title: Development and application of a method for counterselectable in-frame deletion in Clostridium perfringens.
abstract: 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, ΗΝ1303 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 ΗΝ1303 as a host for the large-scale production of recombinant proteins.
authors.affiliation: Department of Microbiology, Faculty of Medicine, Kagawa University, 1750-1 Miki-cho, Kita-gun, Kagawa 761-0793, Japan. microbio@med.kagawa-u.ac.jp
authors.forename: Hirofumi
authors.initials: H
authors.lastname: Nariya
authors.forename: Shigeru
authors.initials: S
authors.lastname: Miyata
authors.forename: Motoo
authors.initials: M
authors.lastname: Suzuki
authors.forename: Eiji
authors.initials: E
authors.lastname: Tamai
authors.forename: Akinobu
authors.initials: A
authors.lastname: Okabe
mesh_terms: D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics; D001427:Bacterial Toxins / Q000096:biosynthesis / Q000235:genetics*; D001483:Base Sequence; D015152:Blotting, Northern; D002135:Calcium-Binding Proteins / Q000096:biosynthesis / Q000235:genetics*; D003016:Clostridium perfringens / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism / Q000472:pathogenicity; D004591:Electrophoresis, Polyacrylamide Gel; D006460:Hemolysin Proteins / Q000096:biosynthesis / Q000235:genetics; D003012:Microbial Collagenase / Q000096:biosynthesis / Q000235:genetics; D016296:Mutagenesis; D010957:Plasmids; D016364:Reading Frames; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D020539:Sequence Analysis, Protein; D017384:Sequence Deletion; D010738:Type C Phospholipases / Q000096:biosynthesis / Q000235:genetics*; D014774:Virulence / Q000235:genetics; D037521:Virulence Factors / Q000235:genetics* / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D001427:Bacterial Toxins; D002135:Calcium-Binding Proteins; D006460:Hemolysin Proteins; D011994:Recombinant Proteins; D037521:Virulence Factors; C020452:Clostridium perfringens theta-toxin; D010738:Type C Phospholipases; C040649:alpha toxin, Clostridium perfringens; D003012:Microbial Collagenase; C460361:kappa-toxin, Clostridium perfringens