Aureispira anguillae sp. nov., isolated from Japanese eel Anguilla japonica leptocephali.

A novel filamentous eel-leptocephalus pathogenic marine bacterium, designated strain EL160426T, was isolated from Japanese eel, Anguilla japonica, leptocephali reared at a laboratory in Mie, Japan. In experimental infection studies on eel larvae, the strain EL160426T caused massive larval mortality and was reisolated from moribund leptocephali. Characteristically, observations of infected larvae found that EL160426T forms columnar colonies on the cranial surface of larvae. The novel isolate exhibited growth at 15-30 °C, pH 7-9, and seawater concentrations of 60-150% (W/V). Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain EL160426T was most closely related to Aureispira maritima 59SAT with 97.7% sequence similarity. The whole genome sequence analysis of the strain EL160426T showed that the strain maintained a circular chromosome with a size of approximately 7.58 Mbp and the DNA G + C content was 36.2%. The major respiratory quinone was MK-7 and the predominant cellular fatty acids were 16:0, 20:4 w6c (arachidonic acid), 17:0 iso and 16:0 N alcohol. DNA relatedness between the closest phylogenetic neighbor strain EL160426T and A. maritima (JCM23207T) was less than 13%. On the basis of the polyphasic taxonomic data, the strain represents a novel species of the genus Aureispira, for which the name Aureispira anguillae sp. nov. is proposed. The type strain is EL160426T (= JCM 35024 T = TSD-286 T).

Non-Toxin-Producing Bacillus cereus Strains Belonging to the B. anthracis Clade Isolated from the International Space Station.

In an ongoing Microbial Observatory investigation of the International Space Station (ISS), 11 Bacillus strains (2 from the Kibo Japanese experimental module, 4 from the U.S. segment, and 5 from the Russian module) were isolated and their whole genomes were sequenced. A comparative analysis of the 16S rRNA gene sequences of these isolates showed the highest similarity (>99%) to the Bacillus anthracis-B. cereus-B. thuringiensis group. The fatty acid composition, polar lipid profile, peptidoglycan type, and matrix-assisted laser desorption ionization-time of flight profiles were consistent with the B. cereus sensu lato group. The phenotypic traits such as motile rods, enterotoxin production, lack of capsule, and resistance to gamma phage/penicillin observed in ISS isolates were not characteristics of B. anthracis. Whole-genome sequence characterizations showed that ISS strains had the plcR non-B. anthracis ancestral "C" allele and lacked anthrax toxin-encoding plasmids pXO1 and pXO2, excluding their identification as B. anthracis. The genetic identities of all 11 ISS isolates characterized via gyrB analyses arbitrarily identified them as members of the B. cereus group, but traditional DNA-DNA hybridization (DDH) showed that the ISS isolates are similar to B. anthracis (88% to 90%) but distant from the B. cereus (42%) and B. thuringiensis (48%) type strains. The DDH results were supported by average nucleotide identity (>98.5%) and digital DDH (>86%) analyses. However, the collective phenotypic traits and genomic evidence were the reasons to exclude the ISS isolates from B. anthracis. Nevertheless, multilocus sequence typing and whole-genome single nucleotide polymorphism analyses placed these isolates in a clade that is distinct from previously described members of the B. cereus sensu lato group but closely related to B. anthracis. IMPORTANCE The International Space Station Microbial Observatory (Microbial Tracking-1) study is generating a microbial census of the space station's surfaces and atmosphere by using advanced molecular microbial community analysis techniques supported by traditional culture-based methods and modern bioinformatic computational modeling. This approach will lead to long-term, multigenerational studies of microbial population dynamics in a closed environment and address key questions, including whether microgravity influences the evolution and genetic modification of microorganisms. The spore-forming Bacillus cereus sensu lato group consists of pathogenic (B. anthracis), food poisoning (B. cereus), and biotechnologically useful (B. thuringiensis) microorganisms; their presence in a closed system such as the ISS might be a concern for the health of crew members. A detailed characterization of these potential pathogens would lead to the development of suitable countermeasures that are needed for long-term future missions and a better understanding of microorganisms associated with space missions.

