Structure of the O-antigen of a halophilic bacterium Salinicola salarius HO-14.

The structure of the O-polysaccharide of an aerobic halophilic bacterium Salinicola salarius HO-14 isolated from a heavy oil reservoir with highly mineralized water was determined. The neutral O-polysaccharide of strain HO-14 was isolated from the lipopolysaccharide and studied by sugar analysis and NMR spectroscopy. The linear tetrasaccharide repeating unit was found to have the following structure: →2)-α-l-Rhap-(1 â†’ 3)-ß-l-Rhap-(1 â†’ 2)-α-l-Rhap-(1 â†’ 2)-α-d-Manp-(1→.

Phytohalomonas tamaricis gen. nov., sp. nov., an endophytic bacterium isolated from Tamarix ramosissima roots growing in Kumtag desert.

A gram-stain-negative, aerobic, non-spore-forming, rod-shaped, non-motile bacterium strain R4HLG17T was isolated from Tamarix ramosissima roots growing in Kumtag desert. The strain grew at salinities of 0-16% (w/v) NaCl (optimum 5-6%), pH 5-9 (optimum 7) and at 16-45 °C. Based on 16S rRNA gene sequence similarity, strain R4HLG17T belonged to the family Halomonadaceae and was most closely related to Halomonas lutea DSM 23508T(95.1%), followed by Halotalea alkalilenta AW-7T(94.8%), Salinicola acroporae S4-41T(94.8%), Salinicola halophilus CG4.1T(94.6%), and Larsenimonas salina M1-18T(94.4%). Multilocus sequence analysis (MLSA) based on the partial sequences of 16S rRNA, atpA, gyrB, rpoD, and secA genes indicated that the strain R4HLG17T formed an independent and monophyletic branch related to other genera of Halomonadaceae, supporting its placement as a new genus in this family. The draft genome of strain R4HLG17T was 3.6 Mb with a total G + C content of 55.1%. The average nucleotide identity to Halomonas lutea DSM 23508T was 83.5%. Q-9 was detected as the major respiratory quinone and summed feature 8 (C18:1ω7c/C18:1ω6c), summed feature 3 (C16:1ω7c/C16:1ω6c), and C16:0 as predominant cellular fatty acids. On the basis of chemotaxonomic, phylogenetic, and phenotypic evidence, strain R4HLG17T is concluded to represent a novel species of a new genus within Halomonadaceae, for which the name Phytohalomonas tamaricis gen. nov., sp. nov., is proposed. The type strain is R4HLG17T (=ACCC 19929T=KCTC 52415T).

Crystal structure of pyruvate decarboxylase from Zymobacter palmae.

Pyruvate decarboxylase (PDC; EC 4.1.1.1) is a thiamine pyrophosphate- and Mg(2+) ion-dependent enzyme that catalyses the non-oxidative decarboxylation of pyruvate to acetaldehyde and carbon dioxide. It is rare in bacteria, but is a key enzyme in homofermentative metabolism, where ethanol is the major product. Here, the previously unreported crystal structure of the bacterial pyruvate decarboxylase from Zymobacter palmae is presented. The crystals were shown to diffract to 2.15 Šresolution. They belonged to space group P21, with unit-cell parameters a = 204.56, b = 177.39, c = 244.55 Šand Rr.i.m. = 0.175 (0.714 in the highest resolution bin). The structure was solved by molecular replacement using PDB entry 2vbi as a model and the final R values were Rwork = 0.186 (0.271 in the highest resolution bin) and Rfree = 0.220 (0.300 in the highest resolution bin). Each of the six tetramers is a dimer of dimers, with each monomer sharing its thiamine pyrophosphate across the dimer interface, and some contain ethylene glycol mimicking the substrate pyruvate in the active site. Comparison with other bacterial PDCs shows a correlation of higher thermostability with greater tetramer interface area and number of interactions.

The new sulfated O-specific polysaccharide from marine bacterium Cobetia pacifica KMM 3878, containing 3,4-O-[(S)-1-carboxyethylidene]-D-galactose and 2,3-O-disulfate-D-galactose.

The O-specific polysaccharide was isolated from the lipopolysaccharide of Cobetia pacifica KMM 3878 and studied by chemical methods along with (1)H and (13)C NMR spectroscopy, including, 1D TOCSY and 2D (1)H, (1)H COSY, (1)H, (13)C HSQC, (1)H, (1)H ROESY, (1)H, (13)C HMBC and (1)H, (13)C H2BC experiments. The following new structure of the sulfated O-polysaccharide from C. pacifica KMM 3878 containing 3,4-O-[(S)-1-carboxyethylidene]-D-galactose and 2,3-O-disulfate-D-galactose was established: →4)-ß-D-Gal2,3R-(1→6)-ß-D-Gal3,4(S-Pyr)-(1→6)-ß-D-Gal-(1→ Where R is -SO3H.

