Tohru Shimizu

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.

The luxS gene is involved in cell–cell signalling for toxin production in Clostridium perfringens

A Gram‐positive anaerobic pathogen, Clostridium perfringens, causes clostridial myonecrosis or gas gangrene in humans by producing numerous extracellular toxins and enzymes that act in concert to degrade host tissues. C. perfringens possesses a homologue of the luxS gene that is reported to be responsible for the production of autoinducer 2 (AI‐2), which participates in quorum sensing in bacteria. The luxS mutant was constructed using C. perfringens strain 13, and the role of the luxS gene in toxin production was examined. The cell‐free culture supernatant from wild‐type strain 13 greatly stimulated the luminescence of Vibrio harveyi BB170, whereas that from the luxS mutant caused no significant stimulation, indicating that the luxS gene is necessary for AI‐2 production in C. perfringens. The luxS mutant showed a reduced level of production of alpha‐, kappa‐ and theta‐toxins. In the luxS mutant, the transcription of the theta‐toxin gene (pfoA) was lower at mid‐exponential growth phase, whereas alpha‐ and kappa‐toxin gene transcription was not significantly affected. The production of toxins in the luxS mutant was stimulated by the addition of the culture supernatant from the wild‐type cells, possibly because of the presence of AI‐2. Moreover, the expression of the pfoA gene in the luxS mutant was apparently activated when the mutant cells were cultured in the presence of culture supernatants from the wild‐type C. perfringens, Escherichia coli DH5α carrying the luxS gene of C. perfringens. A deletion analysis of the luxS operon showed that the luxS gene alone is responsible for cell–cell signalling, and that the metB or cysK genes located upstream of luxS are not involved in regulating toxin production. Our results indicate that cell–cell signalling by AI‐2 plays an important role in the regulation of toxin production in C. perfringens.

Identification of novel VirR/VirS-regulated genes in Clostridium perfringens.

Novel genes that are regulated in Clostridium perfringens by the two‐component regulatory system, VirR/VirS, were identified using a differential display method. A plasmid library was constructed from C. perfringens chromosomal DNA, and the plasmids were hybridized with cDNA probes prepared from total RNA of wild‐type strain 13 and its virR mutant derivative TS133. Three clones were identified that carry newly identified VirR/VirS‐regulated genes, two of which were positively regulated and one of which was negatively regulated. Genes located on the identified clones were deduced by nucleotide sequencing, and the target genes of the VirR/VirS system were identified with a set of Northern hybridizations. A 4.9u2003kb mRNA transcribing the metB (cystathionine gamma‐synthase), cysK (cysteine synthase) and ygaG (hypothetical protein) genes was negatively regulated, whereas 1.6 and 6.0u2003kb transcripts encoding ptp (protein tyrosine phosphatase) and cpd (2′,3′‐cyclic nucleotide 2′‐phosphodiesterase) respectively, were shown to be positively regulated by the VirR/VirS system. The other gene, hyp7, whose transcript was positively regulated by the VirR/VirS system, was shown to activate the transcription of the colA (kappa‐toxin) and plc (alpha‐toxin) genes, but not the pfoA (theta‐toxin) gene in C. perfringens. These results suggested that the global regulatory system VirR/VirS could regulate various genes, other than toxin genes, both positively and negatively and that the hyp7 gene might encode a novel regulatory factor for toxin production in C. perfringens.

The VirR/VirS regulatory cascade affects transcription of plasmid-encoded putative virulence genes in Clostridium perfringens strain 13

We analyzed the transcriptional regulation of the putative virulence genes encoded on the plasmid pCP13 from Clostridium perfringens strain 13. The transcription of the beta2-toxin (cpb2) and possible collagen adhesin (cna) genes were regulated in both a positive and negative manner, respectively, by the two-component VirR/VirS system. The secondary regulator of the VirR/VirS system, VR-RNA, also affects the expression of both of these genes in the same fashion as the VirR/VirS system. This indicates that the global regulatory cascade of the VirR/VirS system controls the expression of virulence genes located on the plasmid, as well as those found chromosomally in C. perfringens strain 13.

Sequence heterogeneity of the ten rRNA operons in Clostridium perfringens.

We have cloned and sequenced rRNA operons of Clostridium perfringens strain 13 and analyzed the sequence structure in view of the phylogenesis. The organism had ten copies of rRNA operons all of that comprised of 16S, 23S and 5S rDNAs except for one operon. The operons clustered around the origin of replication, ranging within one-third of the whole genome sequence as it is arranged in a circle. Seven operons were transcribed in clockwise direction, and the remaining three were transcribed in counter clockwise direction assuming that the gyrA was transcribed in clockwise direction. Two of the counter clockwise operons contained tRNA(Ile) genes between the 16S and 23S rDNAs, and the other had a tRNA(Ile) genes between the 16S and 23S rDNAs and a tRNA(Asn) gene in the place of the 5S rDNA. Microheterogeneity was found within the rRNA structural genes and spacer regions. The length of each 16S, 23S and 5S rDNA were almost identical among the ten operons, however, the intergenic spacer region of 16S-23S and 23S-5S were variable in the length depending on loci of the rRNA operons on the chromosome. Nucleotide sequences of the helix 19, helix 19a, helix 20 and helix 21 of 23S rDNA were divergent and the diversity appeared to be correlated with the loci of the rRNA operons on the chromosome.

