Yoshiyuki Ohtsubo

Complete Genome Sequence of Acidovorax sp. Strain KKS102, a Polychlorinated-Biphenyl Degrader

We report the complete genome sequence of Acidovorax sp. strain KKS102, a polychlorinated-biphenyl-degrading strain isolated from a soil sample in Tokyo. The genome contains a single circular 5,196,935-bp chromosome and no plasmids.

Design and Experimental Application of a Novel Non-Degenerate Universal Primer Set that Amplifies Prokaryotic 16S rRNA Genes with a Low Possibility to Amplify Eukaryotic rRNA Genes

The deep sequencing of 16S rRNA genes amplified by universal primers has revolutionized our understanding of microbial communities by allowing the characterization of the diversity of the uncultured majority. However, some universal primers also amplify eukaryotic rRNA genes, leading to a decrease in the efficiency of sequencing of prokaryotic 16S rRNA genes with possible mischaracterization of the diversity in the microbial community. In this study, we compared 16S rRNA gene sequences from genome-sequenced strains and identified candidates for non-degenerate universal primers that could be used for the amplification of prokaryotic 16S rRNA genes. The 50 identified candidates were investigated to calculate their coverage for prokaryotic and eukaryotic rRNA genes, including those from uncultured taxa and eukaryotic organelles, and a novel universal primer set, 342F-806R, covering many prokaryotic, but not eukaryotic, rRNA genes was identified. This primer set was validated by the amplification of 16S rRNA genes from a soil metagenomic sample and subsequent pyrosequencing using the Roche 454 platform. The same sample was also used for pyrosequencing of the amplicons by employing a commonly used primer set, 338F-533R, and for shotgun metagenomic sequencing using the Illumina platform. Our comparison of the taxonomic compositions inferred by the three sequencing experiments indicated that the non-degenerate 342F-806R primer set can characterize the taxonomic composition of the microbial community without substantial bias, and is highly expected to be applicable to the analysis of a wide variety of microbial communities.

Conjugal Transfer of Polychlorinated Biphenyl/Biphenyl Degradation Genes in Acidovorax sp. Strain KKS102, Which Are Located on an Integrative and Conjugative Element

A polychlorinated biphenyl (PCB)/biphenyl degradation gene cluster in Acidovorax sp. strain KKS102, which is very similar to that in Tn4371 from Cupriavidus oxalaticus A5, was transferred to several proteobacterial strains by conjugation. The mobilized DNA fragment consisted of 61,807 bp and carried genes for mating-pair formation (mpf), DNA transfer (dtr), integrase (int), and replication-partition proteins (rep-parAB). In the transconjugants, transferred DNA was integrated at ATTGCATCAG or similar sequences. The circular-form integrative and conjugative element (ICE) was detected by PCR, and quantitative PCR analyses revealed that, in KKS102 cells, the ratio of the circular form to the integrated form was very low (approximately 10(-5)). The circular form was not detected in a mutant of the int gene, which was located at the extreme left and transcribed in the inward direction, and the level of int transcriptional activity was much higher in the circular form than in the integrated form. These findings clearly demonstrated that the genes for PCB/biphenyl degradation in KKS102 cells are located on an ICE, which was named ICE(KKS102)4677. Comparisons of similar ICE-like elements collected from the public database suggested that those of beta- and gammaproteobacteria were distinguishable from other ICE-like elements, including those in alphaproteobacteria, with respect to the gene composition and gene organization.

Complete Genome Sequence of the γ-Hexachlorocyclohexane-Degrading Bacterium Sphingomonas sp. Strain MM-1

ABSTRACT γ-Hexachlorocyclohexane (γ-HCH) is a man-made chlorinated insecticide that has caused serious environmental problems. Here, we report the complete genome sequence of the γ-HCH-degrading bacterium Sphingomonas sp. strain MM-1, which consists of one chromosome and five plasmids. All the specific lin genes that are almost identical to those of Sphingobium japonicum UT26 for the conversion of γ-HCH to β-ketoadipate are dispersed on four out of the five plasmids.

Time-series metagenomic analysis reveals robustness of soil microbiome against chemical disturbance.

Soil microbial communities have great potential for bioremediation of recalcitrant aromatic compounds. However, it is unclear which taxa and genes in the communities, and how they contribute to the bioremediation in the polluted soils. To get clues about this fundamental question here, time-course (up to 24 weeks) metagenomic analysis of microbial community in a closed soil microcosm artificially polluted with four aromatic compounds, including phenanthrene, was conducted to investigate the changes in the community structures and gene pools. The pollution led to drastic changes in the community structures and the gene sets for pollutant degradation. Complete degradation of phenanthrene was strongly suggested to occur by the syntrophic metabolism by Mycobacterium and the most proliferating genus, Burkholderia. The community structure at Week 24 (∼12 weeks after disappearance of the pollutants) returned to the structure similar to that before pollution. Our time-course metagenomic analysis of phage genes strongly suggested the involvement of the ‘kill-the-winner’ phenomenon (i.e. phage predation of Burkholderia cells) for the returning of the microbial community structure. The pollution resulted in a decrease in taxonomic diversity and a drastic increase in diversity of gene pools in the communities, showing the functional redundancy and robustness of the communities against chemical disturbance.

