Acaryochloris spp. are unique cyanobacteria which contain chlorophyll d as the predominant pigment. The phylogenetic diversity of Acaryochloris spp. associated with 7 Prochloron- or Synechocystis-containing didemnid ascidians and 1 Synechococcus-containing sponge obtained from the coast of the Republic of Palau was analyzed; we established a PCR primer set designed to selectively amplify the partial 16S rRNA gene of Acaryochloris spp. even in DNA samples containing a large amount of other cyanobacterial and algal DNAs. Polymerase chain reaction-denaturing gradient gel electrophoresis with this primer set enabled detection of the phyogenetic diversity of Acaryochloris spp. All the ascidian and sponge samples contained Acaryochloris spp. Fourteen phylotypes that were highly homologous (98–100%) with A. marina MBIC11017 were detected, while only 2 phylotypes were detected with our previously developed method for detecting cyanobacteria. The results also revealed that many uncultured phylotypes of Acaryochloris spp. were associated with those didemnid ascidians, since a clonal culture of only 1 phylotype has been established thus far. No specific relationship was found among the Acaryochloris phylotypes and the genera of the ascidians even when sample localities were identical; therefore, these invertebrates may provide a favorable habitat for Acaryochloris spp. rather than hosts showing any specific symbiotic relationships.

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Clarifying the identity and enzymatic activities of microorganisms associated with the decomposition of organic materials is expected to contribute to the evaluation and improvement of composting processes. In this study, we examined the cellulolytic and hemicellulolytic abilities of bacteria isolated from sawdust compost (SDC) and coffee residue compost (CRC). Cellulolytic bacteria were isolated using Dubos mineral salt agar containing azurine cross-linked (AZCL) HE-cellulose. Bacterial identification was performed based on the sequence analysis of 16S rRNA genes, and cellulase, xylanase, β-glucanase, mannanase, and protease activities were characterized using insoluble AZCL-linked substrates. Eleven isolates were obtained from SDC and 10 isolates from CRC. DNA analysis indicated that the isolates from SDC and CRC belonged to the genera Streptomyces, Microbispora, and Paenibacillus, and the genera Streptomyces, Microbispora, and Cohnella, respectively. Microbispora was the most dominant genus in both compost types. All isolates, with the exception of two isolates lacking mannanase activity, showed cellulase, xylanase, β-glucanase, and mannanase activities. Based on enzyme activities expressed as the ratio of hydrolysis zone diameter to colony diameter, it was suggested that the species of Microbispora (SDCB8, SDCB9) and Paenibacillus (SDCB10, SDCB11) in SDC and Microbispora (CRCB2, CRCB6) and Cohnella (CRCB9, CRCB10) in CRC contribute to efficient cellulolytic and hemicellulolytic processes during composting.

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The mining industry generates huge amounts of wastewater, containing toxic heavy metals. Treatment to remove heavy metals is necessary and recent work has been focused on finding more environmentally friendly materials for removing heavy metals from wastewater. Biosorption can be an effective process for heavy metal removal from aqueous solutions. Our objectives were to investigate the removal of copper (II) from aqueous solutions using dead cells of Mesorhizobium amorphae CCNWGS0123 under differing levels of pH, agitation speed, temperature, initial copper concentration, biosorbent dose and contact time using flame atomic absorption spectroscopy for metal estimation. The maximum copper removal rate was achieved at pH 5.0, agitation speed 150×g, temperature 28°C and initial Cu (II) concentration of 100 mg L^-1. Maximum biosorption capacity was at 0.5 g L^-1 and equilibrium was attained within 30 min. Langmuir and Freundlich isotherms showed correlation coefficients of 0.958 and 0.934, respectively. Fourier transform-infrared spectroscopy (FT-IR) analysis indicated that many functional groups, such as O-H, N-H, C-H, C=O, -NH, -CN, C-N, C-O, amide -I, -II, -III and unsaturated alkenes, alkyls and aromatic groups on the cell surface were involved in the interaction between CCNWGS0123 and Cu. Scanning electron microscope and energy dispersive X-ray scanning results showed deformation, aggregation, and cell-surface damage due to the precipitation of copper on the cell surface. Dead cells of CCNWGS0123 showed potential as an efficient biosorbent for the removal of Cu²⁺ from aqueous solutions.