Genetic analysis of the ycgJ-metB-cysK-ygaG operon negatively regulated by the VirR/VirS system in Clostridium perfringens.

The 5′‐flanking region of the metB‐cysK‐ygaG operon, whose expression is negatively regulated by the VirR/VirS system in C. perfringens, was analyzed. The region contained the ycgJ, mscL, and colA genes encoding a hypothetical protein, a large conductance mechanosensitive channel protein, and kappa‐toxin (collagenase), respectively. Northern analysis revealed that the ycgJ gene was transcribed as a 4.9‐kb operon together with the metB‐cysK‐ygaG genes and that this operon was negatively regulated by the VirR/VirS system. It is indicated that the pfoA (theta‐toxin or perfringolysin O), colA, and ycgJ‐metB‐cysK‐ygaG genes that belong to the VirR/VirS regulon are situated very close together in a 26.5‐kb region of the chromosome, but do not form a pathogenic island.

Genomic map of Clostridium perfringens strain 13.

A physical and genetic map of Clostridium perfringens strain 13 was constructed. C. perfringens strain 13 was found to have a 3.1‐Mb chromosome and a large 50‐kb plasmid, indicating that strain 13 has a relatively small genome among C. perfringens strains. A total of 313 genetic markers were mapped on the chromosome of strain 13. Compared with the physical and genetic map of C. perfringens CPN50, strain 13 had a quite similar genome organization, but with a large deletion (~400 kb) in a particular segment of the chromosome. Among several toxin genes, a beta2 toxin gene that is a novel virulence gene in C. perfringens was found to be located on the 50‐kb plasmid.

Regulation of Toxin Production in Clostridium perfringens

The Gram-positive anaerobic bacterium Clostridium perfringens is widely distributed in nature, especially in soil and the gastrointestinal tracts of humans and animals. C. perfringens causes gas gangrene and food poisoning, and it produces extracellular enzymes and toxins that are thought to act synergistically and contribute to its pathogenesis. A complicated regulatory network of toxin genes has been reported that includes a two-component system for regulatory RNA and cell-cell communication. It is necessary to clarify the global regulatory system of these genes in order to understand and treat the virulence of C. perfringens. We summarize the existing knowledge about the regulatory mechanisms here.

Sequence analysis of flanking regions of the pfoA gene of Clostridium perfringens: beta-galactosidase gene (pbg) is located in the 3'-flanking region.

The 3′‐flanking region of the perfringolysin O (theta‐toxin) gene (pfoA) of Clostridium perfringens was analyzed by chromosome walking. A total of 5,363 bp of the downstream region of the pfoA gene was sequenced and four open reading frames were found. ORF54 and ORF80 were found to be homologous to genes coding for membrane‐bound transporter proteins of other bacteria and the β‐galactosidase gene (bgaB) of Bacillus stearothermophilus, respectively. ORF80 was named the pbg gene. Clones which showed β‐galactosidase activities were selected from a λFIXII genomic library of C. perfringens by blue plaque screening using X‐Gal as a substrate. Four clones whose plaques showed blue appearances were obtained. Two of the four clones hybridized with the pbg probe but the others did not, indicating that there are two distinct β‐galactosidase genes in C. perfringens. The pbg gene was subcloned into pBR322 and was successfully expressed in Escherichia coli, suggesting that the pbg gene codes for a β‐galactosidase of C. perfringens.

Isolation and sequence of rat testis cDNA for a calcium binding polypeptide similar to the regulatory subunit of calcineurin.

We have cloned and sequenced rat testis cDNAs coding for a calcium binding polypeptide similar to calcineurin beta subunit, the Ca(2+)-binding subunit of the Ca2+/calmodulin stimulated protein phosphatase. Rat testis cDNA library was screened with a monoclonal antibody Va1 raised against bovine brain calcineurin beta subunit. The deduced amino acid sequence is similar to that of human brain calcineurin beta subunit with respect to containing four putative calcium binding sites. However, distinct differences were found: 1) The cloned cDNA had six amino acids polypeptide tail at carboxy-terminal which is absent in human brain calcineurin beta subunit. This amino acids tail makes the carboxy-terminal highly hydrophilic in contrast to the human brain beta subunit which is hydrophobic at carboxy-terminal; 2) eleven amino acids at the N terminal of the cloned cDNA were completely different from the corresponding region of the brain calcineurin beta subunit.