Gilvimarinus japonicus sp. nov., a cellulolytic and agarolytic marine bacterium isolated from coastal debris.

A cellulolytic and agarolytic bacterial strain, designated 12-2T, was isolated from a piece of cotton rope fragment washed ashore on a beach and was studied phenotypically, genotypically and phylogenetically. Analyses of 16S rRNA and gyrB gene sequences and DNA base composition suggested that the strain is a member of the genus Gilvimarinus. However, levels of 16S rRNA and gyrB gene sequence similarity between it and the type strains of Gilvimarinus species were no higher than 97.9 and 78.7 %, respectively, suggesting that the strain is distinct. Moreover, the results of DNA-DNA hybridization experiments and physiological characterization clearly differentiated the strain from its closest neighbours. The strain is therefore considered to represent a novel species of the genus Gilvimarinus, for which the name Gilvimarinus japonicus sp. nov. is proposed. The type strain is 12-2T (=NBRC 111987T=KCTC 52141T).

Sulfitobacter pacificus sp. nov., isolated from the red alga Pyropia yezoensis.

Three Gram-negative, aerobic, halophilic bacterial strains, SCM2-10(T), SCM-4, and 14C-6, were isolated from the algal medium of the red alga Pyropia yezoensis (previously classified as Porphyra yezoensis) grown in laboratory experiments. Phylogenetic analysis based on 16S rRNA gene sequences revealed that the organisms with high similarities to the three isolates are Sulfitobacter geojensis MM-124(T) (98.7%), Sulfitobacter noctilucicola NB-77(T) (98.7%), Sulfitobacter noctilucae NB-68(T) (98.6%), Sulfitobacter mediterraneus CH-B427(T) (97.6%), and Sulfitobacter porphyrae SCM-1(T) (97.6%), and that the three isolates belong to the genus Sulfitobacter, within the class Alphaproteobacteria. The DNA G+C contents of the three isolates were found to be in the range of 56.5-57.1 mol%. DNA-DNA hybridization experiments demonstrated that the genomic relatedness between strain SCM2-10(T) and type strains of other Sulfitobacter species was in the range of 6.2-27.1%. The predominant respiratory quinone of the three isolates was identified as ubiquinone-10. The dominant polar lipids in the three isolates were found to be phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, and an unidentified amino lipid. The major fatty acid in the three isolates is C(18:1)ω7c. Strain SCM2-10(T) demonstrated unique phenotypic characteristics, which differed from those of type strains of other Sulfitobacter species. Based on the phylogenetic, genetic, phenotypic, and chemotaxonomic data, we propose a novel species of the genus Sulfitobacter, which we named as Sulfitobacter pacificus sp. nov. The type strain of this species is strain SCM2-10(T) (=LMG 27113(T) = NBRC 109915(T)).

Occurrence, molecular characterization, and antimicrobial susceptibility of Aeromonas spp. in marine species of shrimps cultured at inland low salinity ponds.

