Takahiro Kanagawa

Bias and artifacts in multitemplate polymerase chain reactions (PCR).

Polymerase chain reaction (PCR) is often used for the amplification of a mixture of homologous genes. PCR bias and artifact formation can occur in multitemplate PCR, and provide incorrect information on the abundance and diversity of genes. PCR bias and artifact formation occur at a higher rate during the last few cycles of the reaction, and therefore can be avoided by stopping the PCR earlier.

Bacteria Mediate Methylation of Iodine in Marine and Terrestrial Environments

ABSTRACT Methyl iodide (CH3I) plays an important role in the natural iodine cycle and participates in atmospheric ozone destruction. However, the main source of this compound in nature is still unclear. Here we report that a wide variety of bacteria including terrestrial and marine bacteria are capable of methylating the environmental level of iodide (0.1 μM). Of the strains tested, Rhizobium sp. strain MRCD 19 was chosen for further analysis, and it was found that the cell extract catalyzed the methylation of iodide withS-adenosyl-l-methionine as the methyl donor. These results strongly indicate that bacteria contribute to iodine transfer from the terrestrial and marine ecosystems into the atmosphere.

Burkholderia kururiensis sp. nov., a trichloroethylene (TCE)-degrading bacterium isolated from an aquifer polluted with TCE.

A trichloroethylene (TCE)-degrading bacterium was isolated from an aquifer sample collected at a TCE-polluted site in Japan by enriching with phenol as sole carbon source. The isolate, designated strain KP23T, was a Gram-negative, oval-shaped micro-organism. A phylogenetic study based on 16S rRNA gene sequences indicated that strain KP23T should be placed in the genus Burkholderia. Cellular fatty acids of the strain were mainly composed of C16:0, cyclopropanic acid C17:0 and cyclopropanic acid C19:0. Strain KP23T also contained notable amounts of C13:1 and C17:1. The G + C content of total DNA was 64.8 mol%. Strain KP23T oxidized various sugars and sugar alcohols as sole carbon source such as galactose, glucose, mannose, maltose, glycerol, inositol and mannitol. Comparisons of its phenotypic and genotypic characteristics with other known species belonging to the genus Burkholderia suggested that strain KP23T represents a new species in the genus. The name Burkholderia kururiensis is proposed for this species, with strain KP23T as the type strain (= JCM 10599T).

Phylogenetic Analysis of and Oligonucleotide Probe Development for Eikelboom Type 021N Filamentous Bacteria Isolated from Bulking Activated Sludge

ABSTRACT Fifteen filamentous strains, morphologically classified as Eikelboom type 021N bacteria, were isolated from bulking activated sludges. Based on comparative 16S ribosomal DNA (rDNA) sequence analysis, all strains form a monophyletic cluster together with all recognized Thiothrix species (88.3 to 98.7% 16S rDNA sequence similarity) within the gamma-subclass ofProteobacteria. The investigated Eikelboom type 021N isolates were subdivided into three distinct groups (I to III) demonstrating a previously unrecognized genetic diversity hidden behind the uniform morphology of the filaments. For in situ detection of these bacteria, 16S rRNA-targeted oligonucleotide probes specific for the entire Eikelboom type 021N-Thiothrix cluster and the Eikelboom type 021N groups I, II, and III, respectively, were designed, evaluated, and successfully applied in activated sludge.

In situ identification of polyphosphate-accumulating bacteria in activated sludge by dual staining with rRNA-targeted oligonucleotide probes and 4'6-diamidino-2-phenylindol (DAPI) at a polyphosphate-probing concentration

Polyphosphate-accumulating bacteria in activated sludge samples from an anaerobic/aerobic batch-type reactor for enhanced biological phosphate removal (EBPR) were identified by dual staining with ribosomal RNA-targeted oligonucleotide probes and 4′,6-diamidino-2-phenylindol dihydrochloride (DAPI) at a polyphosphate-probing concentration. The staining with the oligonucleotide probes showed that there were four major bacterial groups in the sludge: the beta subclass of Proteobacteria, Gram-positive bacteria with a high G+C content, the alpha subclass of Proteobacteria, and bacteria belonging to the Cytophaga-Flavobacterium cluster of the Cytophaga-Flavobacterium-Bacteroides phylum. By staining with DAPI, many of the Gram-positive bacteria with a high G+C content and the alpha subclass of Proteobacteria fluoresced bright yellow. These bacteria were considered to accumulate a large amount of polyphosphate.

Reevaluation and Reduction of a PCR Bias Caused by Reannealing of Templates

ABSTRACT We reevaluated the bias toward a 1:1 ratio of products in multitemplate PCR used in ecological studies and showed that the template reannealing at the annealing step would not cause the bias; however, the preferential homoduplex formation during temperature decrease from denaturation to annealing step would cause the bias.

Roseateles depolymerans gen. nov., sp. nov., a new bacteriochlorophyll a-containing obligate aerobe belonging to the beta-subclass of the Proteobacteria.

