Development and Application of a Method for Counterselectable In-Frame Deletion in Clostridium perfringens

• Published in: Applied and Environmental Microbiology 2011-02, Vol.77 (4), p.1375-1382
• Main Authors: Nariya, Hirofumi, Miyata, Shigeru, Suzuki, Motoo, Tamai, Eiji, Okabe, Akinobu
• Format: Article
• Language: English
• Subjects: Background levels; Bacteria; Bacterial Proteins - biosynthesis; Bacterial Proteins - genetics; Bacterial Toxins - biosynthesis; Bacterial Toxins - genetics; Base Sequence; Biological and medical sciences; Blotting, Northern; Calcium-Binding Proteins - biosynthesis; Calcium-Binding Proteins - genetics; Cloning; Clostridium perfringens; Clostridium perfringens - enzymology; Clostridium perfringens - genetics; Clostridium perfringens - metabolism; Clostridium perfringens - pathogenicity; Electrophoresis, Polyacrylamide Gel; Fundamental and applied biological sciences. Psychology; Genes; Genomes; Hemolysin Proteins - biosynthesis; Hemolysin Proteins - genetics; Methods; Microbial Collagenase - biosynthesis; Microbial Collagenase - genetics; Microbiology; Mutagenesis; Plasmids; Proteins; Reading Frames; Recombinant Proteins - biosynthesis; Recombinant Proteins - metabolism; Sequence Analysis, Protein; Sequence Deletion; Toxins; Type C Phospholipases - biosynthesis; Type C Phospholipases - genetics; Virulence - genetics; Virulence Factors - genetics; Virulence Factors - metabolism
• ISSN: 0099-2240; 1098-5336; 1098-6596
• DOI: 10.1128/AEM.01572-10

Many pathogenic clostridial species produce toxins and enzymes. To facilitate genome-wide identification of virulence factors and biotechnological application of their useful products, we have developed a markerless in-frame deletion method for Clostridium perfringens which allows efficient counterselection and multiple-gene disruption. The system comprises a galKT gene disruptant and a suicide galK plasmid into which two fragments of a target gene for in-frame deletion are cloned. The system was shown to be accurate and simple by using it to disrupt the alpha-toxin gene of the organism. It was also used to construct of two different virulence-attenuated strains, HN1303 and HN1314: the former is a disruptant of the virRS operon, which regulates the expression of virulence factors, and the latter is a disruptant of the six genes encoding the α, θ, and κ toxins; a clostripain-like protease; a 190-kDa secretory protein; and a putative cell wall lytic endopeptidase. Comparison of the two disruptants in terms of growth ability and the background levels of secreted proteins showed that HN1314 is more useful than HN1303 as a host for the large-scale production of recombinant proteins.