Complementation of a Delta ccpA mutant of Lactobacillus casei with CcpA mutants affected in the DNA- and cofactor-binding domains.
In low-G+C Gram-positive bacteria, the regulatory protein CcpA has been shown to play a major part in the so-called carbon catabolite repression (CCR) process, as well as in the induction of basic metabolic genes, for which it is considered a global regulator. A strain of Lactobacillus casei that carried a complete deletion of ccpA has been constructed and used to test the effect of CCR on N-acetylglucosaminidase activity and growth performance of a collection of seven CcpA mutations obtained by site-directed mutagenesis. The replaced amino acids were located in the DNA- and cofactor (P-Ser-HPr)-binding domains. Mutations in the DNA-binding domain lacked CCR, as found in Bacillus megaterium. However, mutations in the cofactor-binding domain of L. casei CcpA had a different phenotype to that observed in the previous studies with B. megaterium. Two of them, S80L and T307I, displayed a significant hyper-repression, an effect never reported before for CcpA. Comparison of growth capabilities provided by the different mutants and their ability to sustain CCR demonstrated that CCR, at least on the enzymic activity tested, and the growth defect caused by the CcpA mutations are unrelated features.
Departamento de Biotecnología, Instituto de Agroquímica y Tecnología de Alimentos (IATA-CSIC), Polígono de la Coma s/n, Apartado de Correos (PO Box) 73, 46100-Burjassot, Valencia, Spain.
Carlos D
CD
Esteban
Lehrstuhl für Mikrobiologie, Friedrich-Alexander-Universität Erlangen-Nürnberg, 91058 Erlangen, Germany.
Kerstin
K
Mahr
Departamento de Biotecnología, Instituto de Agroquímica y Tecnología de Alimentos (IATA-CSIC), Polígono de la Coma s/n, Apartado de Correos (PO Box) 73, 46100-Burjassot, Valencia, Spain.
Vicente
V
Monedero
Lehrstuhl für Mikrobiologie, Friedrich-Alexander-Universität Erlangen-Nürnberg, 91058 Erlangen, Germany.
Wolfgang
W
Hillen
Departamento de Biotecnología, Instituto de Agroquímica y Tecnología de Alimentos (IATA-CSIC), Polígono de la Coma s/n, Apartado de Correos (PO Box) 73, 46100-Burjassot, Valencia, Spain.
Gaspar
G
Pérez-Martínez
Lehrstuhl für Mikrobiologie, Friedrich-Alexander-Universität Erlangen-Nürnberg, 91058 Erlangen, Germany.
Fritz
F
Titgemeyer
D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D017353:Gene Deletion; D005798:Genes, Bacterial; D005816:Genetic Complementation Test; D007780:Lacticaseibacillus casei / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D017434:Protein Structure, Tertiary; D012097:Repressor Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D012097:Repressor Proteins; C069286:catabolite control proteins, bacteria