The sulfated O-specific polysaccharide from the marine bacterium Cobetia pacifica KMM 3879(T.).

The O-specific polysaccharide was isolated from the lipopolysaccharide of Cobetia pacifica KMM 3879(T) and studied by chemical methods along with (1)H and (13)C NMR spectroscopy, including 1D TOCSY and 2D (1)H, (1)H-COSY, ROESY, (1)H, (13)C-HSQC, HMBC, H2BC and HMQC-TOCSY experiments. The following new structure of the sulfated O-polysaccharide from the C. pacifica KMM 3879(T) containing rhamnose (Rha), glucose (Glc), and galactose (Gal) was established: where R is -SO3H.

Induction of apoptosis in cancer cell lines by the Red Sea brine pool bacterial extracts.

BACKGROUND: Marine microorganisms are considered to be an important source of bioactive molecules against various diseases and have great potential to increase the number of lead molecules in clinical trials. Progress in novel microbial culturing techniques as well as greater accessibility to unique oceanic habitats has placed the marine environment as a new frontier in the field of natural product drug discovery. METHODS: A total of 24 microbial extracts from deep-sea brine pools in the Red Sea have been evaluated for their anticancer potential against three human cancer cell lines. Downstream analysis of these six most potent extracts was done using various biological assays, such as Caspase-3/7 activity, mitochondrial membrane potential (MMP), PARP-1 cleavage and expression of γH2Ax, Caspase-8 and -9 using western blotting. RESULTS: In general, most of the microbial extracts were found to be cytotoxic against one or more cancer cell lines with cell line specific activities. Out of the 13 most active microbial extracts, six extracts were able to induce significantly higher apoptosis (>70%) in cancer cells. Mechanism level studies revealed that extracts from Chromohalobacter salexigens (P3-86A and P3-86B(2)) followed the sequence of events of apoptotic pathway involving MMP disruption, caspase-3/7 activity, caspase-8 cleavage, PARP-1 cleavage and Phosphatidylserine (PS) exposure, whereas another Chromohalobacter salexigens extract (K30) induced caspase-9 mediated apoptosis. The extracts from Halomonas meridiana (P3-37B), Chromohalobacter israelensis (K18) and Idiomarina loihiensis (P3-37C) were unable to induce any change in MMP in HeLa cancer cells, and thus suggested mitochondria-independent apoptosis induction. However, further detection of a PARP-1 cleavage product, and the observed changes in caspase-8 and -9 suggested the involvement of caspase-mediated apoptotic pathways. CONCLUSION: Altogether, the study offers novel findings regarding the anticancer potential of several halophilic bacterial species inhabiting the Red Sea (at the depth of 1500-2500 m), which constitute valuable candidates for further isolation and characterization of bioactive molecules.

Adhesion of marine fouling organisms on hydrophilic and amphiphilic polysaccharides.

Polysaccharides are a promising material for nonfouling surfaces because their chemical composition makes them highly hydrophilic and able to form water-storing hydrogels. Here we investigated the nonfouling properties of hyaluronic acid (HA) and chondroitin sulfate (CS) against marine fouling organisms. Additionally, the free carboxyl groups of HA and CS were postmodified with the hydrophobic trifluoroethylamine (TFEA) to block free carboxyl groups and render the surfaces amphiphilic. All coatings were tested with respect to their protein resistance and against settlement and adhesion of different marine fouling species. Both the settlement and adhesion strength of a marine bacterium (Cobetia marina), zoospores of the seaweed Ulva linza, and cells of a diatom (Navicula incerta) were reduced compared to glass control surfaces. In most cases, TFEA capping increased or maintained the performance of the HA coatings, whereas for the very well performing CS coatings the antifouling performance was reduced after capping.

Absence of detectable arsenate in DNA from arsenate-grown GFAJ-1 cells.

A strain of Halomonas bacteria, GFAJ-1, has been claimed to be able to use arsenate as a nutrient when phosphate is limiting and to specifically incorporate arsenic into its DNA in place of phosphorus. However, we have found that arsenate does not contribute to growth of GFAJ-1 when phosphate is limiting and that DNA purified from cells grown with limiting phosphate and abundant arsenate does not exhibit the spontaneous hydrolysis expected of arsenate ester bonds. Furthermore, mass spectrometry showed that this DNA contains only trace amounts of free arsenate and no detectable covalently bound arsenate.