We aimed to document the risk of Aeromonas spp. in marine shrimp species cultured in inland low salinity ponds in Thailand. In 14 of 18 shrimp samples retrieved from inland grow-up ponds, Aeromonas spp. were detected at ranges from 4667 to 1,500,000 CFU/g body weight. The phylogenetic tree constructed with the gyrB and cpn60 concatenated sequences indicated that the 87 isolates consisted of Aeromonas veronii (70%), Aeromonas aquariorum (18%), Aeromonas caviae (7%), Aeromonas jandaei (2%), and Aeromonas schubertii (2%). The potential virulence of the isolates was examined by phenotypic and PCR assays. Hemolytic activity and the extracellular activity of lipase, DNase, and gelatinase were observed in most isolates (94-99%). PCR revealed the presence of 9 genes related to virulence in the 87 isolates: act (75%), aer (74%), alt (30%), ast (1%), ascV (34%), aexT (24%), fla (92%), ela (34%), and lip (24%). The susceptibility profiles to 14 antimicrobial agents of isolates were typical for the genus, but resistance to cefotaxime, a third-generation cephalosporin, and imipenem were found in two A. aquariorum and in three A. veronii isolates, respectively. These resistances were confirmed by determining minimum inhibitory concentrations. Our results indicate that the microbiological risk posed by Aeromonas should be considered for marine shrimp species that are cultured in low-salinity ponds. These shrimps may also be a vehicle for the transfer of different genotypes of Aeromonas and antibiotic-resistant determinants to regions worldwide through trade.

Corrigendum: Updating the Vibrio clades defined by multilocus sequence phylogeny: proposal of eight new clades, and the description of Vibrio tritonius sp. nov.

[This corrects the article on p. 414 in vol. 4, PMID: 24409173.].

Isolation of hyphomonas strains that induce normal morphogenesis in protoplasts of the marine red alga Pyropia yezoensis.

Marine macroalgae cannot develop normal morphology under axenic conditions although normal morphogenesis can be sustained when certain bacteria are present. In this study, bacteria that induced normal morphogenesis in the red alga Pyropia yezoensis (Nori) were identified. The bacteria were isolated from algal media, thalli, tissue debris, and purified protoplasts during protoplast isolation from P. yezoensis laboratory cultures. 16S rRNA gene sequence analysis showed these bacterial isolates belonged to α-Proteobacteria (12 groups), γ-Proteobacteria (3 groups), and Flavobacteria (2 groups). Axenic protoplasts of P. yezoensis generated by removing epiphytic bacteria were co-cultured along with the bacterial isolates. Most axenic protoplasts showed irregular morphogenetic and anaplastic cells; cells with normal morphology were scarce. However, inoculation with 11 strains of Hyphomonas (α-Proteobacteria) led to significantly higher normal morphogenetic rates (4.5-7.3 %, P < 0.01 or 0.05) compared to axenic protoplasts (0.06 %). These Hyphomonas strains were recovered from all experiments; thus, certain Hyphomonas strains can induce normal morphogenesis in P. yezoensis protoplasts. Direct inoculation of the Hyphomonas strain exhibited higher morphogenetic activity than inoculation of its extracellular and intracellular products. This is the first study demonstrating the influence of specific bacteria on protoplast morphology in marine macroalgae.

Sulfitobacter porphyrae sp. nov., isolated from the red alga Porphyra yezoensis.

Gram-stain-negative, aerobic, halophilic bacteria, designated SCM-1(T), LCM10-1 and CTBL-B-147, were isolated from modified half-strength SWM-III medium, PES medium and thalli after laboratory cultivation of a red alga, Porphyra yezoensis. Phylogenetic analysis of 16S rRNA gene sequences indicated that the new isolates were affiliated to the genus Sulfitobacter of the class Alphaproteobacteria, and the 16S rRNA gene sequence similarity of the new isolates with the closest related species, Sulfitobacter mediterraneus CH-B427(T), was 98.8%. The DNA G+C contents of the new isolates were in the range of 61.4-62.3 mol%. DNA-DNA relatedness values of strain SCM-1(T) with other type strains of the genus Sulfitobacter were less than 15.9%. The new isolates contained Q-10 as the predominant ubiquinone, phosphatidylcholine, phosphatidylglycerol, an unidentified amino lipid and an unidentified lipid as the main polar lipids, and C(18 : 1)ω7c, C(19 : 1)ω7c and C(16 : 0) as the major fatty acids (>10% of the total). Strain SCM-1(T) could be differentiated from Sulfitobacter mediterraneus JCM 21792(T) by 35 morphological and phenotypic characteristics. On the basis of the phylogenetic, genetic and phenotypic properties of the new isolates, the name Sulfitobacter porphyrae sp. nov. is proposed, with strain SCM-1(T) ( = LMG 27110(T) = NBRC 109054(T)) as the type strain.