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The amount of oxygen released by Phragmites roots and the community structure and in situ activity of nitrifying bacteria in the root biofilms were analyzed by the combined use of 16S rRNA gene-cloning analysis, quantitative PCR (qPCR) assay and microelectrodes. Axial and radial O₂ microprofiles were obtained for individual roots of Phragmites in a horizontal flow reactor fed with artificial medium continuously. Axial O₂ profiles revealed that O₂ was released at a rate of 0.21 μmol O₂ cm^-2 (root surface area) h^-1 only in the apical region (up to ca. 40 mm from the root apex), where there was a high abundance (10⁷ to 10⁸ copies g^-1 biomass) of Nitrosomonas-like AOB and Nitrospira-like NOB. This abundance, however, sharply declined to the detection limit at positions more basal than 80 mm. Phylogenetic analysis based on 16S rRNA gene identified strains related to Nitrosomonas oligotropha and Nitrosomonas cryotolerans as the predominant AOB and strains related to Nitrospira marina and Nitrospira moscoviensis as the predominant NOB in the root biofilms. Based on radial O₂ microprofiles, the oxic region only extended about 0.5 mm into the surrounding sediment due to a high rate of O₂ consumption in the rhizosphere. The net NH₄⁺ and O₂ consumption rates in the apical region were higher than those determined at the oxic sediment surface in which the abundance of AOB and NOB was one order of magnitude lower than in the rhizosphere. These results clearly indicated that Phragmites root biofilms played an important role in nitrification in the waterlogged anoxic sediment.

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In Vibrio cholerae, the etiological agent of cholera, most of the virulence genes are located in two pathogenicity islands, named TCP (Toxin-Co-regulated Pilus) and CTX (Cholera ToXins). For each V. cholerae pathogenicity gene, we retrieved every primer published since 1990 and every known allele in order to perform a complete in silico survey and assess the quality of the PCR primers used for amplification of these genes. Primers with a melting temperature in the range 55–60°C against any target sequence were considered valid. Our survey clearly revealed that two thirds of the published primers are not able to properly detect every genetic variant of the target genes. Moreover, the quality of primers did not improve with time. Their lifetime, i.e. the number of times they were cited in the literature, is also not a factor allowing the selection of valid primers. We were able to improve some primers or design new primers for the few cases where no valid primer was found. In conclusion, many published primers should be avoided or improved for use in molecular detection tests, in order to improve and perfect specificity and coverage. This study suggests that bioinformatic analyses are important to validate the choice of primers.

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There are increasing applications of diazotrophic rhizobacteria in the sustainable agriculture system. A field experiment on young immature oil palm was conducted to quantify the uptake of N derived from N₂ fixation by the diazotroph Bacillus sphaericus strain UPMB-10, using the ¹⁵N isotope dilution method. Eight months after ¹⁵N application, young immature oil palms that received 67% of standard N fertilizer application together with B. sphaericus inoculation had significantly lower ¹⁵N enrichment than uninoculated palms that received similar N fertilizers. The dilution of labeled N served as a marker for the occurrence of biological N₂ fixation. The proportion of N uptake that was derived from the atmosphere was estimated as 63% on the whole plant basis. The inoculation process increased the N and dry matter yields of the palm leaflets and rachis significantly. Field planting of young, immature oil palm in soil inoculated with B. sphaericus UPMB-10 might mitigate inorganic fertilizer-N application through supplementation by biological nitrogen fixation. This could be a new and important source of nitrogen biofertilizer in the early phase of oil palm cultivation in the field.

