Cloning and Characterization of a Novel Oligoalginate Lyase from a Newly Isolated Bacterium Sphingomonas sp. MJ-3

A bacterium possessing alginate-degrading activity was isolated from marine brown seaweed soup liquefied by salted and fermented anchovy. The isolated strain was designated as Sphingomonas sp. MJ-3 based on the analyses of 16S ribosomal DNA sequences, 16S-23S internal transcribed spacer region sequences, biochemical characteristics, and cellular fatty acid composition. A novel alginate lyase gene was cloned from genomic DNA library and then expressed in Escherichia coli. When the deduced amino acid sequence was compared with the sequences on the databases, interestingly, the cloned gene product was predicted to consist of AlgL (alginate lyase L)-like and heparinase-like protein domain. The MJ-3 alginate lyase gene shared below 27.0% sequence identity with exolytic alginate lyase of Sphingomonas sp. A1. The optimal pH and temperature for the recombinant MJ-3 alginate lyase were 6.5 and 50°C, respectively. The final degradation products of alginate oligosaccharides were analyzed by electrospray ionization mass spectrometry and proved to be alginate monosaccharides. Based on the results, the recombinant alginate lyase from Sphingomonas sp. MJ-3 is regarded as an oligoalginate lyase that can degrade oligoalginate and alginate into alginate monosaccharides.     

Polycyclic Aromatic Hydrocarbon Degradation of Phytoplankton-Associated Arenibacter spp. and Description of Arenibacter algicola sp. nov., an Aromatic Hydrocarbon-Degrading Bacterium

Pyrosequencing of the bacterial community associated with a cosmopolitan marine diatom during enrichment with crude oil revealed several Arenibacter phylotypes, of which one (OTU-202) had become significantly enriched by the oil. Since members of the genus Arenibacter have not been previously shown to degrade hydrocarbons, we attempted to isolate a representative strain of this genus in order to directly investigate its hydrocarbon-degrading potential. Based on 16S rRNA sequencing, one isolate (designated strain TG409^T) exhibited >99% sequence identity to three type strains of this genus. On the basis of phenotypic and genotypic characteristics, strain TG409^T represents a novel species in the genus Arenibacter, for which the name Arenibacter algicola sp. nov. is proposed. We reveal for the first time that polycyclic aromatic hydrocarbon (PAH) degradation is a shared phenotype among members of this genus, indicating that it could be used as a taxonomic marker for this genus. Kinetic data for PAH mineralization rates showed that naphthalene was preferred to phenanthrene, and its mineralization was significantly enhanced in the presence of glass wool (a surrogate for diatom cell surfaces). During enrichment on hydrocarbons, strain TG409^T emulsified n-tetradecane and crude oil, and cells were found to be preferentially attached to oil droplets, indicating an ability by the strain to express cell surface amphiphilic substances (biosurfactants or bioemulsifiers) as a possible strategy to increase the bioavailability of hydrocarbons. This work adds to our growing knowledge on the diversity of bacterial genera in the ocean contributing to the degradation of oil contaminants and of hydrocarbon-degrading bacteria found living in association with marine eukaryotic phytoplankton.     

Cloning, Expression, and Characterization of the katG Gene, Encoding Catalase-Peroxidase, from the Polycyclic Aromatic Hydrocarbon-Degrading Bacterium Mycobacterium sp. Strain PYR-1

A 81-kDa protein from Mycobacterium sp. strain PYR-1 was expressed in response to exposure of the strain to the polycyclic aromatic hydrocarbon pyrene and recovered by two-dimensional gel electrophoresis. The N-terminal sequence of the protein indicated that it was similar to catalase-peroxidase. An oligonucleotide probe designed from this sequence was used to screen a genomic library of Mycobacterium sp. strain PYR-1, and a positive clone, containing a part of the gene encoding the 81-kDa protein, was isolated. A gene-walking technique was used to sequence the entire gene, which was identified as katG for catalase-peroxidase. The deduced KatG protein sequence showed significant homology to KatGII of Mycobacterium fortuitum and clustered with catalase-peroxidase proteins from other Mycobacterium species in a phylogenetic tree. The katG gene was expressed in Escherichia coli to produce a protein with catalase-peroxidase activity. Since the induction of this catalase-peroxidase occurred in pyrene-induced cultures and the exposure of these cultures to hydrogen peroxide reduced pyrene metabolism, our data suggest that this enzyme plays a role in polycyclic aromatic hydrocarbon metabolism by strain PYR-1.     

