Uncovering potential interspecies signaling factors in plant-derived mixed microbial culture.

Microbes use signaling factors for intraspecies and interspecies communications. While many intraspecies signaling factors have been found and characterized, discovery of factors for interspecies communication is lagging behind. To facilitate the discovery of such factors, we explored the potential of a mixed microbial culture (MMC) derived from wheatgrass, in which heterogeneity of this microbial community might elicit signaling factors for interspecies communication. The stability of Wheatgrass MMC in terms of community structure and metabolic output was first characterized by 16S ribosomal RNA amplicon sequencing and liquid chromatography/mass spectrometry (LC/MS), respectively. In addition, detailed MS analyses led to the identification of 12-hydroxystearic acid (12-HSA) as one of the major metabolites produced by Wheatgrass MMC. Stereochemical analysis revealed that Wheatgrass MMC produces mostly the (R)-isomer, although a small amount of the (S)-isomer was also observed. Furthermore, 12-HSA was found to modulate planktonic growth and biofilm formation of various marine bacterial strains. The current study suggests that naturally derived MMCs could serve as a simple and reproducible platform to discover potential signaling factors for interspecies communication. In addition, the study indicates that hydroxylated long-chain fatty acids, such as 12-HSA, may constitute a new class of interspecies signaling factors.

Characterization of membrane vesicles secreted by seaweed associated bacterium Alteromonas macleodii KS62.

Recent studies have reported abundant presence of bacterial extracellular membrane vesicles in the marine environment. However, the ecological significance of these bacterial vesicles in the marine environment is only beginning to be explored. In present study, for the first time we report and characterize membrane vesicles secreted by a seaweed associated bacterium, Alteromonas macleodii KS62. Proteomics studies revealed that the vesicle proteome was rich in hydrolytic enzymes (30%) like glycoside hydrolases, proteases, sulphatases, lipases, nucleases and phosphatases. Zymography experiments and enzyme assays established that the vesicles carry active κ-carrageenan degrading enzymes. κ-carrageenan is a major polysaccharide of cell walls of certain red seaweeds like Kappaphycus. Purified membrane vesicles were successfully able to degrade Kappaphycus biomass. Based on these results, we discuss how the hydrolase-rich vesicles may play a role in red seaweed cell wall degradation so that the bacteria can invade and colonise the seaweed biomass establishing a saprophytic lifestyle. We also discuss the role of these vesicles in nutrient acquisition and their ecological significance in the marine environment.

Alteromonas addita sp. nov.

On the basis of phenotypic, genotypic characteristics and analysis of 16S rRNA gene sequences, a novel species belonging to the genus Alteromonas is described. A non-pigmented, motile, Gram-negative bacterium designated R10SW13(T) was isolated from sea water samples collected in Chazhma Bay (Sea of Japan, Pacific Ocean). The novel organism mainly grew between 4 degrees C and 37 degrees C, was neutrophilic and slightly halophilic, tolerating up to 10 % NaCl. Strain R10SW13(T) was haemolytic and was able to degrade starch and Tween 80 and to degrade gelatin and agar weakly, but did not degrade casein. Phosphatidylethanolamine (44.3 +/- 0.9 %) and phosphatidylglycerol (55.7 +/ -0.9 %) were the predominant phospholipids. The major fatty acids formed were typical for the genus Alteromonas, including 16 : 0, 16 : 1omega-7 and 18 : 1omega-7. The G + C content of the DNA was 43.4 mol%. DNA-DNA hybridization experiments showed 38-53 % binding with the DNAs of type strains of phylogenetically related species of the genus Alteromonas, namely: Alteromonas macleodii, Alteromonas marina, Alteromonas stellipolaris, Alteromonas litorea, 'Alteromonas macleodii subsp. fijiensis' and 'Alteromonas infernus'. Based on these results, a novel species, Alteromonas addita sp. nov., is proposed, with strain R10SW13(T) (=KMM 3600(T) = KCTC 12195(T) = LMG 22532(T)) as the type strain.

Alteromonas stellipolaris sp. nov., a novel, budding, prosthecate bacterium from Antarctic seas, and emended description of the genus Alteromonas.

Seven novel, cold-adapted, strictly aerobic, facultatively oligotrophic strains, isolated from Antarctic sea water, were investigated by using a polyphasic taxonomic approach. The isolates were Gram-negative, chemoheterotrophic, motile, rod-shaped cells that were psychrotolerant and moderately halophilic. Buds were produced on mother and daughter cells and on prosthecae. Prostheca formation was peritrichous and prosthecae could be branched. Phylogenetic analysis based on 16S rRNA gene sequences indicated that these strains belong to the gamma-Proteobacteria and are related to the genus Alteromonas, with 98.3% sequence similarity to Alteromonas macleodii and 98.0% to Alteromonas marina, their nearest phylogenetic neighbours. Whole-cell fatty acid profiles of the isolates were very similar and included C(16 : 0), C(16 : 1)omega7c, C(17 : 1)omega8c and C(18 : 1)omega8c as the major fatty acid components. These results support the affiliation of these isolates to the genus Alteromonas. DNA-DNA hybridization results and differences in phenotypic characteristics show that the strains represent a novel species with a DNA G+C content of 43-45 mol%. The name Alteromonas stellipolaris sp. nov. is proposed for this novel species; the type strain is ANT 69aT (=LMG 21861T=DSM 15691T). An emended description of the genus Alteromonas is given.

