Masataka Satomi

Molecular microbial diversity of a spacecraft assembly facility.

In ongoing investigations to map and archive the microbial footprints in various components of the spacecraft and its accessories, we have examined the microbial populations of the Jet Propulsion Laboratorys Spacecraft Assembly Facility (JPL-SAF). Witness plates made up of spacecraft materials, some painted with spacecraft qualified paints, were exposed for approximately 7 to 9 months at JPL-SAF and examined the particulate materials collected for the incidence of total cultivable aerobic heterotrophs and heat-tolerant (80 degrees C for 15-min.) spore-formers. The results showed that the witness plates coated with spacecraft qualified paints attracted more dust particles than the non-coated stainless steel witness plates. Among the four paints tested, witness plates coated with NS43G accumulated the highest number of particles, and hence attracted more cultivable microbes. The conventional microbiological examination revealed that the JPL-SAF harbors mainly Gram-positive microbes and mostly spore-forming Bacillus species. Most of the isolated microbes were heat resistant to 80 degrees C and proliferate at 60 degrees C. The phylogenetic relationships among 23 cultivable heat-tolerant microbes were examined using a battery of morphological, physiological, molecular and chemotaxonomic characterizations. By 16S rDNA sequence analysis, the isolates fell into seven clades: Bacillus licheniformis, B. pumilus, B. cereus, B. circulans, Staphylococcus capitis, Planococcus sp. and Micrococcus lylae. In contrast to the cultivable approach, direct DNA isolation, cloning and 16S rDNA sequencing analysis revealed equal representation of both Gram-positive and Gram-negative microorganisms.

Identification of Shewanella baltica as the Most Important H2S-Producing Species during Iced Storage of Danish Marine Fish

ABSTRACT Shewanella putrefaciens has been considered the main spoilage bacteria of low-temperature stored marine seafood. However, psychrotropic Shewanella have been reclassified during recent years, and the purpose of the present study was to determine whether any of the new Shewanella species are important in fish spoilage. More than 500 H2S-producing strains were isolated from iced stored marine fish (cod, plaice, and flounder) caught in the Baltic Sea during winter or summer time. All strains were identified as Shewanella species by phenotypic tests. Different Shewanella species were present on newly caught fish. During the warm summer months the mesophilic human pathogenic S. algae dominated the H2S-producing bacterial population. After iced storage, a shift in the Shewanella species was found, and most of the H2S-producing strains were identified as S. baltica. The 16S rRNA gene sequence analysis confirmed the identification of these two major groups. Several isolates could only be identified to the genus Shewanella level and were separated into two subgroups with low (44%) and high (47%) G+C mol%. The low G+C% group was isolated during winter months, whereas the high G+C% group was isolated on fish caught during summer and only during the first few days of iced storage. Phenotypically, these strains were different from the type strains of S. putrefaciens, S. oneidensis, S. colwelliana, and S. affinis, but the high G+C% group clustered close to S. colwelliana by 16S rRNA gene sequence comparison. The low G+C% group may constitute a new species. S. baltica, and the low G+C% group of Shewanella spp. strains grew well in cod juice at 0°C, but three high G+C Shewanella spp. were unable to grow at 0°C. In conclusion, the spoilage reactions of iced Danish marine fish remain unchanged (i.e., trimethylamine-N-oxide reduction and H2S production); however, the main H2S-producing organism was identified as S. baltica.

Tetragenococcus muriaticus sp. nov., a New Moderately Halophilic Lactic Acid Bacterium Isolated from Fermented Squid Liver Sauce

A total of 11 strains of moderately halophilic histamine-producing bacteria isolated from fermented squid liver sauce were studied phenotypically, genotypically, and phylogenetically. These strains are considered members of the genus Tetragenococcus based on their physiological, morphological, and chemotaxonomic characteristics. A 16S rRNA gene sequence analysis showed that these strains clustered with, but were separate from, Tetragenococcus halophilus. The results of DNA-DNA hybridization experiments indicated that the new isolates represent a new Tetragenococcus species, for which we propose the name Tetragenococcus muriaticus; strain X-1 (= JCM 10006) is the type strain of this species.

