Hoeflea prorocentri sp. nov., isolated from a culture of the marine dinoflagellate Prorocentrum mexicanum PM01.

A Gram-stain negative, aerobic, rod-shaped, non-motile, yellow-pigmented and non-spore-forming bacterial strain, designated PM5-8T, was isolated from a culture of a marine toxigenic dinoflagellate Prorocentrum mexicanum PM01. Strain PM5-8T grew at 15-35 °C (optimum, 25-30 °C) and pH 6-11 (optimum, 7.5-8). Cells required at least 1.5% (w/v) NaCl for growth, and can tolerate up to 7.0% with the optimum of 4%. Phylogenetic analysis based on 16S rRNA gene sequence revealed that the strain PM5-8T is closely related to members of the genus Hoeflea, with high sequence similarities with Hoeflea halophila JG120-1T (97.06%) and Hoeflea alexandrii AM1V30T (97.01%). DNA-DNA hybridization values between the isolate and other type strains of recognized species of the genus Hoeflea were between 11.8 and 25.2%, which is far below the value of 70% threshold for species delineation. The DNA G + C content was 50.3 mol%. The predominant cellular fatty acids of the strain were identified as summed feature 8 (C16:1 ω7c and/or C16:1 ω6c; 51.5%), C18:1 ω7c 11-methyl (20.7%), C16:0 (17.2%) and C18:0 (5.7%). The major respiratory quinone was Q-10. Polar lipids profiles contained phosphatidylcholine, phosphatidylglycerol, sulfoquinovosyl diacylglycerol, phosphatidylmono- methylethanolamine, phosphatidylethanolamine and four unidentified lipids. On the basis of the polyphasic taxonomic data presented, strain PM5-8T (= CCTCC AB 2016294T = KCTC 62490T) represents a novel species of the genus Hoeflea, for which the name Hoeflea prorocentri sp. nov. is proposed.

Fatty Acids of Chthonomonas calidirosea, of a novel class Chthonomonadetes from a recently described phylum Armatimonadetes.

A Gram-negative, aerobic, pink-pigmented, rod-shaped bacterium Chthonomonas calidirosea (strain T49(T)) with an optimal temperature for growth of 68 °C, isolated from soil samples from Hell's Gate in the Tikitere geothermal system (New Zealand), was the first cultivated bacterium of the novel phylum Armatimonadetes (formerly candidate division OP10). The lipid composition of C. calidirosea presents a number of unusual features both in the fatty acids and polar lipids. This contribution reports on the fatty acid profile of C. calidirosea. Transmethylation of bacterial biomass yielded fatty acid methyl esters and hydrocarbons, including squalene, partially hydrogenated squalenes, and diploptene. The only type of unsaturation found in C. calidirosea fatty acids was cis-Δ5, as revealed by GCMS of dimethyl disulfide (DMDS) adducts, and the lack of trans-unsaturation absorbance at 960-980 cm(-1) in the IR spectrum of fatty acids methyl esters. An unidentified component X with ECL 16.86 (BP1) and ECL 17.27 (BP20) was also observed, with molecular ion at m/z 282 ("17:1"). X did not form DMDS adducts, nor was affected by mild hydrogenation conditions, indicating the likely presence of a ring rather than unsaturation. The presence of a cyclopropane ring with cis-stereochemistry was confirmed by the (1)H-NMR spectrum. Hydrogenation of X in acetic acid resulted in formation of straight chain 17:0, 5-methyl- and 6-methyl-16:0 fatty acid methyl esters, thus confirming the structure of a novel 5,6-methylene hexadecanoic acid. The major fatty acids of a solid media-grown C. calidirosea were as follows (in weight % of total fatty acids): 16:0 (25.8), i17:0 (19.3), ai17:0 (13.5), 16:1∆5 (8.8), i17:1∆5 (6.8), 5,6-methylene 16:0 (5.2), i16:0 (4.4), 18:0 (3.6), 18:1∆5 (3.2).

Petrobactin sulfonate, a new siderophore produced by the marine bacterium Marinobacter hydrocarbonoclasticus.

Culture of the oil-degrading marine bacterium Marinobacter hydrocarbonoclasticus gave the known siderophore petrobactin (1) and the new metabolite petrobactin sulfonate (2), the first marine siderophore containing a sulfonated 3,4-dihydroxy aromatic ring. The structure of petrobactin sulfonate was elucidated from spectral data, resulting in a revision of the NMR assignments of petrobactin.

