Masahira Hattori

Complete genome sequence of Clostridium perfringens, an anaerobic flesh-eater

Clostridium perfringens is a Gram-positive anaerobic spore-forming bacterium that causes life-threatening gas gangrene and mild enterotoxaemia in humans, although it colonizes as normal intestinal flora of humans and animals. The organism is known to produce a variety of toxins and enzymes that are responsible for the severe myonecrotic lesions. Here we report the complete 3,031,430-bp sequence of C. perfringens strain 13 that comprises 2,660 protein coding regions and 10 rRNA genes, showing pronounced low overall G + C content (28.6%). The genome contains typical anaerobic fermentation enzymes leading to gas production but no enzymes for the tricarboxylic acid cycle or respiratory chain. Various saccharolytic enzymes were found, but many enzymes for amino acid biosynthesis were lacking in the genome. Twenty genes were newly identified as putative virulence factors of C. perfringens, and we found a total of five hyaluronidase genes that will also contribute to virulence. The genome analysis also proved an efficient method for finding four members of the two-component VirR/VirS regulon that coordinately regulates the pathogenicity of C. perfringens. Clearly, C. perfringens obtains various essential materials from the host by producing several degradative enzymes and toxins, resulting in massive destruction of the host tissues.

Genomic and proteomic characterization of the large Myoviridae bacteriophage ϕTMA of the extreme thermophile Thermus thermophilus

A lytic phage, designated as ϕTMA, was isolated from a Japanese hot spring using Thermus thermophilus HB27 as an indicator strain. Electron microscopic examination showed that ϕTMA had an icosahedral head and a contractile tail. The circular double-stranded DNA sequence of ϕTMA was 151,483 bp in length, and its organization was essentially same as that of ϕYS40 except that the ϕTMA genome contained genes for a pair of transposase and resolvase, and a gene for a serine to asparagine substituted ortholog of the protein involved in the initiation of the ϕYS40 genomic DNA synthesis. The different host specificities of ϕTMA and ϕYS40 could be explained by the sequence differences in the C-terminal regions of their distal tail fiber proteins. The ΔpilA knockout strains of T. thermophilus showed simultaneous loss of sensitivity to their cognate phages, pilus structure, twitching motility and competence for natural transformation, thus suggesting that the phage infection required the intact host pili. Pulsed-field gel electrophoresis analysis of the ϕTMA and ϕYS40 genomes revealed that the length of their DNA exceeded 200 kb, indicating that the terminal redundancy is more than 30% of the closed circular form. Proteomic analysis of the ϕTMA virion using a combination of N-terminal sequencing and mass spectrometric analysis of peptide fragments suggested that the maturation of several proteins involved in the phage assembly process was mediated by a trypsin-like protease. The gene order of the phage structural proteins was also discussed.