Streptomyces abietis sp. nov., a cellulolytic bacterium isolated from soil of a pine forest.

Cellulolytic bacteria A191(T), A192 and A193 isolated from the soil of Sakhalin fir forest in Hokkaido, Japan were studied phenotypically, genotypically and phylogenetically. Analysis of their 16S rRNA gene and gyrB sequences and DNA base composition suggested that these isolates were conspecific and members of the genus Streptomyces. However, levels of 16S rRNA gene and gyrB sequence similarity between the isolates and the type strains of their closest relatives in the genus Streptomyces were no higher than 97.9 and 95.0 %, respectively, implying that these isolates were distinctive. Moreover, the results of DNA-DNA hybridization experiments and physiological characterization clearly differentiated these isolates from their closest neighbours. It is therefore concluded that these isolates represent a novel species of the genus Streptomyces, for which the name Streptomyces abietis is proposed. The type strain is A191(T) ( = NBRC 109094(T) = DSM 42080(T)).

Algimonas ampicilliniresistens sp. nov., isolated from the red alga Porphyra yezoensis, and emended description of the genus Algimonas.

Three strains (14A-2-7(T), 14A-3-1 and 14A-3) of Gram-stain-negative, prosthecate, motile bacteria were isolated from an algal medium supplemented with 10 mg ampicillin l(-1) (w/v), in which the red alga Porphyra yezoensis had been cultured. Based on the 16S rRNA gene sequence analysis, the three isolates formed a cluster with the genus Algimonas of the family Hyphomonadaceae. The sequences of the three isolates had high similarity with those of Algimonas porphyrae 0C-2-2(T) (97.6 % similarity) and Litorimonas taeanensis G5(T) (95.6 % similarity). The DNA G+C contents of the three isolates ranged from 54.3 to 55.0 mol%, which were more similar to that of A. porphyrae 0C-2-2(T) (58.5 mol%) than to that of L. taeanensis G5(T) (47.1 mol%). The DNA-DNA relatedness showed that the three isolates were representatives of the same species (88.1-94.0 % relatedness) and that strain 14A-2-7(T) was a representative of a different species from A. porphyrae 0C-2-2(T) and L. taeanensis G5(T) (1.2-8.6 % relatedness). The phenotypic characteristics of strain 14A-2-7(T) differed by 20 results and 30 results from A. porphyrae 0C-2-2(T) and L. taeanensis G5(T), respectively. The three isolates contained ubiquinone-10 as the predominant quinone and C18 : 1ω7c as the major fatty acid. Based on the polyphasic taxonomic analysis, the three isolates represent a novel species of the genus Algimonas, for which the name Algimonas ampicilliniresistens sp. nov. is proposed. The type strain is 14A-2-7(T) ( = LMG 26421(T) = NBRC 108219(T)). An emended description of the genus Algimonas is also proposed.

Draft Genome Sequence of Agarivorans albus Strain MKT 106T, an Agarolytic Marine Bacterium.

Agarivorans albus is a Gram-negative, strictly aerobic, and agar-hydrolyzing marine bacterium. We present the draft genome sequence of the A. albus strain MKT 106(T), which is composed of 67 contigs (>500 bp) totaling 4,734,285 bp and containing 4,397 coding DNA sequences (CDSs), four rRNAs, and 64 tRNA sequences.

The first symbiont-free genome sequence of marine red alga, Susabi-nori (Pyropia yezoensis).