New neoverrucosane diterpenoids produced by the marine gliding bacterium Saprospira grandis.

Chemical examination of the culture broth extracts of the marine gliding bacterium Saprospira grandis (ATCC 23116) has resulted in the isolation of four new diterpenoids of the neoverrucosane class. The structures of the new diterpenoids, compounds 1-4, were assigned by combined spectroscopic methods emphasizing 2D NMR experiments. The relative stereochemistry of 1 was determined by 2D ROESY NMR methods, while the absolute stereochemistry was assigned by application of the modified Mosher method. This study adds to the rare observation of terpene production by prokaryotic microorganisms and suggests that marine gliding bacteria may be a significant source for new terpenoid secondary metabolites.

Structural analysis of the extracellular polysaccharide produced by Sphaerotilus natans.

An extracellular polysaccharide (EPS) was recovered and purified from the culture fluid of a sheathed bacterium, Sphaerotilus natans. Glucose, rhamnose, and aldobiouronic acid were detected in the acid hydrolysate of EPS by thin-layer chromatography (TLC). The aldobiouronic acid was found to be composed of glucuronic acid and rhamnose by TLC and gas-liquid chromatography analyses of the corresponding neutral disaccharide. The structure of EPS was identified by methylation linkage analysis and nuclear magnetic resonance. Additionally, partial acid hydrolysates of EPS were prepared and put through fast atom bombardment-mass spectrometry to determine the sugar sequence of EPS. The resulting data showed that EPS produced by S. natans is a new gellan-like polysaccharide constructed from a tetrasaccharide repeating unit, as shown below. -->4)-alpha-D-Glcp-(1-->2)-beta-D-GlcA p-(1-->2)-alpha-L-Rha p-(1-->3)-beta-L-Rha p-(1-->.

Isolation and phylogenetic characterization of acidophilic microorganisms indigenous to acidic drainage waters at an abandoned Norwegian copper mine.

The biodiversity of culturable acidophilic microbes in three acidic (pH 2.7-3.7), metal-rich waters at an abandoned subarctic copper mine in central Norway was assessed. Acidophilic bacteria were isolated by plating on selective solid media, and dominant isolates were identified from their physiological characteristics and 16S rRNA gene sequences. The dominant iron-oxidizing acidophile in all three waters was an Acidithiobacillus ferrooxidans-like eubacterium, which shared 98% 16S rDNA identity with the type strain. A strain of Leptospirillum ferrooxidans was obtained from one of the waters after enrichment in pyrite medium, but this iron oxidizer was below detectable levels in the acidic waters themselves. In two sites, there were up to six distinct heterotrophic acidophiles, present at 10(3) ml(-1). These included Acidiphilium-like isolates (one closely related to Acidiphilium rubrum, a second to Acidiphilium cryptum and a third apparently novel isolate), an Acidocella-like isolate (96% 16S rDNA identity to Acidocella facilis) and a bacterium that shared 94.5% 16S rDNA identity to Acidisphaera rubrifaciens. The other numerically significant heterotrophic isolate was not apparently related to any known acidophile, with the closest match (96% 16S rDNA sequence identity) to an acetogen, Frateuria aurantia. The results indicated that the biodiversity of acidophilic bacteria, especially heterotrophs, in acidic mine waters may be much greater than previously recognized.

Polyphasic evidence for the reclassification of Rhodothermus obamensis Sako et al. 1996 as a member of the species Rhodothermus marinus Alfredsson et al. 1988.

DNA-DNA reassociation studies, 16S rRNA gene sequence comparisons and fatty acid analysis were used to reassess the taxonomic status of the type strain of Rhodothermus obamensis and several strains of the genus Rhodothermus isolated from widely distributed shallow marine hot springs. The results show that the type strain of R. obamensis, JCM 9785T, has a DNA-DNA reassociation value of 78% with the type strain of R. marinus, DSM 4252T. The other strains examined had DNA-DNA reassociation values that varied between about 68 and 94% with R. marinus. The 165 rRNA gene sequence was determined for the type strain of R. obamensis and found to share 99.5% similarity with the type strain of R. marinus. The fatty acid composition of R. obamensis was slightly different from that of the other strains examined, but indicated that this strain is very closely related to the other strains examined in this study. On the basis of DNA-DNA reassociation values, 16S rRNA gene sequence comparison and fatty acid profiles, it was concluded that R. obamensis and R. marinus represent the same species and that the name Rhodothermus obamensis should be regarded as a junior synonym of Rhodothermus marinus.