Bacterial clade with the ribosomal RNA operon on a small plasmid rather than the chromosome

Significance In bacterial genomes, chromosomes are distinguished from plasmids by the localization of essential genes. It has been taken for granted that fundamental genes such as the rRNA (rrn) operon should be transmitted faithfully on the chromosome. Here, we found a striking exception: A plant-associated bacterium, Aureimonas sp. AU20, and its close relatives harbor the rrn operon only on a small, high-copy-number replicon but not on the chromosome. Our findings show the existence of novel genome organization in bacteria. rRNA is essential for life because of its functional importance in protein synthesis. The rRNA (rrn) operon encoding 16S, 23S, and 5S rRNAs is located on the “main” chromosome in all bacteria documented to date and is frequently used as a marker of chromosomes. Here, our genome analysis of a plant-associated alphaproteobacterium, Aureimonas sp. AU20, indicates that this strain has its sole rrn operon on a small (9.4 kb), high-copy-number replicon. We designated this unusual replicon carrying the rrn operon on the background of an rrn-lacking chromosome (RLC) as the rrn-plasmid. Four of 12 strains close to AU20 also had this RLC/rrn-plasmid organization. Phylogenetic analysis showed that those strains having the RLC/rrn-plasmid organization represented one clade within the genus Aureimonas. Our finding introduces a previously unaddressed viewpoint into studies of genetics, genomics, and evolution in microbiology and biology in general.

Isolation of oxygenase genes for indigo-forming activity from an artificially polluted soil metagenome by functional screening using Pseudomonas putida strains as hosts

Metagenomes contain the DNA from many microorganisms, both culturable and non-culturable, and are a potential resource of novel genes. In this study, a 5.2-Gb metagenomic DNA library was constructed from a soil sample (artificially polluted with four aromatic compounds, i.e., biphenyl, phenanthrene, carbazole, and 3-chlorobenzoate) in Escherichia coli by using a broad-host-range cosmid vector. The resultant library was introduced into naphthalene-degrading Pseudomonas putida-derived strains having deficiencies in their naphthalene dioxygenase components, and indigo-forming clones on the indole-containing agar plates were screened. Cosmids isolated from 29 positive clones were classified by their various properties (original screening hosts, hosts showing indigo-forming activity, and digestion patterns with restriction enzymes), and six representative cosmids were chosen. Sequencing and in vitro transposon mutagenesis of the six cosmids resulted in the identification of genes encoding putative class B and D flavoprotein monooxygenases, a multicomponent hydroxylase, and a reductase that were responsible for the indigo-forming activity in the host cells. Among them, the genes encoding the multicomponent hydroxylase were demonstrated to be involved in phenol degradation. Furthermore, two genes encoding ring-cleavage dioxygenases were also found adjacent to the genes responsible for the indigo formation, and their functions were experimentally confirmed.

Properties and biotechnological applications of natural and engineered haloalkane dehalogenases.

Haloalkane dehalogenases (HLDs) convert halogenated compounds to corresponding alcohols, halides, and protons. They belong to α/β-hydrolases, and their principal catalytic mechanism is SN2 nucleophilic substitution followed by the addition of water. Since HLDs generally have broad and different substrate specificities, they have various biotechnological applications. HLDs have previously been believed to be present only in bacterial strains that utilize xenobiotic halogenated compounds, and three archetypal HLDs, i.e., DhlA, DhaA, and LinB, have been intensively investigated by biochemical, structural, and computational analyses. Furthermore, by using the resulting data and target-selected random mutagenesis approaches, these HLDs have been successfully engineered to improve their substrate specificities and activities. In addition, important insights into protein evolution have been obtained by studying these HLDs. At the same time, the genome and metagenome information has revealed that HLD homologues are widely distributed in many bacterial strains, including ones that have not been reported to degrade halogenated compounds. Some of these cryptic HLD homologues have been experimentally confirmed to be “true” HLDs with unique substrate specificities and enantioselectivities. Although their biological functions and physiological roles remain mysterious, these potential HLDs are considered promising materials for the development of new biocatalysts.

Cointegrate-resolution of toluene-catabolic transposon Tn4651: Determination of crossover site and the segment required for full resolution activity

Tn3-family transposon Tn4651 from Pseudomonas putida mt-2 plasmid pWW0 carries two divergently transcribed genes, tnpS and tnpT, for cointegrate-resolution. While tnpS encodes a tyrosine recombinase, tnpT encodes a protein that shows no homology to any other characterized protein. The Tn4651 resolution site was previously mapped within the 203-bp fragment that covered the tnpS and tnpT promoter region. To better understand the molecular mechanisms underlying the Tn4651 cointegrate-resolution, we determined the extent of the functional resolution site (designated the rst site) of Tn4651 and the location of the crossover site for the cointegrate-resolution. Deletion analysis of the rst region localized the fully functional rst site to a 136-bp segment. The analysis of the site-specific recombination between Tn4651 rst and a rst variant from the Tn4651-related transposon, Tn4661, indicated that the crossover occurs in the 33-bp inverted repeat region, which separates the 136-bp functional rst site into the tnpS- and tnpT-proximal segments. Electrophoretic mobility shift assays demonstrated specific binding of TnpT to the 20-bp inverted repeat region in the tnpT-proximal segment. The requirement for accessory sequences on both sides of the crossover site and the involvement of the unique DNA-binding protein TnpT suggest that the Tn4651-specified resolution system uses a different mechanism than other known resolution systems. Furthermore, comparative sequence analysis for Tn4651-related transposons revealed the occurrence of DNA exchange at the rst site among different transposons, suggesting an additional role of the TnpS-TnpT-rst system in the evolution of Tn4651-related transposons.

Complete Genome Sequence of a Propionibacterium acnes Isolate from a Sarcoidosis Patient

ABSTRACT Propionibacterium acnes is a human skin commensal that resides preferentially within sebaceous follicles and is the only microorganism that has been isolated from sarcoid lesions. We report the complete genome sequence of P. acnes, which was isolated from a Japanese patient with sarcoidosis.