Nori, a marine red alga, is one of the most profitable mariculture crops in the world. However, the biological properties of this macroalga are poorly understood at the molecular level. In this study, we determined the draft genome sequence of susabi-nori (Pyropia yezoensis) using next-generation sequencing platforms. For sequencing, thalli of P. yezoensis were washed to remove bacteria attached on the cell surface and enzymatically prepared as purified protoplasts. The assembled contig size of the P. yezoensis nuclear genome was approximately 43 megabases (Mb), which is an order of magnitude smaller than the previously estimated genome size. A total of 10,327 gene models were predicted and about 60% of the genes validated lack introns and the other genes have shorter introns compared to large-genome algae, which is consistent with the compact size of the P. yezoensis genome. A sequence homology search showed that 3,611 genes (35%) are functionally unknown and only 2,069 gene groups are in common with those of the unicellular red alga, Cyanidioschyzon merolae. As color trait determinants of red algae, light-harvesting genes involved in the phycobilisome were predicted from the P. yezoensis nuclear genome. In particular, we found a second homolog of phycobilisome-degradation gene, which is usually chloroplast-encoded, possibly providing a novel target for color fading of susabi-nori in aquaculture. These findings shed light on unexplained features of macroalgal genes and genomes, and suggest that the genome of P. yezoensis is a promising model genome of marine red algae.

Polaribacter porphyrae sp. nov., isolated from the red alga Porphyra yezoensis, and emended descriptions of the genus Polaribacter and two Polaribacter species.

Three Gram-negative, non-motile, strictly aerobic strains, designated LNM-20(T), LCM-1 and LAM-13, were isolated from thalli of the marine red alga Porphyra yezoensis. Phylogenetic analysis based on 16S rRNA gene sequences revealed that the isolates were associated with the genus Polaribacter in the family Flavobacteriaceae and were most closely related to Polaribacter dokdonensis DSW-5(T) (96.2 % 16S rRNA gene sequence similarity) and Polaribacter gangjinensis K17-16(T) (95.0 %). The DNA G+C content of the isolates was 28.6-29.2 mol%. DNA-DNA hybridization analysis showed that the isolates belonged to a single species distinct from both of their closest relatives. The only isoprenoid quinone detected was menaquinone-6. The main polar lipids were phosphatidylethanolamine, two unidentified aminolipids and two unidentified lipids. The major fatty acids were iso-C15 : 0, iso-C15 : 1ω10c and iso-C15 : 0 3-OH. The phenotypic features of strain LNM-20(T) differed from those of their closest relatives in several regards (colony colour, growth with 1 % NaCl and on TSA plus 2.5 % NaCl, hydrolysis of Tweens 40 and 80, and oxidization of five carbon compounds). On the basis of phylogenetic, chemotaxonomic and phenotypic analysis, the isolates represent a novel species in the genus Polaribacter, for which the name Polaribacter porphyrae sp. nov. is proposed. The type strain is LNM-20(T) ( = LMG 26671(T)  = NBRC 108759(T)). Emended descriptions of the genus Polaribacter and P. dokdonensis and P. gangjinensis are also proposed.

Algimonas porphyrae gen. nov., sp. nov., a member of the family Hyphomonadaceae, isolated from the red alga Porphyra yezoensis.

Three Gram-negative, stalked, motile bacteria, designated 0C-2-2(T), 0C-17 and LNM-3, were isolated from the red alga Porphyra yezoensis. 16S rRNA gene sequence analysis revealed that the three novel strains belonged to the family Hyphomonadaceae, and were closely related to Litorimonas taeanensis G5(T) (96.5 % 16S rRNA gene sequence similarity) and Hellea balneolensis 26III/A02/215(T) (94.3 %). The DNA G+C contents of the novel isolates (58.5-60.2 mol%) were clearly distinguished from those of L. taeanensis G5(T) (47.1 mol%) and H. balneolensis DSM 19091(T) (47.9 mol%). The G+C content of L. taeanensis G5(T) obtained in this study was quite different from a previous report (63.6 mol%). DNA-DNA hybridization experiments showed that the novel strains constituted a single species. Eleven phenotypic features of the three isolates differed from those of both related genera. The predominant respiratory quinone was ubiquinone-10 and the major fatty acid was C(18 : 1)ω7c. On the basis of this polyphasic taxonomic analysis, the novel strains represent a novel genus and species, for which the name Algimonas porphyrae gen. nov., sp. nov. is proposed. The type strain of Algimonas porphyrae is 0C-2-2(T) (= LMG 26424(T) = NBRC 108216(T)).