Emendation of the description of Blastomonas natatoria (Sly 1985) Sly and Cahill 1997 as an aerobic photosynthetic bacterium and reclassification of Erythromonas ursincola Yurkov et al. 1997 as Blastomonas ursincola comb. nov.

Photosynthetic properties of Blastomonas natatoria (Sly 1985) Sly and Cahill 1997, which had been recognized as being non-photosynthetic, were examined and compared with those of its close relative, the aerobic photosynthetic bacterium, Erythromonas ursincola Yurkov et al. 1997. HPLC experiments demonstrated that bacteriochlorophyll a was present in a detectable amount in the lipid extract from B. natatoria DSM 3183T as well as that from E. ursincola DSM 9006T. The puf genes, encoding the proteins of the photosynthetic reaction centre and core light-harvesting complexes, were detected by PCR from both the organisms. 16S rDNA sequence comparisons and DNA-DNA hybridization studies confirmed that B. natatoria and E. ursincola were closely related genetically in a single genus. On the basis of phenotypic, chemotaxonomic and phylogenetic data, it is proposed that the description of B. natatoria is emended as a species of aerobic photosynthetic bacteria and that E. ursincola is reclassified as Blastomonas ursincola comb. nov.

Thermobacillus xylanilyticus gen. nov., sp. nov., a new aerobic thermophilic xylan-degrading bacterium isolated from farm soil.

An aerobic, thermophilic, xylanolytic, spore-forming bacterium, XETP (T = type strain; P = patent strain), has been isolated from farm soil situated underneath a manure heap in northern France. Strain XETP, which stained negative in the Gram test, occurs as short rods which sometimes form chains. Its spores are ellipsoidal, central to subterminal and occur in swollen sporangia. It grows at temperatures up to 63 degrees C and in the pH range 6.5-8.5. When grown on glucose in optimal conditions, its doubling time was found to be 33 min. CO2 was observed to have a growth-stimulating effect at the start of the culture. In addition to glucose, the isolate utilizes xylose, arabinose, mannose, cellobiose, galactose, maltose, sucrose, xylan and starch. Growth is inhibited by 5% NaCl. The G+C content of strain XETP is 57.5 mol%. The 16S rDNA sequence analysis indicated that strain XETP falls into the radiation of the Bacillus-Lactobacillus-Streptococcus subdivision of the Gram-positive phylum. Its three closest phylogenetic relatives are 'Bacillus viscosus', Paenibacillus curdlanolyticus and Bacillus popilliae with identity values of 91.15, 90.94 and 90.92%, respectively. The major cellular fatty acids are 14-methyl pentadecanoic acid (16:0 iso), hexadecanoic acid (16:0) and 14-methyl hexadecanoic acid (17:0 anteiso). On the basis of 16S rRNA sequence and chemotaxonomic characteristics, the isolate is different enough for it to be considered as a member of a new genus. It is therefore proposed that this isolate represents a new genus and species: Thermobacillus xylanilyticus. Strain XETP, the type strain of Thermobacillus xylanilyticus, has been deposited in the Collection Nationale de Cultures Microbiennes (CNCM I-1017) as a patent strain.

Proposal of Sphingomonas suberifaciens (van Bruggen, Jochimsen and Brown 1990) comb. nov., sphingomonas natatoria (Sly 1985) comb. nov., Sphingomonas ursincola (Yurkov et al. 1997) comb. nov., and emendation of the genus Sphingomonas.

Based on the results of a phylogenetic analysis of 16S rRNA and the presence of sphingoglycolipid in cellular lipids of the type strains, transfer of "Rhizomonas" suberifaciens, Blastomonas natatoria and Erythromonas ursincola to the genus Sphingomonas as Sphingomonas suberifaciens (van Bruggen et al 1990) comb. nov., Sphingomonas natatoria (Sly 1985) comb. nov., and Sphingomonas ursincola (Yurkov et al 1997) comb. nov. are herein proposed together with the emendation of genus Sphingomonas. The type strain of S. suberifaciens is van Bruggen Cal=ATCC 49382=NCPPB 3629=IFO 15211=JCM 8521, that of S. natatoria is ATCC 35951 =DSM 3183=NCIMB 12085=JCM10396, and that of S. ursincola is DSM 9006= KR-99.