Influence of surface-energy components of Ni-P-TiO2-PTFE nanocomposite coatings on bacterial adhesion.

The influence of total surface energy on bacterial adhesion has been investigated intensively with the frequent conclusion that bacterial adhesion is less on low-energy surfaces. However, there are also a number of contrary findings that high-energy surfaces have a smaller biofouling tendency. Recently, it was found that the CQ ratio, which is defined as the ratio of Lifshitz-van der Waals (LW) apolar to electron donor surface-energy components of substrates, has a strong correlation to bacterial adhesion. However, the electron donor surface-energy components of substrates varied over only a very limited range. In this article, a series of Ni-P-TiO(2)-PTFE nanocomposite coatings with wide range of surface-energy components were prepared using an electroless plating technique. The bacterial adhesion and removal on the coatings were evaluated with different bacteria under both static and flow conditions. The experimental results demonstrated that there was a strong correlation between bacterial attachment (or removal) and the CQ ratio. The coatings with the lowest CQ ratio had the lowest bacterial adhesion or the highest bacterial removal, which was explained using the extented DLVO theory.

Comment on "A bacterium that can grow by using arsenic instead of phosphorus".

Wolfe-Simon et al. (Research Articles, 3 June 2011, p. 1163; published online 2 December 2010) reported that the bacterial strain GFAJ-1 can grow by using arsenic (As) instead of phosphorus (P), noting that the P content in bacteria grown in +As/-P culture medium was far below the quantity needed to support growth. However, low P content is a common phenotype across a broad range of environmental bacteria that experience P limitation.

The extremely halophilic bacterium Salicola marasensis IC10 accumulates the compatible solute betaine.

The extremely halophilic bacterium strain IC10 was isolated from a solar saltern on Isla Cristina (southern Spain). Phylogenetic, genotypic and phenotypic data supported the inclusion of this strain in the species Salicola marasensis. An analysis of intracellular organic osmotic solutes showed glycine betaine to be present, contributing to the overall osmotic balance, and this was the only compatible solute accumulated when S. marasensis IC10 was grown over a wide range of external NaCl concentrations (10-25%, w/v).

Description of Kushneria aurantia gen. nov., sp. nov., a novel member of the family Halomonadaceae, and a proposal for reclassification of Halomonas marisflavi as Kushneria marisflavi comb. nov., of Halomonas indalinina as Kushneria indalinina comb. nov. and of Halomonas avicenniae as Kushneria avicenniae comb. nov.

An aerobic, moderately halophilic, Gram-negative, motile, non-sporulating rod-shaped bacterium, designated strain A10(T), was isolated from the surface of leaves of the black mangrove Avicennia germinans and was subjected to a polyphasic taxonomic study. Strain A10(T) was able to grow at NaCl concentrations in the range 5-17.5 % (w/v) with optimum growth at 10 % (w/v) NaCl. Growth occurred at temperatures of 20-40 degrees C (optimal growth at 37 degrees C) and pH 5.5-8.5 (optimal growth at pH 7.0-8.0). The major respiratory quinone was ubiquinone 9. The major fatty acids were C(16 : 0), C(18 : 1)omega7c, C(19 : 0) cyclo omega8c and C(12 : 0) 3-OH. The polar lipids were phosphatidylglycerol, diphosphatidylglycerol, phosphatidylethanolamine, and unidentified phospholipids, glycolipids and an aminoglycolipid. Phylogenetic analysis based on 16S rRNA gene sequence comparisons revealed that strain A10(T) is closely related to Halomonas avicenniae MW2a(T) (95.7 % sequence similarity), Halomonas marisflavi SW32(T) (95.2 %) and Halomonas indalinina GC2.1(T) (95.0 %). Strain A10(T) formed a coherent phylogenetic branch with these three species, separated from other species of Halomonas and closely related genera (with 16S rRNA gene sequence similarities below 94.0 %). A complete 23S rRNA gene sequence comparison of strain A10(T) with closely related species confirmed the phylogenetic position of the novel isolate, forming a branch with the species Halomonas avicenniae, Halomonas indalinina and Halomonas marisflavi, separated from other species of the genera belonging to the family Halomonadaceae (showing sequence similarities below 91.7 %). DNA-DNA hybridization studies between strain A10(T) and Halomonas avicenniae MW2a(T), Halomonas marisflavi DSM 15357(T) and Halomonas indalinina CG2.1(T) were 21, 17 and 10 %, respectively. These levels of DNA-DNA relatedness were low enough to classify strain A10(T) as representing a genotypically distinct species. Overall, the phenotypic, genotypic, chemotaxonomic and phylogenetic results demonstrated that strain A10(T) represents a new genus and species. The name Kushneria aurantia gen. nov., sp. nov. is proposed, with strain A10(T) (=CCM 7415(T)=CECT 7220(T)) as the type strain. This is the type species of the new proposed genus, which belongs to the family Halomonadaceae. In addition, our data support the placement of the species Halomonas marisflavi, Halomonas indalinina and Halomonas avicenniae within this new genus, as Kushneria marisflavi comb. nov., Kushneria indalinina comb. nov. and Kushneria avicenniae comb. nov., respectively.