Updating the Vibrio clades defined by multilocus sequence phylogeny: proposal of eight new clades, and the description of Vibrio tritonius sp. nov.

To date 142 species have been described in the Vibrionaceae family of bacteria, classified into seven genera; Aliivibrio, Echinimonas, Enterovibrio, Grimontia, Photobacterium, Salinivibrio and Vibrio. As vibrios are widespread in marine environments and show versatile metabolisms and ecologies, these bacteria are recognized as one of the most diverse and important marine heterotrophic bacterial groups for elucidating the correlation between genome evolution and ecological adaptation. However, on the basis of 16S rRNA gene phylogeny, we could not find any robust monophyletic lineages in any of the known genera. We needed further attempts to reconstruct their evolutionary history based on multilocus sequence analysis (MLSA) and/or genome wide taxonomy of all the recognized species groups. In our previous report in 2007, we conducted the first broad multilocus sequence analysis (MLSA) to infer the evolutionary history of vibrios using nine housekeeping genes (the 16S rRNA gene, gapA, gyrB, ftsZ, mreB, pyrH, recA, rpoA, and topA), and we proposed 14 distinct clades in 58 species of Vibrionaceae. Due to the difficulty of designing universal primers that can amplify the genes for MLSA in every Vibrionaceae species, some clades had yet to be defined. In this study, we present a better picture of an updated molecular phylogeny for 86 described vibrio species and 10 genome sequenced Vibrionaceae strains, using 8 housekeeping gene sequences. This new study places special emphasis on (1) eight newly identified clades (Damselae, Mediterranei, Pectenicida, Phosphoreum, Profundum, Porteresiae, Rosenbergii, and Rumoiensis); (2) clades amended since the 2007 proposal with recently described new species; (3) orphan clades of genomospecies F6 and F10; (4) phylogenetic positions defined in 3 genome-sequenced strains (N418, EX25, and EJY3); and (5) description of V. tritonius sp. nov., which is a member of the "Porteresiae" clade.

Bacterial communities in fish sauce mash using culture-dependent and -independent methods.

In fish sauce production, microorganisms are associated with the fermentation process; however, the sequential changes in the bacterial communities have never been examined throughout the period of fermentation. In this study, we determined the bacterial floras in a fish sauce mash over 8 months, using three different culture media and 16S rRNA gene clone library analysis. During the first 4 weeks, viable counts of non-halophilic and halophilic bacteria decreased and were dominated by Staphylococcus species. Between 4 and 6 weeks, halophilic and highly halophilic bacterial counts markedly increased from 10(7) to 10(8) cfu/g, and the predominant species changed to Tetragenococcus halophilus. The occurrence of T. halophilus was associated with an increase of lactic acid and a reduction of pH values. In contrast, non-halophilic bacterial counts decreased to 10(6) cfu/g by 6 weeks with Bacillus subtilis as the dominant isolate. Clone library analysis revealed that the dominant bacterial group also changed from Staphylococcus spp. to T. halophilus, and the changes were consistent with those of the floras of halophilic and highly halophilic isolates. This is the first report describing a combination approach of culture and clone library methods for the analysis of bacterial communities in fish sauce mash.

Maritalea porphyrae sp. nov., isolated from a red alga (Porphyra yezoensis), and transfer of Zhangella mobilis to Maritalea mobilis comb. nov.

