas-like bacteria associated with deep-sea invertebrates six Stenotrophomonas strains were isolated from sponge, sea urchin, and ophiura specimens collected from differing Pacific areas, including the Philippine Sea, the Fiji Sea and the Bering Sea. 16S rRNA gene sequence analysis confirmed an assignment of marine isolates to the genus Stenotrophomonas as it placed four strains into the S. maltophilia CIP 60.77T cluster and two related to the S. rhizophila DSM 14405T. Together with a number of common characteristics typical of S. maltophilia and S. rhizophila marine isolates exhibited differences in pigmentation, a NaCl tolerance, a range of temperatures, which supported their growth, substrate utilization pattern, and antibiotics resistance. Strains displayed hemolytic and remarkable inhibitory activity against a number of fungal cultures and Gram-positive microorganisms, but very weak or none against Candida albicans. This is the first report on isolation, taxonomic characterization and antimicrobial activity of Stenotrophomonas strains isolated from deep-sea invertebrates.

Retrieval of the species Alteromonas tetraodonis Simidu et al. 1990 as Pseudoalteromonas tetraodonis comb. nov. and emendation of description.

A polyphasic taxonomy study was undertaken of three strains of Pseudoalteromonas haloplanktis subsp. tetraodonis (Simidu et al. 1990) Gauthier et al. 1995. DNA was prepared from each of the strains and genomic relatedness was measured by DNA-DNA hybridization. Strains KMM 458T and IAM 14160T shared 99% genetic relatedness, but were only 48-49% related to the type strain of Pseudoalteromonas haloplanktis subsp. haloplanktis, IAM 12915T. The third strain, P. haloplanktis subsp. tetraodonis A-M, showed 83% genetic similarity with P. haloplanktis subsp. haloplanktis IAM 12915T and 32% with KMM 458T. From these results, it is concluded that strains KMM 458T and IAM 14160T comprise a separate species, originally described as Alteromonas tetraodonis, whereas strain A-M belongs to the species Pseudoalteromonas haloplanktis. Based on phenotypic and chemotaxonomic data, genomic fingerprint patterns, DNA-DNA hybridization data and phylogenetic analysis of 16S rRNA, it is proposed that the species Alteromonas tetraodonis be retrieved and recognized as Pseudoalteromonas tetraodonis comb. nov. (type strain IAM 14160T = KMM 458T).

Reclassification of Alteromonas distincta Romanenko et al. 1995 as Pseudoalteromonas distincta comb. nov.

The 16S rRNA gene of Alteromonas distincta KMM 638T was amplified, cloned and sequenced. The nucleotide sequence was aligned with sequences of representative strains of Alteromonas, Moritella, Pseudoalteromonas and Shewanella. Results of phylogenetic analysis, using neighbour-joining and Fitch-Margoliash methods, clearly indicated that this species should be assigned to the genus Pseudoalteromonas. On the basis of polyphasic data obtained from previous work and this study, it is proposed that the species Alteromonas distincta be reclassified as Pseudoalteromonas distincta comb. nov. with type strain KMM 638T (= ATCC 700518T).

An Enzyme Linked Immunosorbent Assay for Detecting Palytoxin-producing Bacteria

A competitive ELISA using the intact toxin as a coating antigen for detecting palytoxin was developed. This immunoassay allows palytoxin (PTX) to be determined in the range of 6–250 ng/ml. In sensitivity, this determination is comparable with RIA but is three times inferior to ELISA using monoclonal antibodies. Inhibition experiments using some toxins of marine invertebrates proved the serological specificity of the palytoxin binding to antibodies. Both the indirect and competitive ELISA were used to find PTX-producing bacteria among 420 isolates of sea bacteria. It was found that gram-negative bacteriaAeromonas sp. andVibrio sp. associated with toxic samples of the soft coralPalythoa sp. produced compounds antigenically related to PTX.

Litoreibacter albidus gen. nov., sp. nov. and Litoreibacter janthinus sp. nov., members of the class Alphaproteobacteria isolated from the seashore

Two Gram-negative, strictly aerobic, non-motile bacteria designated strains KMM 3851(T) and KMM 3842(T) were respectively isolated from a marine snail specimen (Umbonium costatum) and from surrounding sediments collected simultaneously from the shore of the Sea of Japan. Phylogenetic analysis based on 16S rRNA gene sequences showed that strains KMM 3851(T) and KMM 3842(T) were affiliated with the Roseobacter lineage of the class Alphaproteobacteria as a separate phylogenetic line adjacent to the members of the genus Thalassobacter. These novel isolates shared 98.5 % 16S rRNA gene sequence similarity and 15 % DNA-DNA relatedness. The major isoprenoid quinone of both strains was Q-10 and polar lipids consisted of phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol and aminophospholipids. In addition, strain KMM 3851(T) contained two unknown lipids, whereas strain KMM 3842(T) contained diphosphatidylglycerol. Fatty acid analysis revealed C(18 : 1)ω7c and C(16 : 0) as major components and small amounts of C(18 : 2). The DNA G+C contents were 60.4 mol% (KMM 3851(T)) and 58.5 mol% (KMM 3842(T)). Based on distinctive phenotypic characteristics, DNA-DNA hybridization data and phylogenetic distance, strains KMM 3851(T) and KMM 3842(T) should be classified as representatives of two novel species in a new genus, Litoreibacter gen. nov., with the type species Litoreibacter albidus sp. nov. (type strain KMM 3851(T) =NRIC 0773(T) =JCM 16493(T)) and a second species Litoreibacter janthinus sp. nov. (type strain KMM 3842(T) =NRIC 0772(T) =JCM 16492(T)).

Pseudomonas marincola sp. nov., isolated from marine environments

An aerobic, Gram-negative, motile, non-pigmented bacterium, strain KMM 3042(T), isolated from a deep-sea brittle star in the Fiji Sea, was subjected to a polyphasic taxonomic study. Phylogenetic analysis based on 16S rRNA gene sequences revealed marine Mn(II)-oxidizing isolate S185-2B as the closest neighbour of strain KMM 3042(T) (99.9 % sequence similarity). The two strains formed a distinct lineage within the genus Pseudomonas adjacent to the members of the Pseudomonas borbori cluster, sharing highest sequence similarity of 97.4 and 97.0 %, respectively, with P. borbori DSM 17834(T) and Pseudomonas flavescens DSM 12071(T). The DNA-DNA hybridization value (71 %) between strains KMM 3042(T) and S185-2B confirmed their assignment to the same species. On the basis of phylogenetic analysis, DNA-DNA hybridization and physiological and biochemical characterization, strains KMM 3042(T) and S185-2B should be assigned to a novel species of the genus Pseudomonas, for which the name Pseudomonas marincola sp. nov. is proposed. The type strain is KMM 3042(T) (=NRIC 0729(T) =JCM 14761(T)).