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The emergence of drug-resistant bacteria is a severe problem in aquaculture. The ability of drug resistance genes to transfer from a bacterial cell to another is thought to be responsible for the wide dissemination of these genes in the aquaculture environment; however, little is known about the gene transfer mechanisms in marine bacteria. In this study, we show that a tetracycline-resistant strain of Photobacterium damselae subsp. damselae, isolated from seawater at a coastal aquaculture site in Japan, harbors a novel multiple drug resistance plasmid. This plasmid named pAQU1 can be transferred to Escherichia coli by conjugation. Nucleotide sequencing showed that the plasmid was 204,052 base pairs and contained 235 predicted coding sequences. Annotation showed that pAQU1 did not have known repA, suggesting a new replicon, and contained seven drug resistance genes: bla_CARB-9-like, floR, mph(A)-like, mef(A)-like, sul2, tet(M) and tet(B). The plasmid has a complete set of genes encoding the apparatus for the type IV secretion system with a unique duplication of traA. Phylogenetic analysis of the deduced amino acid sequence of relaxase encoded by traI in pAQU1 demonstrated that the conjugative transfer system of the plasmid belongs to MOB_H12, a sub-group of the MOB_H plasmid family, closely related to the IncA/C type of plasmids and SXT/R391 widely distributed among species of Enterobacteriaceae and Vibrionaceae. Our data suggest that conjugative transfer is involved in horizontal gene transfer among marine bacteria and provide useful insights into the molecular basis for the dissemination of drug resistance genes among bacteria in the aquaculture environment.

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A novel strain of p-xylene-degrading sulfate reducer was isolated in pure culture. Strain PP31 was obtained from a p-xylene-degrading enrichment culture established from polluted marine sediment. Analyses of the 16S rRNA gene and two functional genes involved in sulfate respiration and anaerobic degradation of aromatic compounds revealed that the isolate was closely related to members of the genus Desulfosarcina. Strain PP31 was capable of growing on p-xylene under sulfate-reducing conditions, and the ratio of generated sulfide and consumed p-xylene suggested complete oxidation by the novel isolate. The strain could not grow on benzene, toluene, ethylbenzene, m-xylene o-xylene, or n-hexane as an electron donor. Strain PP31 is the first isolated bacterium that degrades p-xylene anaerobically, and will be useful to understanding the mechanism of anaerobic degradation of p-xylene.

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The diversity of cultivable methane-oxidizing bacteria (MOB) in the rice paddy field ecosystem was investigated by combined culture-dependent and fluorescence in situ hybridization (FISH) techniques. Seven microsites of a Japanese rice paddy field were the focus of the study: floodwater, surface soil, bulk soil, rhizosphere soil, root, basal stem of rice plant, and rice stumps of previous harvest. Based on pmoA gene analysis and transmission electron microscopy (TEM), four type I, and nine type II MOB isolates were obtained from the highest dilution series of enrichment cultures. The type I MOB isolates included a novel species in the genus Methylomonas from floodwater and this is the first type I MOB strain isolated from floodwater of a rice paddy field. In the type I MOB, two isolates from stumps were closely related to Methylomonas spp.; one isolate obtained from rhizosphere soil was most related to Methyloccocus-Methylocaldum-Methylogaea clade. Almost all the type II MOB isolates were related to Methylocystis methanotrophs. FISH confirmed the presence of both types I and II MOB in all the microsites and in the related enrichment cultures. The study reported, for the first time, the diversity of cultivable methanotrophs including a novel species of type I MOB in rice paddy field compartments. Refining growth media and culture conditions, in combination with molecular approaches, will allow us to broaden our knowledge on the MOB community in the rice paddy field ecosystem and consequently to implement strategies for mitigating CH4 emission from this ecosystem.

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Hand, foot and mouth disease (HFMD), caused by a group of enteric viruses such as Enterovirus 71 (EV71), Coxsackievirus A16 (CVA16) and Coxsackievirus A10 (CVA10), is heavily epidemic in East Asia. This research focused on investigating the occurrence of HFMD pathogens in domestic sewage and secondary effluent before disinfection in a wastewater treatment plant (WWTP) in Xi’an, the largest megacity in northwest China. In order to simultaneously detect all three HFMD pathogens, a semi-nested RT-PCR assay was constructed with a newly designed primer set targeting conservative gene regions from the 5′ untranslated region (UTR) to VP2. As a result, 86% of raw sewage samples and 29% of the secondary effluent samples were positive for the HFMD viral gene, indicating that HFMD pathogens were highly prevalent in domestic wastewater and that they could also persist, even with lower probability, in the secondary effluent before disinfection. Of the three HFMD pathogens, CVA10 was positive in 48% of the total samples, while the occurrences of CVA16 and EV71 were 12% and 2%, respectively. It could thus be stated that CVA10 is the main HFMD pathogen prevailing in the study area, at least during the investigation period. High genetic diversity in the conservative gene region among the same serotype of the HFMD pathogen was identified by phylogenetic analysis, implying that this HFMD pathogen replicates frequently among the population excreting the domestic sewage.