Three Gram-negative, motile, aerobic bacteria were isolated from cultures of the marine red alga Porphyra yezoensis. Phylogenetic analysis based on 16S rRNA gene sequences revealed that the novel strains were closely related to Maritalea myrionectae CL-SK30(T) (97.9% 16S rRNA gene sequence similarity) and Zhangella mobilis E6(T) (96.2 %). 16S rRNA gene sequence similarity between Z. mobilis E6(T) and M. myrionectae CL-SK30(T) was 97.9%. The DNA G+C contents of the isolates (49.4-50.0 mol%) were similar to those of M. myrionectae DSM 19524(T) (52.3 mol%) and Z. mobilis JCM 15144(T) (50.3 mol%). From these results, it was difficult to differentiate the genus Zhangella from the genus Maritalea. DNA-DNA hybridization demonstrated that the isolates belonged to a single species. The isolates could also be distinguished from M. myrionectae and Z. mobilis on the basis of chemotaxonomic and phenotypic features, including fatty acid composition (particularly C(16:1)ω7c), growth with 6-9% (w/v) NaCl, carbon utilization, oxidation patterns and so on. A novel species of the genus Maritalea is proposed to accommodate the three isolates, with the name Maritalea porphyrae sp. nov. The type strain is LCM-3(T) (=LMG 25872(T)=NBRC 107169(T)). Furthermore, it is proposed that Zhangella mobilis should be transferred from the genus Zhangella to the genus Maritalea, with the name Maritalea mobilis comb. nov. (type strain E6(T)=CGMCC 1.7002(T)=JCM 15144(T)).

Diversity of plasmids encoding histidine decarboxylase gene in Tetragenococcus spp. isolated from Japanese fish sauce.

Nineteen isolates of histamine producing halophilic bacteria were isolated from four fish sauce mashes, each mash accumulating over 1000 ppm of histamine. The complete sequences of the plasmids encoding the pyruvoyl dependent histidine decarboxylase gene (hdcA), which is harbored in histamine producing bacteria, were determined. In conjunction, the sequence regions adjacent to hdcA were analyzed to provide information regarding its genetic origin. As reference strains, Tetragenococcus halophilus H and T. muriaticus JCM10006(T) were also studied. Phenotypic and 16S rRNA gene sequence analyses identified all isolates as T. halophilus, a predominant histamine producing bacteria present during fish sauce fermentation. Genetic analyses (PCR, Southern blot, and complete plasmid sequencing) of the histamine producing isolates confirmed that all the isolates harbored approximately 21-37 kbp plasmids encoding a single copy of the hdc cluster consisting of four genes related to histamine production. Analysis of hdc clusters, including spacer regions, indicated >99% sequence similarity among the isolates. All of the plasmids sequenced encoded traA, however genes related to plasmid conjugation, namely mob genes and oriT, were not identified. Two putative mobile genetic elements, ISLP1-like and IS200-like, respectively, were identified in the up- and downstream region of the hdc cluster of all plasmids. Most of the sequences, except hdc cluster and two adjacent IS elements, were diverse among plasmids, suggesting that each histamine producers harbored a different histamine-related plasmid. These results suggested that the hdc cluster was not spread by clonal dissemination depending on the specific plasmid and that the hdc cluster in tetragenococcal plasmid was likely encoded on transformable elements.

Characterization of the histidine decarboxylase gene of Staphylococcus epidermidis TYH1 coded on the staphylococcal cassette chromosome.