Massilia sp. BS-1, a Novel Violacein-Producing Bacterium Isolated from Soil

A novel bacterium, Massilia sp. BS-1, producing violacein and deoxyviolacein was isolated from a soil sample collected from Akita Prefecture, Japan. The 16S ribosomal DNA of strain BS-1 displayed 93% homology with its nearest violacein-producing neighbor, Janthinobacterium lividum. Strain BS-1 grew well in a synthetic medium, but required both L-tryptophan and a small amount of L-histidine to produce violacein.     

Identification of opsA,a Gene Involved in Solute Stress Mitigation and Survival in Soil,in the Polycyclic Aromatic Hydrocarbon-Degrading Bacterium Novosphingobium sp. Strain LH128

The aim of this study was to identify genes involved in solute and matric stress mitigation in the polycyclic aromatic hydrocarbon (PAH)-degrading Novosphingobium sp. strain LH128. The genes were identified using plasposon mutagenesis and by selection of mutants that showed impaired growth in a medium containing 450 mM NaCl as a solute stress or 10% (wt/vol) polyethylene glycol (PEG) 6000 as a matric stress. Eleven and 14 mutants showed growth impairment when exposed to solute and matric stresses, respectively. The disrupted sequences were mapped on a draft genome sequence of strain LH128, and the corresponding gene functions were predicted. None of them were shared between solute and matric stress-impacted mutants. One NaCl-affected mutant (i.e., NA7E1) with a disruption in a gene encoding a putative outer membrane protein (OpsA) was susceptible to lower NaCl concentrations than the other mutants. The growth of NA7E1 was impacted by other ions and nonionic solutes and by sodium dodecyl sulfate (SDS), suggesting that opsA is involved in osmotic stress mitigation and/or outer membrane stability in strain LH128. NA7E1 was also the only mutant that showed reduced growth and less-efficient phenanthrene degradation in soil compared to the wild type. Moreover, the survival of NA7E1 in soil decreased significantly when the moisture content was decreased but was unaffected when soluble solutes from sandy soil were removed by washing. opsA appears to be important for the survival of strain LH128 in soil, especially in the case of reduced moisture content, probably by mitigating the effects of solute stress and retaining membrane stability.     

Spirosoma radiotolerans sp. nov., a Gamma-Radiation-Resistant Bacterium Isolated from Gamma Ray-Irradiated Soil

A Gram-negative, short-rod-shaped bacterial strain with gliding motility, designated as DG5A^T, was isolated from a rice field soil in South Korea. Phylogenic analysis using 16S rRNA gene sequence of the new isolate showed that strain DG5A^T belong to the genus Spirosoma in the family Spirosomaceae, and the highest sequence similarities were 95.5 % with Spirosoma linguale DSM 74^T, 93.4 % with Spirosoma rigui WPCB118^T, 92.8 % with Spirosoma luteum SPM-10^T, 92.7 % with Spirosoma spitsbergense SPM-9^T, and 91.9 % with Spirosoma panaciterrae Gsoil 1519^T. Strain DG5A^T revealed resistance to gamma and UV radiation. Chemotaxonomic data showed that the most abundant fatty acids were summed feature C_16:1 ω7c/C_16:1 ω6c (36.90 %), C_16:1 ω5c (29.55 %), and iso-C_15:0 (14.78 %), and the major polar lipid was phosphatidylethanolamine (PE). The DNA G+C content of strain DG5A^T was 49.1 mol%. Together, the phenotypic, phylogenetic, and chemotaxonomic data supported that strain DG5A^T presents a novel species of the genus Spirosoma, for which the name Spirosoma radiotolerans sp. nov., is proposed. The type strain is DG5A^T (=KCTC 32455^T = JCM19447^T).     