[Hydroxyectoine synthesis and release under osmotic shock in Cobetia marina CICC10367].

OBJECTIVE: To obtain hydroxyectoine-producing strain with tolerance to osmotic shock and improve hydroxyectoine productivity by adopting "bacteria milking" process. METHODS: We isolated a strain from salt lake, and then carried out the identifications of morphology, physiological and biochemical characteristics of the strain. We also investigated the effects of the medium and its NaCl concentration on the hydroxyectoine synthesis of this strain. Under optimal condition, hydroxyectoine was produced by adopting "bacteria milking" process. RESULTS: A hydroxyectoine-producing strain was obtained and identified as Cobetia marina CICC10367 (C. marina CICC10367). It could enhance hydroxyeceoine synthesis when the medium adopting monosodium glutamate as the sole source of carbon and nitrogen at 90 g/L NaCl. The highest synthesized hydroxyectoine concentration was 694.5 mg/L in the above described medium. After the "bacteria milking" process of six osmotic shock, the total concentration of hydroxyectoine was 4179.0 mg/L, the productivity was 597.0 mg/L/d, and the conversion rate of hydroxyectoine on substrate was 80.2 mg/g. CONCLUSION: C. marina CICC10367 was able to synthesize hydroxyectoine under NaCl induction and tolerant to osmotic stress. The hydroxyectoine productivity and conversion rate of hydroxyectoine on substrate were both significantly improved by adopting "bacteria milking" process.

Chromohalobacter japonicus sp. nov., a moderately halophilic bacterium isolated from a Japanese salty food.

A Gram-negative, non-spore-forming, rod-shaped, motile bacterium, designated strain 43(T), was isolated from a Japanese salty food and then subjected to a polyphasic taxonomic study. Strain 43(T) is moderately halophilic, growing at NaCl concentrations in the range 5-25 % (w/v), with optimum growth between 7.5 and 12.5 % (w/v) NaCl. Growth occurs at temperatures from 15 to 42 degrees C (optimally at 28-37 degrees C) and at pH 5.5-9.0 (optimally at pH 7.0-8.0). A phylogenetic analysis based on 16S rRNA gene sequence comparisons revealed that strain 43(T) belongs to the genus Chromohalobacter. The closest relatives were Chromohalobacter canadensis ATCC 43984(T) (99.3 % 16S rRNA gene sequence similarity), Chromohalobacter beijerinckii ATCC 19372(T) (99.1 %), Chromohalobacter sarecensis LV4(T) (98.3 %), Chromohalobacter nigrandesensis LTS-4N(T) (97.9 %) and Chromohalobacter marismortui ATCC 17056(T) (97.9 %). The DNA G+C content was 62.9 mol%, which is within the range described for the genus Chromohalobacter. DNA-DNA hybridization studies between strain 43(T) and C. canadensis CECT 5385(T) and C. beijerinckii DSM 7218(T) showed 38 and 49 % relatedness, respectively; lower DNA-DNA hybridization percentages were obtained with respect to other related Chromohalobacter species. The major fatty acids of strain 43(T) were C(16 : 0), C(19 : 0) cyclo omega8c and C(12 : 0) 3-OH. Overall, the phenotypic, genotypic and phylogenetic results demonstrated that strain 43(T) represents a novel species within the genus Chromohalobacter. The name Chromohalobacter japonicus sp. nov. is proposed, with strain 43(T) (=CECT 7219(T) =CCM 7416(T)) as the type strain.

[Salinicola socius gen. nov., sp. nov., a moderately halophilic bacterium from a naphthalene-utilizing microbial association].

