Akira Hosoyama

Whole genome sequencing of meticillin-resistant Staphylococcus aureus

BACKGROUND Staphylococcus aureus is one of the major causes of community-acquired and hospital-acquired infections. It produces numerous toxins including superantigens that cause unique disease entities such as toxic-shock syndrome and staphylococcal scarlet fever, and has acquired resistance to practically all antibiotics. Whole genome analysis is a necessary step towards future development of countermeasures against this organism. METHODS Whole genome sequences of two related S aureus strains (N315 and Mu50) were determined by shot-gun random sequencing. N315 is a meticillin-resistant S aureus (MRSA) strain isolated in 1982, and Mu50 is an MRSA strain with vancomycin resistance isolated in 1997. The open reading frames were identified by use of GAMBLER and GLIMMER programs, and annotation of each was done with a BLAST homology search, motif analysis, and protein localisation prediction. FINDINGS The Staphylococcus genome was composed of a complex mixture of genes, many of which seem to have been acquired by lateral gene transfer. Most of the antibiotic resistance genes were carried either by plasmids or by mobile genetic elements including a unique resistance island. Three classes of new pathogenicity islands were identified in the genome: a toxic-shock-syndrome toxin island family, exotoxin islands, and enterotoxin islands. In the latter two pathogenicity islands, clusters of exotoxin and enterotoxin genes were found closely linked with other gene clusters encoding putative pathogenic factors. The analysis also identified 70 candidates for new virulence factors. INTERPRETATION The remarkable ability of S aureus to acquire useful genes from various organisms was revealed through the observation of genome complexity and evidence of lateral gene transfer. Repeated duplication of genes encoding superantigens explains why S aureus is capable of infecting humans of diverse genetic backgrounds, eliciting severe immune reactions. Investigation of many newly identified gene products, including the 70 putative virulence factors, will greatly improve our understanding of the biology of staphylococci and the processes of infectious diseases caused by S aureus.

Genome sequencing and analysis of Aspergillus oryzae

The genome of Aspergillus oryzae, a fungus important for the production of traditional fermented foods and beverages in Japan, has been sequenced. The ability to secrete large amounts of proteins and the development of a transformation system have facilitated the use of A. oryzae in modern biotechnology. Although both A. oryzae and Aspergillus flavus belong to the section Flavi of the subgenus Circumdati of Aspergillus, A. oryzae, unlike A. flavus, does not produce aflatoxin, and its long history of use in the food industry has proved its safety. Here we show that the 37-megabase (Mb) genome of A. oryzae contains 12,074 genes and is expanded by 7–9 Mb in comparison with the genomes of Aspergillus nidulans and Aspergillus fumigatus. Comparison of the three aspergilli species revealed the presence of syntenic blocks and A. oryzae-specific blocks (lacking synteny with A. nidulans and A. fumigatus) in a mosaic manner throughout the genome of A. oryzae. The blocks of A. oryzae-specific sequence are enriched for genes involved in metabolism, particularly those for the synthesis of secondary metabolites. Specific expansion of genes for secretory hydrolytic enzymes, amino acid metabolism and amino acid/sugar uptake transporters supports the idea that A. oryzae is an ideal microorganism for fermentation.

Whole-genome analyses reveal genetic instability of Acetobacter pasteurianus

Acetobacter species have been used for brewing traditional vinegar and are known to have genetic instability. To clarify the mutability, Acetobacter pasteurianus NBRC 3283, which forms a multi-phenotype cell complex, was subjected to genome DNA sequencing. The genome analysis revealed that there are more than 280 transposons and five genes with hyper-mutable tandem repeats as common features in the genome consisting of a 2.9-Mb chromosome and six plasmids. There were three single nucleotide mutations and five transposon insertions in 32 isolates from the cell complex. The A. pasteurianus hyper-mutability was applied for breeding a temperature-resistant strain grown at an unviable high-temperature (42°C). The genomic DNA sequence of a heritable mutant showing temperature resistance was analyzed by mutation mapping, illustrating that a 92-kb deletion and three single nucleotide mutations occurred in the genome during the adaptation. Alpha-proteobacteria including A. pasteurianus consists of many intracellular symbionts and parasites, and their genomes show increased evolution rates and intensive genome reduction. However, A. pasteurianus is assumed to be a free-living bacterium, it may have the potentiality to evolve to fit in natural niches of seasonal fruits and flowers with other organisms, such as yeasts and lactic acid bacteria.