Deinococcus soli sp. nov., a Gamma-Radiation-Resistant Bacterium Isolated from Rice Field Soil

A Gram-negative, non-motile, short rod-shaped bacterial strain, designated N5^T, was isolated from a rice field soil in South Korea. Phylogenetic analysis based on the 16S rRNA gene sequence of the new isolate showed that strain N5^T belongs to the genus Deinococcus, family Deinococcaceae, showing the highest sequence similarity to Deinococcus grandis KACC 11979^T (98.4 %) and Deinococcus daejeonensis KCTC 13751^T (97.5 %). Strain N5^T exhibits resistance to gamma-radiation similar to that of other members of the genus Deinococcus, with a D₁₀ value in excess of 4 kGy. Chemotaxonomic data showed that the most abundant fatty acids are C_16:1 ω7c (25.25 %), C_15:1 ω6c (19.77 %), C_17:1 ω6c (11.87 %), and C_17:0 (9.41 %), and the major polar lipid is an unknown phosphoglycolipid. The predominant respiratory quinone is menaquinone MK-8. The DNA G+C content is 71.4 mol%. Phenotypic, phylogenetic, and chemotaxonomic data support designation of strain N5^T as a novel species of the genus Deinococcus, for which the name Deinococcus soli sp. nov. is proposed. The type strain is N5^T (=KCTC 33153^T = JCM 19176^T).     

Isolation of an Exopolysaccharide-producing Bacterium,Sphingomonas sp. CS101, Which Forms an Unusual Type of Sphingan

An exopolysaccharide-producing Gram negative bacterium was isolated and determined to be a Sphingomonas sp. (CS101). A sugar composition analysis of an exopolysaccharide indicated that the Sphingomonas sp. CS101 secreted an exopolysaccharide composed of glucose, mannose, fucose, and rhamnose in the ratio of 2.1:1.1:1.0:0.1, suggesting that this exoplysaccharide is an unusual type of sphingan family. The mean molecular weight of the exopolysaccharide was determined to be 4.2×10⁵ Da by size exclusion chromatography coupled with multi-angle laser-light scattering (SEC/MALLS) analysis. An exopolysaccharide was produced up to 17 g/l (pH 7; 30 °C) with the optimal medium condition over 4 days of cultivation.     

A Novel Protein-Deamidating Enzyme from Chryseobacterium proteolyticum sp. nov., a Newly Isolated Bacterium from Soil

A novel protein-deamidating enzyme, which has potential for industrial applications, was purified from the culture supernatant of Chryseobacterium proteolyticum strain 9670^T isolated from rice field soil in Tsukuba, Japan. The deamidating activities on carboxybenzoxy (Cbz)-Gln-Gly and caseins and protease activity were produced synchronously by the isolate. Both deamidating activities were eluted as identical peaks separated from several proteases by phenyl-Sepharose chromatography of the culture supernatant. The enzyme catalyzed the deamidation of native caseins with no protease and transglutaminase activities. Phenotypic characterization and DNA analyses of the isolate were performed to determine its taxonomy. Physiological and biochemical characteristics, 16S rRNA gene sequence analysis, and DNA-DNA relatedness data indicated that the isolate should be placed as a new species belonging to the genus Chryseobacterium. The isolate showed no growth on MacConkey agar and produced acid from sucrose. The levels of DNA-DNA relatedness between the isolate and other related strains were less than 17%. The name Chryseobacterium proteolyticum is proposed for the new species; strain 9670 is the type strain (=FERM P-17664).     

Genome Sequence of Enterobacter sp. Strain SP1, an Endophytic Nitrogen-Fixing Bacterium Isolated from Sugarcane

Enterobacter sp. strain SP1 is an endophytic nitrogen-fixing bacterium isolated from a sugarcane stem and can promote plant growth. The draft genome sequence of strain SP1 presented here will promote comparative genomic studies to determine the genetic background of interactions between endophytic enterobacteria and plants.     