Cycloprodigiosin hydrochloride obtained from Pseudoalteromonas denitrificans is a potent antimalarial agent.

Cycloprodigiosin hydrochloride (cPrG*HCl) is a stable fluorescent red pigment obtained from the marine bacterium Pseudoalteromonas denitrificans. It was found that the compound was incorporated into Plasmodium falciparum cells upon incubation and exhibited a potent antimalarial activity with the concentration required for 50% of the activity being 11 nM, which is stronger than that of chloroquine, a well-known antimalarial agent. The compound did not affect growth rate of mammalian cells. Antimalarial activity of cPrG*HCl was also observed in vivo. These results indicate that cPrG*HCl is a potent antimalarial drug.

Fundibacter jadensis gen. nov., sp. nov., a new slightly halophilic bacterium, isolated from intertidal sediment.

A moderately halophilic hydrocarbon-degrading bacterium was isolated from continuous cultures containing a suspension of intertidal sediment from the German North Sea coast with hexadecane as the sole carbon source. On the basis of phenotypic characteristics, fatty acid analysis and 16S rDNA sequence analysis, it was considered to be a new species belonging to a new genus. It is a Gram-negative, aerobic, rod-shaped bacterium, whose cell size varies. It grows at concentrations of 0.5-15% (w/v) NaCl and utilizes a restricted spectrum of carbon sources. The G + C content of the DNA is 63.6 mol%. Comparative 16S rDNA studies show a clear affiliation of this bacterium to the gamma subclass of the class Proteobacteria. Comparison of phylogenetic data indicate that it is most closely related to Marinobacter hydrocarbonoclasticus (88.9% similarity in 16S rRNA gene sequence). Since it is impossible to find a sufficiently closely related species, we propose the name Fundibacter jadensis gen. nov., sp. nov. for the bacteria. The type strain is T9T (= DSM 12178T).

Structural studies of an exopolysaccharide produced by Alteromonas macleodii subsp. fijiensis originating from a deep-sea hydrothermal vent.

The structure of the exopolysaccharide produced by Alteromonas macleodii subsp. fijiensis recovered from a deep-sea hydrothermal vent has been investigated. By means of chemical analysis and NMR studies, the repeating unit of the polymer was deduced to be a branched hexasaccharide with the structure shown. [formula: see text]

Detection and partial characterization of bacteriocin in the methanotrophic bacterium Methylobacter bovis.

The strain Methylobacter bovis 98 was selected among methanotrophic bacteria as one of the most active producers of secretory bacteriocin-like compounds. In the above strain this compound was shown to be a protein with a molecular weight of about 70 kD, relatively thermostable, having a bactericidal effect on closely related organisms. Its properties as a whole are consistent with the accepted definition of bacteriocins, which so far have not been found in this group of microorganisms. A methodical approach that combines electrophoretic separation of secretory proteins and testing their antibacterial activity directly in polyacrylamide gel allowed us for the first time to identify bacteriocin in methanotrophic bacterial culture.

Structure of sulfated O-specific polysaccharide of the marine bacterium Pseudoalteromonas marinoglutinosa KMM 232.

A sulfated O-specific polysaccharide containing D-mannose, L-rhamnose, and the sulfate group was obtained by mild acid hydrolysis of lipopolysaccharide (S-form) of the marine bacterium Pseudoalteromonas marinoglutinosa KMM 232. Based on analysis of methylation and 13C-NMR spectroscopy of native and desulfated polysaccharides, the following structure of disaccharide repeat unit in the O-specific polysaccharide has been established: [scheme]. This is the first report of a sulfated O-specific polysaccharide isolated from gram-negative bacteria.

Structure of the O-specific polysaccharide from Pseudoalteromonas elyakovii sp. nov. CMM 162.

The structure of the O-specific polysaccharide from lipopolysaccharide of Pseudoalteromonas elyakovii sp. nov. CMM 162 on the basis of NMR data, Smith degradation and methylation study was elucidated as follows:-->2)-alpha-D-Glcp-(1-->4)-beta-D-GalpNAc-(1--> 3)-alpha-D-Galp-(1-->3)-beta-D-Galp-NAc-(1-->6)-alpha-D-Glcp-(1-->.