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Microbial mats containing the filamentous anoxygenic photosynthetic bacterium Chloroflexus aggregans develop at Nakabusa hot spring in Japan. Under anaerobic conditions in these mats, interspecies interaction between sulfate-reducing bacteria as sulfide producers and C. aggregans as a sulfide consumer has been proposed to constitute a sulfur cycle; however, the electron donor utilized for microbial sulfide production at Nakabusa remains to be identified. In order to determine this electron donor and its source, ex situ experimental incubation of mats was explored. In the presence of molybdate, which inhibits biological sulfate reduction, hydrogen gas was released from mat samples, indicating that this hydrogen is normally consumed as an electron donor by sulfate-reducing bacteria. Hydrogen production decreased under illumination, indicating that C. aggregans also functions as a hydrogen consumer. Small amounts of hydrogen may have also been consumed for sulfur reduction. Clone library analysis of 16S rRNA genes amplified from the mats indicated the existence of several species of hydrogen-producing fermentative bacteria. Among them, the most dominant fermenter, Fervidobacterium sp., was successfully isolated. This isolate produced hydrogen through the fermentation of organic carbon. Dispersion of microbial cells in the mats resulted in hydrogen production without the addition of molybdate, suggesting that simultaneous production and consumption of hydrogen in the mats requires dense packing of cells. We propose a cyclic electron flow within the microbial mats, i.e., electron flow occurs through three elements: S (elemental sulfur, sulfide, sulfate), C (carbon dioxide, organic carbon) and H (di-hydrogen, protons).

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Marine invertebrates interact with various microorganisms ranging from pathogens to symbionts. One-to-one symbiosis between a single microbial species and a single host animal has served as a model for the study of host-microbe interactions. In addition, increasing attention has recently been focused on the complex symbiotic associations, e.g., associations between sponges and their symbionts, due to their biotechnological potential; however, relatively little is known about the microbial diversity associated with members of the phylum Echinodermata. Here, for the first time, we investigated microbial communities associated with a commercially important holothurian species, Apostichopus japonicus, using culture-dependent and -independent methods. Diverse and abundant heterotrophs, mostly Gammaproteobacteria members, were cultured semi-quantitatively. Using the cloning and sequencing technique, different microbial communities were found in different holothurian tissues. In the holothurian coelomic fluid, potentially metabolically active and phylogenetically unique members of Epsilonproteobacteria and Rickettsiales were discovered. This study suggests that coelomic fluids of marine invertebrates, at least those inhabiting intertidal areas where physical and chemical conditions fluctuate, provide microbes with unique and stable habitats.

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Bradyrhizobium sp. S23321 is an oligotrophic bacterium isolated from paddy field soil. Although S23321 is phylogenetically close to Bradyrhizobium japonicum USDA110, a legume symbiont, it is unable to induce root nodules in siratro, a legume often used for testing Nod factor-dependent nodulation. The genome of S23321 is a single circular chromosome, 7,231,841 bp in length, with an average GC content of 64.3%. The genome contains 6,898 potential protein-encoding genes, one set of rRNA genes, and 45 tRNA genes. Comparison of the genome structure between S23321 and USDA110 showed strong colinearity; however, the symbiosis islands present in USDA110 were absent in S23321, whose genome lacked a chaperonin gene cluster (groELS3) for symbiosis regulation found in USDA110. A comparison of sequences around the tRNA-Val gene strongly suggested that S23321 contains an ancestral-type genome that precedes the acquisition of a symbiosis island by horizontal gene transfer. Although S23321 contains a nif (nitrogen fixation) gene cluster, the organization, homology, and phylogeny of the genes in this cluster were more similar to those of photosynthetic bradyrhizobia ORS278 and BTAi1 than to those on the symbiosis island of USDA110. In addition, we found genes encoding a complete photosynthetic system, many ABC transporters for amino acids and oligopeptides, two types (polar and lateral) of flagella, multiple respiratory chains, and a system for lignin monomer catabolism in the S23321 genome. These features suggest that S23321 is able to adapt to a wide range of environments, probably including low-nutrient conditions, with multiple survival strategies in soil and rhizosphere.