Methanosarcina acetivorans sp. nov., an Acetotrophic Methane-Producing Bacterium Isolated from Marine Sediments

A new acetotrophic marine methane-producing bacterium that was isolated from the methane-evolving sediments of a marine canyon is described. Exponential phase cultures grown with sodium acetate contained irregularly shaped cocci that aggregated in the early stationary phase and finally differentiated into communal cysts that released individual cocci when ruptured or transferred to fresh medium. The irregularly shaped cocci (1.9 ± 0.2 mm in diameter) were gram negative and occurred singly or in pairs. Cells were nonmotile, but possessed a single fimbria-like structure. Micrographs of thin sections showed a monolayered cell wall approximately 10 nm thick that consisted of protein subunits. The cells in aggregates were separated by visible septation. The communal cysts contained several single cocci encased in a common envelope. An amorphous form of the communal cyst that had incomplete septation and internal membrane-like vesicles was also present in late exponential phase cultures. Sodium acetate, methanol, methylamine, dimethylamine, and trimethylamine were substrates for growth and methanogenesis; H₂-CO₂ (80:20) and sodium formate were not. The optimal growth temperature was 35 to 40°C. The optimal pH range was 6.5 to 7.0. Both NaCl and Mg²⁺ were required for growth, with maximum growth rates at 0.2 M NaCl and 0.05 M MgSO₄. The DNA base composition was 41 ± 1% guanine plus cytosine. Methanosarcina acetivorans is the proposed species. C2A is the type strain (DSM 2834, ATCC 35395).     

Characterization of tetrachlorohydroquinone reductive dehalogenase from Sphingomonas sp. UG30

Tetrachlorohydroquinone reductive dehalogenase (PcpC) is the second of three enzymes that catalyze the initial degradation of pentachlorophenol in Sphingomonas sp. UG30 and several other bacterial strains. The UG30 PcpC shares a high degree (94%) of primary sequence identity with the well-studied PcpC from Sphingobium chlorophenolicum ATCC 39723. Significant differences, however, were observed between the two PcpC enzymes in some of their functional and kinetic properties. The temperature optimum of the UG30 PcpC is 10 degrees C higher and the pH optimum is approximately 2 units higher than the S. chlorophenolicum PcpC. In addition, the S. chlorophenolicum PcpC is subject to inhibition by the substrate tetrachlorohydroquinone (TCHQ), and this has necessitated the use of a mutant enzyme, which was not inhibited by TCHQ, for kinetic studies. In contrast, the UG30 PcpC was not inhibited by TCHQ and this may allow detailed kinetic and mechanistic studies using the wild-type enzyme.     

Salinicola zeshunii sp. nov., a Moderately Halophilic Bacterium Isolated from Soil of a Chicken Farm

The taxonomic status of a moderately halophilic bacterium, strain N4^T, isolated from soil of a chicken farm in China was determined. It was Gram-negative, non-spore-forming, motile, and rod-shaped. Phylogenetic analysis based on 16S rRNA gene sequence indicated that this strain belonged to the genus Salinicola, as it showed the highest sequence similarities to Salinicola salaries M27^T (98.3 %), Salinicola socius SMB35^T (98.1 %), and Salinicola halophilus CG4.1^T (98.1 %). The major cellular fatty acids were C_16:0 (25.6 %), C_18:1ω7c (35.0 %), and C_19:0 cyclo ω8c (11.9 %), which are properties shared by members of the genus Salinicola. The DNA G+C content of strain N4^T was 69.1 mol %. The level of DNA–DNA relatedness between strain N4^T and the other three type strains of the genus of Salinicola salaries M27^T, Salinicola socius SMB35^T, and Salinicola halophilus CG4.1^T were 34.3, 28.7, and 26.9 %, respectively. Based on the results of phenotypic, chemotaxonomic, DNA–DNA relatedness, and phylogenetic analysis, strain N4^T should be classified as a novel species of the genus Salinicola, for which the name Salinicola zeshunii sp. nov. is proposed, with strain N4^T (=KACC 16602^T = CCTCC AB 2012912^T) as the type strain.     

Genome Sequence of Pedobacter arcticus sp. nov., a Sea Ice Bacterium Isolated from Tundra Soil

Pedobacter arcticus sp. nov. was originally isolated from tundra soil collected from Ny-Ålesund, in the Arctic region of Norway. It is a Gram-negative bacterium which shows bleb-shaped appendages on the cell surface. Here, we report the draft annotated genome sequence of Pedobacter arcticus sp. nov., which belongs to the genus Pedobacter.