Whole-Genome Sequencing of Sake Yeast Saccharomyces cerevisiae Kyokai no. 7

The term ‘sake yeast’ is generally used to indicate the Saccharomyces cerevisiae strains that possess characteristics distinct from others including the laboratory strain S288C and are well suited for sake brewery. Here, we report the draft whole-genome shotgun sequence of a commonly used diploid sake yeast strain, Kyokai no. 7 (K7). The assembled sequence of K7 was nearly identical to that of the S288C, except for several subtelomeric polymorphisms and two large inversions in K7. A survey of heterozygous bases between the homologous chromosomes revealed the presence of mosaic-like uneven distribution of heterozygosity in K7. The distribution patterns appeared to have resulted from repeated losses of heterozygosity in the ancestral lineage of K7. Analysis of genes revealed the presence of both K7-acquired and K7-lost genes, in addition to numerous others with segmentations and terminal discrepancies in comparison with those of S288C. The distribution of Ty element also largely differed in the two strains. Interestingly, two regions in chromosomes I and VII of S288C have apparently been replaced by Ty elements in K7. Sequence comparisons suggest that these gene conversions were caused by cDNA-mediated recombination of Ty elements. The present study advances our understanding of the functional and evolutionary genomics of the sake yeast.

Genetic basis of the highly efficient yeast Kluyveromyces marxianus: complete genome sequence and transcriptome analyses.

BackgroundHigh-temperature fermentation technology with thermotolerant microbes has been expected to reduce the cost of bioconversion of cellulosic biomass to fuels or chemicals. Thermotolerant Kluyveromyces marxianus possesses intrinsic abilities to ferment and assimilate a wide variety of substrates including xylose and to efficiently produce proteins. These capabilities have been found to exceed those of the traditional ethanol producer Saccharomyces cerevisiae or lignocellulose-bioconvertible ethanologenic Scheffersomyces stipitis.ResultsThe complete genome sequence of K. marxianus DMKU 3-1042 as one of the most thermotolerant strains in the same species has been determined. A comparison of its genomic information with those of other yeasts and transcriptome analysis revealed that the yeast bears beneficial properties of temperature resistance, wide-range bioconversion ability, and production of recombinant proteins. The transcriptome analysis clarified distinctive metabolic pathways under three different growth conditions, static culture, high temperature, and xylose medium, in comparison to the control condition of glucose medium under a shaking condition at 30°C. Interestingly, the yeast appears to overcome the issue of reactive oxygen species, which tend to accumulate under all three conditions.ConclusionsThis study reveals many gene resources for the ability to assimilate various sugars in addition to species-specific genes in K. marxianus, and the molecular basis of its attractive traits for industrial applications including high-temperature fermentation. Especially, the thermotolerance trait may be achieved by an integrated mechanism consisting of various strategies. Gene resources and transcriptome data of the yeast are particularly useful for fundamental and applied researches for innovative applications.

Complete Genome Sequence of NBRC 3288, a Unique Cellulose-Nonproducing Strain of Gluconacetobacter xylinus Isolated from Vinegar

Gluconacetobacter xylinus is involved in the industrial production of cellulose. We have determined the genome sequence of G. xylinus NBRC 3288, a cellulose-nonproducing strain. Comparative analysis of genomes of G. xylinus NBRC 3288 with those of the cellulose-producing strains clarified the genes important for cellulose production in Gluconacetobacter.

Single-Cell Analyses Revealed Transfer Ranges of IncP-1, IncP-7, and IncP-9 Plasmids in a Soil Bacterial Community