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An anammox assay involving a ¹⁵N tracer and gas chromatography-mass spectrometry revealed that the potential anammox activity accounted for 1 to 5% of total N2 production in a ravine paddy field, Japan. Among four 4-cm-deep layers, the top layer showed the highest activity. Clone libraries showed that the DNA in the top layer contained sequences related to those of Candidatus ‘Brocadia fulgida’, Ca. ‘B. anammoxidans’, and Ca. ‘Kuenenia stuttgartiensis’. These results suggest that a specific population of anammox bacteria was present in paddy soils, although a small part of dinitrogen gas was emitted from the soil via anammox.

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Many aerobic microorganisms can colonize at the air-liquid interface and form a multicellular structure, known as a pellicle. In this study, the involvement of motility and attachment traits in the Pseudomonas aeruginosa pellicle formation process was investigated. Flagella- and flagellar-motor-deficient mutants exhibited delayed pellicle formation and unusual pellicle morphology, indicating the large contribution of flagella-driven motility to structural development of the pellicle. A pili-deficient mutant showed normal pellicle formation properties, while the disruption of the pilus gene in the flagella-deficient mutant restored normal pellicle morphology. These results indicate that flagella and pili play key roles in P. aeruginosa pellicle development.

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In this study, a microcolony technique was combined with direct fluorescent antibody staining for the specific detection and enumeration of Legionella pneumophila in freshwater samples with growth activity. This method allowed the detection of active L. pneumophila (within 48 h) in 91 bath water samples collected from 30 bathing facilities, with similar sensitivity of a conventional plate-counting method. These results suggest that the microcolony method combined with fluorescent antibody staining could be useful as a monitoring technique for the prevention of Legionnaires’ disease through the early detection of L. pneumophila in freshwater.

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The diversity of purple phototrophic bacteria in algae-dominated biofilm of a streambed in Tama River, Japan was investigated. Clone library analysis of the pufM gene encoding a subunit of the photochemical reaction center of purple bacteria detected 18 operational taxonomic units (OTUs) in several classes of Proteobacteria. Most of the OTUs showed less than 85% identity to the PufM amino acid sequences of known phototrophic bacteria. These results suggest that phylogenetically divergent and unknown purple phototrophic bacteria are present in the epilithic biofilm of the river.

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N-Acylhomoserine lactone (AHL)-degrading enzyme, AiiM, was identified from the potato leaf-associated Microbacterium testaceum StLB037. In this study, we cloned eight aiiM gene homologues from other AHL-degrading Microbacterium strains. The similarity of the chromosomal locus of the aiiM gene is associated with the phylogenetic classification based on 16S rRNA. Degenerate PCR revealed that the aiiM gene was only conserved in AHL-degrading Microbacterium strains, but not in fifteen Microbacterium type strains or two Microbacterium isolates from other plants. These results suggested that the high level of AHL-degrading activity in Microbacterium strains was caused by the aiiM gene encoded on their chromosome.

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In the present investigation, the abundance and molecular phylogeny of part of the culturable bacterial population involved in the dissolution of “miliolite”, a bioclastic limestone, from Gopnath, India, was studied. Carbonate-dissolving bacteria were isolated, enumerated and screened for their ability to dissolve miliolite. Amplified ribosomal DNA restriction analysis (ARDRA) indicated 14 operational taxonomic units (OTUs) to be distributed in 5 different clades at a similarity coefficient of 0.85. Then, 16S rRNA sequence analysis helped to decipher that the majority of carbonate-dissolving bacteria were affiliated to phyla Firmicutes (Families Bacillaceae and Staphylococcaceae) and Actinobacteria (Family Promicromonosporaceae) indicating their role in miliolite weathering.

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Two morphologically distinct extracellular stalk fibers produced by Gallionella ferruginea were compared by electron microscopy and elemental analysis. The thick- and fine-fiber stalks were different in structure on a micrometer scale and in the site on the mother cell to which they were attached, but on a nanometer scale they were similar in ultrastructure and in the elemental composition of their basic fiber matrix.

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