Purification and characterization of chaperonins 60 and 10 from Methylobacillus glycogenes.

Two proteins belonging to the group I chaperonin family were isolated from an obligate methanotroph, Methylobacillus glycogenes. The two proteins, one a GroEL homologue (cpn60: M. glycogenes 60 kDa chaperonin) and the other a GroES homologue (cpn10: M. glycogenes 10 kDa chaperonin), composed a heteropolymeric complex in the presence of ATP. Both proteins were purified from crude extracts of M. glycogenes by anion-exchange (DEAE-Toyopearl) and gel-filtration (Sephacryl S-400) chromatography. The native molecular weights of each chaperonin protein as determined by high-performance liquid chromatography (HPLC) gel-filtration were 820 000 for cpn60 and 65 000 for cpn10. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the subunit molecular weights of cpn60 and cpn10 were 58 000 and 10 000, respectively. Both cpn60 and cpn10 possessed amino acid sequences which were highly homologous to other group I chaperonins. M. glycogenes cpn60 displayed an ATPase activity which was inhibited in the presence of cpn10. The chaperonins also displayed an ability to interact with and facilitate the refolding of Thermus malate dehydrogenase and yeast enolase in a manner similar to that of GroEL/ES. The similarities between the Escherichia coli GroE proteins are discussed.

Isolation and chemical composition of the sheath of Sphaerotilus natans.

A sheathed bacterium, Sphaerotilus natans, was cultured with vigorous shaking in a medium containing peptone. Then the biomass was harvested and treated with lysozyme, sodium dodecyl sulfate, and protease. With treatment, 1.6 mg of sheaths was obtained from 15 mg of biomass. For the preparation of sheaths of high purity, cultivation must be in the absence of glucose with sufficient aeration to prevent poly(3-hydroxybutyrate) accumulation. Carbohydrate (54.1%), protein (12.2%), and lipid (1-3%) were detected in the sheaths by colorimetric reactions and solvent extraction. Gas-liquid chromatography showed glucose and galactosamine to be present in the molar ratio of 1:4. The most abundant amino acids in the sheath protein were glycine (49.2 mol%) and cysteine (24.6 mol%). The sheaths were resistant to agents that reduce disulfide bonds (dithiothreitol and 2-mercaptoethanol) and to protease. However, sheathes were degraded completely by hydrazine, and a heteropolysaccharide composed of glucose and galactosamine (1:4) was released. The weight-average molecular weight of the polysaccharide was estimated to be 1.2 x 10(5) by gel filtration chromatography with a low-angle laser-light scattering photometer and a rotation index detector. A ladder of 1.5-kDa peptides separable by sodium dodecyl sulfate gel electrophoresis was obtained by partial hydrolysis of sheaths, suggesting the sheath protein has repeating units of 1.5 kDa.

Structure of a highly acidic O-specific polysaccharide of lipopolysaccharide of Pseudoalteromonas haloplanktis KMM 223 (44-1) containing L-iduronic acid and D-QuiNHb4NHb.

An acidic O-specific polysaccharide was obtained by mild acid degradation of the lipopolysaccharide isolated by phenol-water extraction of Pseudoalteromonas haloplanktis strain KMM 223 (44-1). L-Iduronic acid (IdoA) was found to be a component of the polysaccharide and identified by NMR spectroscopy and after carboxyl-reduction followed by acid hydrolysis and acetylation, by GLC-MS as 2,3,4-tri-O-acetyl-1,6-anhydroidose. On the basis of 1H and 13C NMR spectroscopic studies, including 1D NOE, 2D NOESY, HSQC and HMBC experiments, the following structure of the branched pentasaccharide repeating unit of the polysaccharide was established: -->4)-beta-D-GlcpAI-(1-->4)-beta-D-GlcpAII-(1-->3)-beta-D-++ +QuipNHb4NHbII- (1-->2)-alpha-L-IdopA-(-->4 increases 1 alpha-D-QuipNAc4NAcI where QuiNAc4NAc and QuiNHb4NHb are 2,4-diacetamido-2,4,6-trideoxyglucose and 2,4,6-tri-deoxy-2,4- di[(S)-3-hydroxybutyramido]glucose, respectively. This is the first report of L-iduronic acid in a lipopolysaccharide and of D-QuiNHb4NHb in nature.