Degradation of 17β-Estradiol by a Gram-Negative Bacterium Isolated from Activated Sludge in a Sewage Treatment Plant in Tokyo, Japan

ABSTRACT A 17β-estradiol (E2)-degrading bacterium was isolated from activated sludge in a sewage treatment plant in Tokyo, Japan. The isolate was suggested to be a new Novosphingobium species. Gas chromatography-mass spectrometry and 1H nuclear magnetic resonance analyses of the metabolites of E2 degradation suggested that no toxic products accumulated in the culture medium.

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.

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.

Sphingomonas cloacae sp. nov., a nonylphenol-degrading bacterium isolated from wastewater of a sewage-treatment plant in Tokyo

A nonylphenol-assimilating bacterium isolated at a sewage-treatment plant in Tokyo was studied phenotypically, genotypically and phylogenetically. Analysis of the 16S rDNA sequence, the G+C content of the DNA (63 mol%) and the isoprenoid quinone composition, as well as the presence of sphingoglycolipid and the whole-cell fatty acid profile, revealed that the isolate is a member of the genus Sphingomonas. However, the sequence similarity of the 16S rDNA with that of known Sphingomonas spp. was found to be at most 96%, implying that the isolate is distinctive. Furthermore, the results of DNA-DNA hybridization experiments and its physiological characteristics clearly indicated that the isolate represents a new Sphingomonas species, for which the name Sphingomonas cloacae is proposed; strain S-3T (= JCM 10874T = IAM 14885) is the type strain.

Analysis of a 30 kbp plasmid encoding histidine decarboxylase gene in Tetragenococcus halophilus isolated from fish sauce.

In order to analyze the genes related to the histamine production, a strain of histamine producing halophilic bacteria, referred to as strain H, was isolated using enrichment culture and dilution-to-extinction methods with histidine broth inoculated from the fish sauce mashes. The two Japanese fish sauce mashes used, accumulate over 1000 mg/l of histamine. Phenotypic and 16 S rRNA gene sequence analyses identified strain H as Tetragenococcus halophilus, the predominant histamine producing bacteria present during fish sauce fermentation. Genetic analyses (PCR and Southern blot) of the histamine producing strain confirmed that the strain harbored a 30 kbp plasmid (pHDC) encoding a single copy of the pyruvoyl dependent histidine decarboxylase gene (hdc). A comparison of hdcA that is a structural gene of histidine decarboxylase among strain H, Lactobacillus hilgardii 0006, L. sakei LTH2076, Oenococcus oeni 9204, T. halophilus and T. muriaticus JCM10006 (T) indicated >99% sequence similarity. The hdc gene cluster consisted of 4 ORFs, hdcP, hdcA, hdcB, and hdcRS, and were almost identical to that of L. hilgardii 0006 with 99% sequence similarity including the structural hdc spacer region. However, the approximately 500 bp regions upstream and downstream of the hdc gene were different between that of strain H and L. hilgardii 0006. The complete sequence of pHDC revealed 29,924 nucleotides including 28 ORFs, two pairs of IR (inverted repeat), similar sequence of plasmid conjugative elements, and a theta-type replicon. These results suggested that hdc could be encoded on transformable elements among lactic acid bacteria.

Changes in extractive components and microbial proliferation during fermentation of fish sauce from underutilized fish species and quality of final products

To make use of underutilized fish species and produce high-quality condiments substitutable for those of mammalian origin, fish sauces were produced on a small industrial scale from three fish species, the flyingfish Cypselurus agoo agoo, the small dolphinfish Coryphaena hippurus, and the deepsea smelt Glossanodon semifasciatus, using salt and koji mold. During 180 days of fermentation, the pH decreased to approximately 4.5 and total nitrogen and free amino acids increased to approximately 2 g/100 mL and 6000 mg/100 mL, respectively. Halophilic and extreme halophilic bacterial counts dramatically increased from 106 to 108 colony forming units/mL between days 14 and 30 and decreased the reafter. In the final products, the pH and salt concentration decreased to between soy sauce and a Vietnamese fish sauce, Nuoc mam. The total amino acid and organic acid contents were almost the same as those of Nuoc mam and soy sauce. Sensory evaluation found these products to have lower smell, saltiness, and bitterness, and higher sweetness and umami taste than Nuoc mam. During fermentation, the histamine content increased in one or two of three fermentation tanks for each fish species. As a result, the histamine content of the final products was higher than that in Nuoc mam.

Intra‐specific phenotypic and genotypic variation in toxic cyanobacterial Microcystis strains

Aims:  We determined if the intra‐specific genetic diversity of Microcystis aeruginosa correlates with phenotypic characteristics.