Strains 61AT (T = type strain) and 61B2, the first bacteriochlorophyll (BChl) a-containing obligate aerobes to be classified in the beta-subclass of the Proteobacteria, were isolated from river water. The strains were originally isolated as degraders of poly(hexamethylene carbonate) (PHC). The organisms can utilize PHC and some other biodegradable plastics. The strains grow only under aerobic conditions. Good production of BChl a and caroterioid pigments is achieved on PHC agar plates and an equivalent production is observed under oligotrophic conditions on agar medium. Spectrometric results suggest that BChl a is present in light-harvesting complex I and the photochemical reaction centre. The main carotenoids are spirilloxanthin and its precursors. Analysis of the 16S rRNA gene sequence indicated that the phylogenetic positions of the two strains are similar to each other and that their closest relatives are the genera Rubrivivax, ideonella and Leptothrix with similarities of 96.3, 96.2 and 96.1%, respectively. The cells are motile, straight rods and contain poly-beta-hydroxybutyrate granules. Ubiquinone-8 is the predominant quinone. Vitamins are not required for growth. The G + C content of genomic DNA is 66.2-66.3 mol%. Genetic and phenotypic features suggest that the strains represent a new genus in the beta-subclass which is evenly distant from known genera. Consequently, the name Roseateles depolymerans gen. nov., sp. nov. is proposed for the strains; the type strain of Roseateles depolymerans is strain 61AT (= DSM 11813T).

Photosynthetic Apparatus in Roseateles depolymerans 61A Is Transcriptionally Induced by Carbon Limitation

ABSTRACT Production of a photosynthetic apparatus in Roseateles depolymerans 61A, a recently discovered freshwater β-Proteobacterium showing characteristics of aerobic phototrophic bacteria, was observed when the cells were subjected to a sudden decrease in carbon sources (e.g., when cells grown with 0.1 to 0.4% Casamino Acids were diluted or transferred into medium containing ≤0.04% Casamino Acids). Accumulation of bacteriochlorophyll (BChl) a was observed in the presence of oxygen and was enhanced under semiaerobic conditions (2% oxygen) but was reduced in the presence of light. Similarly to what has been reported regarding some aerobic phototrophic bacteria belonging to the α subclass of the Proteobacteria, viability of the cells in the carbon source-free medium was prolonged under aerobic-light (10 W m−2) conditions, possibly due to photosynthetic energy conversion, but was not prolonged under aerobic-dark conditions. The puf operon, which encodes most of the apoproteins of light-harvesting and reaction center complexes, was sequenced, and the effect of changes in Casamino Acids concentrations, oxygen, and light on its expression was estimated by the accumulation of its mRNA. The expression of the puf operon was induced by the decrease in carbon sources, similarly to what was observed for the accumulation of BChl a under aerobic and semiaerobic conditions (≥0.2% O2), and was reduced in the presence of light. Transcription of the R. depolymerans puf operon is considered to be controlled by changes in carbon nutrients in addition to oxygen tension and light intensity.

Phylogenetic analysis of trichloroethylene-degrading bacteria newly isolated from soil polluted with this contaminant

Five methanotrophs (strains 18-2, EB1, KSWIII, KSPIII and KSPIII) and three aromatic compound oxidizers (strains KP22, KP24 and KT1) were isolated from the natural field polluted with trichloroethylene (TCE). Phylogenetic analysis based on 16S rRNA gene sequence suggested that all of the isolates belonged to the class Proteobacteria. Two of the methanotrophic isolates, strains 18-2 and EB1, were closely related to Methylocystis sp. strain M in the α subclass of Proteobacteria with sequence similarities of 98.2–98.4%, while strains KSWIII, KSPIII and KSPII were akin to Methylomonas methanica in the γ subclass of Proteobacteria with sequence similarities of 97.8–98.1%. The aromatic compounds oxidizers, strains KP22, KP24 and KT1, were assigned to the β subclass of Proteobacteria, and classified as Bordetella sp. (97.2–97.8% sequence similarity to species of the genus Bordetella), Burkholderia cepacia (99.2%) and Ralstonia eutropha (99.4%), respectively. All isolates degraded TCE when cells were grown with the appropriate substrate, i.e., methane, phenol or toluene. Detailed kinetic analyses of their TCE degradation revealed that the rates of degradation (k1) among the isolates were 10–36 ml of TCE/mg of dry cell weight/h, and the transformation capacities (Tc) were 0.01–0.13 mg of TCE/mg of dry cell weight.

Identification of a gene essential for sheathed structure formation in Sphaerotilus natans, a filamentous sheathed bacterium

ABSTRACT Sphaerotilus natans, a filamentous bacterium that causes bulking in activated sludge processes, can assume two distinct morphologies, depending on the substrate concentration for growth; in substrate-rich media it grows as single rod-shaped cells, whereas in substrate-limited media it grows as filaments. To identify genes responsible for sheath formation, we carried out transposon Tn5 mutagenesis. Of the approximately 20,000 mutants obtained, 7 did not form sheathed structures. Sequencing of the Tn5-flanking regions showed that five of the seven Tn5 insertions converged at the same open reading frame, designated sthA. The deduced amino acids encoded by sthA were found to be homologous to glycosyltransferase, which is known to be involved in linking sugars to lipid carriers during bacterial exopolysaccharide biosynthesis. Disruption of the gene of the wild-type strain by inserting a kanamycin resistance gene cassette also resulted in sheathless growth under either type of nutrient condition. These findings indicate that sthA is a crucial component responsible for sheath formation.