Histamine production from histidine in fermented food results in food spoilage, and is harmful to consumers. From fish-miso, we have isolated a new bacterial strain Staphylococcus epidermidis TYH1, which produced histamine under acidic condition in the medium supplemented with glucose. Using oligonucleotides deduced from the histidine decarboxylase gene (hdcA) of Lactobacillus hilgardii, about 14-kbp DNA region of the TYH1 genome was cloned and sequenced. This region contained two putative genes hdcA(TYH1) and hdcP(TYH1) encoding proteins HdcA(TYH1) (310 amino acid residues) and HdcP(TYH1) (495 residues), respectively. Nucleotide sequence around this hdc cluster showed similarity to SCCpbp4 region of S. epidermidis ATCC 12228. Downstream of the cluster, ccrA, ccrB (Type II, respectively) and pbp4 were located. The CcrA and CcrB proteins catalyzed excision of the hdc cluster from the TYH1 chromosome, upon introduction into the TYH1 strain via multicopy plasmid. When hdcA(TYH1) was expressed in Staphylococcus warneri M, histamine was extracellularly accumulated in dependence on exogenous histidine. These results indicate that the gene encoding a histidine decarboxylase resides in a movable genetic element, SCC. This new element is designated as SCChdc.

Description of Rummeliibacillus stabekisii gen. nov., sp. nov. and reclassification of Bacillus pycnus Nakamura et al. 2002 as Rummeliibacillus pycnus comb. nov.

Strains of aerobic, Gram-positive, rod-shaped, round-spore-forming bacteria were isolated from different geographical locations and a subsequent polyphasic study was undertaken to clarify the taxonomic position of the round-spore-forming isolates strain KSC-SF6g(T), strain M32 and strain NBRC 12622. 16S rRNA gene sequence similarities demonstrated that these strains were most closely affiliated with Bacillus pycnus NRRL NRS-1691(T) (98 %), with species of Kurthia (96 %) and Viridibacillus (94-96 %) as the next nearest relatives. However, while DNA-DNA hybridization studies showed approx. 70 % reassociation among strains KSC-SF6g(T), M32 and NBRC 12622, DNA-DNA hybridization values between these strains and B. pycnus NRRL NRS-1691(T) never exceeded 13 %. Differences in the molecular structure of the cell-wall peptidoglycan could not differentiate these strains sufficiently from other closely related genera (Viridibacillus and Kurthia). However, Lys-Asp was present in strains KSC-SF6g(T), M32 and NBRC 12622, whereas l-Lys-d-Glu was reported in B. pycnus NRRL NRS-1691(T). The menaquinone MK-7 was dominant in strains KSC-SF6g(T), M32 and NBRC 12622 and members of the genus Kurthia, whereas MK-8 was abundant in Viridibacillus species. Strains KSC-SF6g(T), M32 and NBRC 12622 exhibited fatty acid profiles consisting of major amounts of anteiso-C(15 : 0) ( approximately 50 %) and iso-C(15 : 0) ( approximately 25 %) and moderate amounts of anteiso-C(17 : 0) ( approximately 7 %), which discriminated them from closely related B. pycnus NRRL NRS-1691(T) and species of Viridibacillus (iso-C(15 : 0); 46-74 %). The authors propose that strains KSC-SF6g(T), M32 and NBRC 12622 and B. pycnus NRRL NRS-1691(T) be reclassified into a separate genus based on clear-cut differences in discriminative taxonomic markers and the distant placement of B. pycnus and the novel strains described herein from other species of this clade according to current 16S rRNA gene sequence-based relatedness ( approximately 4 % difference in sequence). We propose the placement of these isolates into the novel genus Rummeliibacillus gen. nov. For the new taxon comprising strains KSC-SF6g(T), M32 and NBRC 12622, we propose the name Rummeliibacillus stabekisii gen. nov., sp. nov. (the type species of Rummeliibacillus), represented by the type strain KSC-SF6g(T) (=NRRL B-51320(T) =NBRC 104870(T)). In addition, Bacillus pycnus, which bears traits distinct from other round-spore-forming species [i.e. absence of growth at high NaCl (7 %), positive reaction for gelatin liquefaction], is reclassified as Rummeliibacillus pycnus comb. nov. (type strain JCM 11075(T) =NRRL NRS-1691(T)) based on phylogenetic affiliations and phenotypic characterization.