ABSTRACT The conjugative transfer ranges of three different plasmids of the incompatibility groups IncP-1 (pBP136), IncP-7 (pCAR1), and IncP-9 (NAH7) were investigated in soil bacterial communities by culture-dependent and culture-independent methods. Pseudomonas putida, a donor of each plasmid, was mated with soil bacteria, and green fluorescent protein (GFP), encoded on the plasmid, was used as a reporter protein for successful transfer. GFP-expressing transconjugants were detected and separated at the single-cell level by flow cytometry. Each cell was then analyzed by PCR and sequencing of its 16S rRNA gene following either whole-genome amplification or cultivation. A large number of bacteria within the phylum Proteobacteria was identified as transconjugants for pBP136 by both culture-dependent and culture-independent methods. Transconjugants belonging to the phyla Actinobacteria, Bacteroidetes, and Firmicutes were detected only by the culture-independent method. Members of the genus Pseudomonas (class Gammaproteobacteria) were identified as major transconjugants of pCAR1 and NAH7 by both methods, whereas Delftia species (class Betaproteobacteria) were detected only by the culture-independent method. The transconjugants represented a minority of the soil bacteria. Although pCAR1-containing Delftia strains could not be cultivated after a one-to-one filter mating assay between the donor and cultivable Delftia strains as recipients, fluorescence in situ hybridization detected pCAR1-containing Delftia cells, suggesting that Delftia was a “transient” host of pCAR1.

Bacterial Lifestyle in a Deep-sea Hydrothermal Vent Chimney Revealed by the Genome Sequence of the Thermophilic Bacterium Deferribacter desulfuricans SSM1

The complete genome sequence of the thermophilic sulphur-reducing bacterium, Deferribacter desulfuricans SMM1, isolated from a hydrothermal vent chimney has been determined. The genome comprises a single circular chromosome of 2 234 389 bp and a megaplasmid of 308 544 bp. Many genes encoded in the genome are most similar to the genes of sulphur- or sulphate-reducing bacterial species within Deltaproteobacteria. The reconstructed central metabolisms showed a heterotrophic lifestyle primarily driven by C1 to C3 organics, e.g. formate, acetate, and pyruvate, and also suggested that the inability of autotrophy via a reductive tricarboxylic acid cycle may be due to the lack of ATP-dependent citrate lyase. In addition, the genome encodes numerous genes for chemoreceptors, chemotaxis-like systems, and signal transduction machineries. These signalling networks may be linked to this bacteriums versatile energy metabolisms and may provide ecophysiological advantages for D. desulfuricans SSM1 thriving in the physically and chemically fluctuating environments near hydrothermal vents. This is the first genome sequence from the phylum Deferribacteres.

A genome sequence-based approach to taxonomy of the genus Nocardia.

The genus Nocardia includes both pathogens and producers of useful secondary metabolites. Although 16S rRNA analysis is required to accurately discriminate among phylogenetic relationships of the Nocardia species, most branches of 16S rRNA-based phylogenetic trees are not reliable. In this study, we performed in silico analyses of the genome sequences of Nocardia species in order to understand their diversity and classification for their identification and applications. Draft genome sequences of 26 Nocardia strains were determined. Phylogenetic trees were prepared on the basis of multilocus sequence analysis of the concatenated sequences of 12 genes (atpD-dnaJ-groL1-groL2-gyrB-recA-rpoA-secA-secY-sodA-trpB-ychF) and a bidirectional best hit. To elucidate the evolutionary relationships of these genes, the genome-to-genome distance was investigated on the basis of the average nucleotide identity, DNA maximal unique matches index, and genome-to-genome distance calculator. The topologies of all phylogenetic trees were found to be essentially similar to each other. Furthermore, whole genome-derived and multiple gene-derived relationships were found to be suitable for extensive intra-genus assessment of the genus Nocardia.

The Impact of Injections of Different Nutrients on the Bacterial Community and Its Dechlorination Activity in Chloroethene-Contaminated Groundwater

Dehalococcoides spp. are currently the only organisms known to completely reduce cis-1,2-dichloroethene (cis-DCE) and vinyl chloride (VC) to non-toxic ethene. However, the activation of fermenting bacteria that generate acetate, hydrogen, and CO2 is considered necessary to enhance the dechlorination activity of Dehalococcoides and enable the complete dechlorination of chloroethenes. In the present study, we stimulated chloroethene-contaminated groundwater by injecting different nutrients prepared from yeast extract or polylactate ester using a semicontinuous culture system. We then evaluated changes in the bacterial community structure and their relationship with dechlorination activity during the biostimulation. The populations of Dehalococcoides and the phyla Bacteroidetes, Firmicutes, and Spirochaetes increased in the yeast extract-amended cultures and chloroethenes were completely dechlorinated. However, the phylum Proteobacteria was dominant in polylactate ester-amended cultures, in which almost no cis-DCE and VC were dechlorinated. These results provide fundamental information regarding possible interactions among bacterial community members involved in the dechlorination process and support the design of successful biostimulation strategies.