A chemoorganotrophic, moderately halophilic bacterium (strain SMB35) has been isolated from a naphthalene-utilizing microbial community obtained from salt mines (Perm region of Russia). Strain SMB35 grows in a wide salinity range, 0.5 to 30% (wt/vol) NaCl. Cells are gram-negative rods motile by means of a single polar flagellum. The predominant fatty acids are 16:1omega7, 16:0, 18:1omega7, and 19 cy. The major lipoquinone is an unsaturated ubiquinone with nine isoprene units (Q-9). The DNA G+C content is 63.0 mol%. The 16S rDNA-based phylogenetic analysis has shown that strain SMB35 formed a separate clade in the cluster of the family Halomonadaceae. The 16S rDNA sequence similarity of the isolate to the members of the family is in the range from 90.6% to 95.1%. The phylogenetic and phenotypic differences from Halomonas elongata (the type species of the genus) and from other members of the family suggest that the isolate represents a novel genus and species, for which the name Salinicola socius gen. nov., sp. nov. is proposed. The type strain is SMB35(T) (=VKM B-2397(T)).

Reclassification of Pseudomonas beijerinckii Hof 1935 as Chromohalobacter beijerinckii comb. nov., and emended description of the species.

Pseudomonas beijerinckii (type strain DSM 7218(T)=ATCC 19372(T)=NCIMB 9041(T)) was isolated from salted beans and was first described by Hof in 1935. 16S rRNA gene sequence comparisons demonstrated its close relatedness (>97-99 %) to species of the genus Chromohalobacter. A recent isolate from salted herrings originating from the Baltic Sea, strain 3b, also clustered phylogenetically within this genus. Phenotypic features, substrate utilization, fatty acid profile, quinone and polar lipid composition and whole-cell protein patterns supported the similarity of strain 3b to P. beijerinckii DSM 7218(T) and confirmed its relatedness to members of the genus Chromohalobacter. The G+C content of the DNA from strain 3b and P. beijerinckii DSM 7218(T) was 60.4 and 60.7 mol%, respectively. DNA-DNA hybridization data showed that the two strains represent the same species, but are separated from Chromohalobacter canadensis, the closest species from a phylogenetic point of view. Therefore, the reclassification of Pseudomonas beijerinckii as Chromohalobacter beijerinckii comb. nov. (type strain DSM 7218(T)=ATCC 19372(T)=NCIMB 9041(T)) is proposed. The species description has been emended considering the new data on both the type strain and strain 3b.

How to be moderately halophilic with broad salt tolerance: clues from the genome of Chromohalobacter salexigens.

We analyzed the amino acid composition of different categories of proteins of the moderately halophilic bacterium Chromohalobacter salexigens, as deduced from its genome sequence. Comparison with non-halophilic representatives of the gamma-Proteobacteria (Escherichia coli, Pseudomonas aeruginosa, Vibrio cholerae) shows only a slight excess of acidic residues in the cytoplasmic proteins, and no significant differences were found in the acidity of membrane-bound proteins. In contrast, a very pronounced difference in mean pI value was observed for the periplasmic binding proteins of the ABC transport systems of C. salexigens and the non-halophiles E. coli and P. aeruginosa. V. cholerae, which is adapted to life in brackish water, showed intermediate values. The findings suggest that there is a major difference between the proteins of the moderate halophile C. salexigens and non-halophilic bacteria in their periplasmic proteins, exemplified by the substrate binding proteins of transport systems. The highly acidic nature of these proteins may enable them to function at high salt concentrations. The evolution of highly salt-tolerant prokaryotes may have depended on an increase in acidity of the proteins located external to the cytoplasmic membrane, enabling effective transport of nutrients into the cell.

Major outer membrane proteins in moderately halophilic eubacteria of genera Chromohalobacter and Halomonas.

Outer and inner membrane fractions of Chromohalobacter marismortui and Halomonas elongata were isolated by differential detergent solubilization, and profiles of membrane proteins, especially major outer membrane proteins, were analyzed. These type strains possessed one extremely abundant outer membrane protein which showed similarity in amino-terminal amino acid sequence with the outer membrane porin proteins in other Gram-negative bacteria. Three halophilic eubacterial strains isolated from saline environments were also characterized. Strains 160 and 43 were found to be Chromohalobacter spp. and strain 40 to be a Halomonas sp. by sequence analysis of their 16 S ribosomal RNA genes. Extremely abundant porin proteins with an apparent molecular mass of 49 kDa were found in Chromohalobacter sp.160 and Halomonas sp. 40, but no major outer membrane protein was detected in Chromohalobacter sp. 43, suggesting strain 43 was most likely a naturally defective porin mutant. Porin proteins from Chromohalobacter spp. and Halomonas spp. showed the same migration on SDS-polyacrylamide gel electrophoresis with or without heat-treatment, indicating that these porin proteins did not form a SDS-resistant trimeric structure, which was detected in most of the Gram-negative bacterial porin proteins.