Alteromonas litorea sp. nov., a slightly halophilic bacterium isolated from an intertidal sediment of the Yellow Sea in Korea.

A Gram-negative, motile, non-spore-forming, rod-shaped bacterium, designated strain TF-22T, was isolated from an intertidal sediment in Korea. This organism grew optimally at 30-37 degrees C and in the presence of 2-5% (w/v) NaCl. It did not grow without NaCl or in the presence of more than 14% (w/v) NaCl. Strain TF-22T was characterized chemotaxonomically as having ubiquinone-8 as the predominant respiratory lipoquinone and C(16 : 0), C(16 : 1) omega7c and/or iso-C(15 : 0) 2-OH and C(18 : 1) omega7c as the major fatty acids. The DNA G+C content of strain TF-22T was 46.0 mol%. Phylogenetic analyses based on 16S rDNA sequences showed that strain TF-22T falls within the gamma-subclass of the Proteobacteria and forms a coherent cluster with Alteromonas macleodii and Alteromonas marina. Levels of 16S rDNA similarity between strain TF-22T and the type strains of two Alteromonas species were in the range 98.1-98.6%. The level of DNA-DNA relatedness between strain TF-22T and the type strains of two Alteromonas species was 15.7-18.5%. Therefore, on the basis of phenotypic properties, phylogeny and genomic distinctiveness, strain TF-22T should be placed in the genus Alteromonas as a novel species, for which the name Alteromonas litorea sp. nov. is proposed. The type strain is TF-22TT (=KCCM 41775T=JCM 12188T).

Differentiation of chitinase-active and non-chitinase-active subpopulations of a marine bacterium during chitin degradation.

The ability of marine bacteria to adhere to detrital particulate organic matter and rapidly switch on metabolic genes in an effort to reproduce is an important response for bacterial survival in the pelagic marine environment. The goal of this investigation was to evaluate the relationship between chitinolytic gene expression and extracellular chitinase activity in individual cells of the marine bacterium Pseudoalteromonas sp. strain S91 attached to solid chitin. A green fluorescent protein reporter gene under the control of the chiA promoter was used to evaluate chiA gene expression, and a precipitating enzyme-linked fluorescent probe, ELF-97-N-acetyl-beta-D-glucosaminide, was used to evaluate extracellular chitinase activity among cells in the bacterial population. Evaluation of chiA expression and ELF-97 crystal location at the single-cell level revealed two physiologically distinct subpopulations of S91 on the chitin surface: one that was chitinase active and remained associated with the surface and another that was non-chitinase active and released daughter cells into the bulk aqueous phase. It is hypothesized that the surface-associated, non-chitinase-active population is utilizing chitin degradation products that were released by the adjacent chitinase-active population for cell replication and dissemination into the bulk aqueous phase.

Assignment of Alteromonas elyakovii KMM 162T and five strains isolated from spot-wounded fronds of Laminaria japonica to Pseudoalteromonas elyakovii comb. nov. and the extended description of the species.

A marine bacterium, Alteromonas elyakovii KMM 162T, which was described recently, and five strains isolated from spot-wounded fronds of Laminaria japonica have been subjected to phylogenetic analysis, and geno- and phenotypic characterization. The phenotypic features of Pseudoalteromonas elyakovii strains were closely related to that of Pseudoalteromonas espejiana IAM 12640T, but utilization of three carbon compounds (D-mannose, L-tyrosine and trehalose) distinguished both species. The G+C content of Pseudoalteromonas elyakovii was between 38.5 and 38.9 mol%. Pseudoalteromonas elyakovii KMM 162T and the five Laminaria isolates constitute a single species different from any other Alteromonas and Pseudoalteromonas species as revealed by DNA-DNA hybridization data, especially Pseudoalteromonas distincta KMM 638T (52.4%), Pseudoalteromonas citrea KMM 216 (49.5%), Pseudoalteromonas carrageenovora NCIMB 302T (46.9%) and Pseudoalteromonas espejiana IAM 12640T (29.9%). All the data indicated that Alteromonas elyakovii KMM 162T should be reclassified as Pseudoalteromonas elyakovii and five strains isolated from Laminaria japonica have to be included in the species. Pseudoalteromonas elyakovii comb. nov. (type strain, KMM 162T = ATCC 700519T) is proposed and a set of phenotypic features which differentiate the Pseudoalteromonas species is described.