The CcpA protein is necessary for efficient sporulation and enterotoxin gene (cpe) regulation in Clostridium perfringens.
Clostridium perfringens is the cause of several human diseases, including gas gangrene (clostridial myonecrosis), enteritis necroticans, antibiotic-associated diarrhea, and acute food poisoning. The symptoms of antibiotic-associated diarrhea and acute food poisoning are due to sporulation-dependent production of C. perfringens enterotoxin encoded by the cpe gene. Glucose is a catabolite repressor of sporulation by C. perfringens. In order to identify the mechanism of catabolite repression by glucose, a mutation was introduced into the ccpA gene of C. perfringens by conjugational transfer of a nonreplicating plasmid into C. perfringens, which led to inactivation of the ccpA gene by homologous recombination. CcpA is a transcriptional regulator known to mediate catabolite repression in a number of low-G+C-content gram-positive bacteria, of which C. perfringens is a member. The ccpA mutant strain sporulated at a 60-fold lower efficiency than the wild-type strain in the absence of glucose. In the presence of 5 mM glucose, sporulation was repressed about 2,000-fold in the wild-type strain and 800-fold in the ccpA mutant strain compared to sporulation levels for the same strains grown in the absence of glucose. Therefore, while CcpA is necessary for efficient sporulation in C. perfringens, glucose-mediated catabolite repression of sporulation is not due to the activity of CcpA. Transcription of the cpe gene was measured in the wild-type and ccpA mutant strains grown in sporulation medium by using a cpe-gusA fusion (gusA is an Escherichia coli gene encoding the enzyme beta-glucuronidase). In the exponential growth phase, cpe transcription was two times higher in the ccpA mutant strain than in the wild-type strain. Transcription of cpe was highly induced during the entry into stationary phase in wild-type cells but was not induced in the ccpA mutant strain. Glucose repressed cpe transcription in both the wild-type and ccpA mutant strain. Therefore, CcpA appears to act as a repressor of cpe transcription in exponential growth but is required for efficient sporulation and cpe transcription upon entry into stationary phase. CcpA was also required for maximum synthesis of collagenase (kappa toxin) and acted as a repressor of polysaccharide capsule synthesis in the presence of glucose, but it did not regulate synthesis of the phospholipase PLC (alpha toxin).
Department of Biology, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061, USA.
John
J
Varga
Veronica L
VL
Stirewalt
Stephen B
SB
Melville
D000222:Adaptation, Physiological; D019677:Artificial Gene Fusion; D016667:Bacterial Capsules / Q000096:biosynthesis / Q000235:genetics; D001426:Bacterial Proteins / Q000235:genetics / Q000502:physiology*; D001427:Bacterial Toxins / Q000096:biosynthesis / Q000235:genetics; D002135:Calcium-Binding Proteins / Q000096:biosynthesis / Q000235:genetics; D003016:Clostridium perfringens / Q000235:genetics / Q000502:physiology*; D004269:DNA, Bacterial / Q000737:chemistry / Q000302:isolation & purification; D004268:DNA-Binding Proteins / Q000235:genetics / Q000502:physiology*; D004768:Enterotoxins / Q000096:biosynthesis* / Q000235:genetics; D017353:Gene Deletion; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D017930:Genes, Reporter; D005947:Glucose / Q000378:metabolism; D005966:Glucuronidase / Q000235:genetics / Q000378:metabolism; D003012:Microbial Collagenase / Q000096:biosynthesis / Q000235:genetics; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D009154:Mutation / Q000235:genetics / Q000502:physiology; D011135:Polysaccharides, Bacterial / Q000096:biosynthesis / Q000235:genetics; D012097:Repressor Proteins / Q000235:genetics / Q000502:physiology*; D017422:Sequence Analysis, DNA; D013171:Spores, Bacterial / Q000235:genetics / Q000502:physiology; D014158:Transcription, Genetic; D010738:Type C Phospholipases / Q000096:biosynthesis / Q000235:genetics
D001426:Bacterial Proteins; D001427:Bacterial Toxins; D002135:Calcium-Binding Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D004768:Enterotoxins; D011135:Polysaccharides, Bacterial; D012097:Repressor Proteins; C069286:catabolite control proteins, bacteria; C035598:enterotoxin, Clostridium; D010738:Type C Phospholipases; C040649:alpha toxin, Clostridium perfringens; D005966:Glucuronidase; D003012:Microbial Collagenase; C460361:kappa-toxin, Clostridium perfringens; D005947:Glucose