Characterization and use of catabolite-repressed promoters from gluconate genes in Corynebacterium glutamicum.
The genes involved in gluconate catabolism (gntP and gntK) in Corynebacterium glutamicum are scattered in the chromosome, and no regulatory genes are apparently associated with them, in contrast with the organization of the gnt operon in Escherichia coli and Bacillus subtilis. In C. glutamicum, gntP and gntK are essential genes when gluconate is the only carbon and energy source. Both genes contain upstream regulatory regions consisting of a typical promoter and a hypothetical cyclic AMP (cAMP) receptor protein (CRP) binding region but lack the expected consensus operator region for binding of the GntR repressor protein. Expression analysis by Northern blotting showed monocistronic transcripts for both genes. The expression of gntP and gntK is not induced by gluconate, and the gnt genes are subject to catabolite repression by sugars, such as glucose, fructose, and sucrose, as was detected by quantitative reverse transcription-PCR (qRT-PCR). Specific analysis of the DNA promoter sequences (PgntK and PgntP) was performed using bifunctional promoter probe vectors containing mel (involved in melanin production) or egfp2 (encoding a green fluorescent protein derivative) as the reporter gene. Using this approach, we obtained results parallel to those from qRT-PCR. An applied example of in vivo gene expression modulation of the divIVA gene in C. glutamicum is shown, corroborating the possible use of the gnt promoters to control gene expression. glxR (which encodes GlxR, the hypothetical CRP protein) was subcloned from the C. glutamicum chromosomal DNA and overexpressed in corynebacteria; we found that the level of gnt expression was slightly decreased compared to that of the control strains. The purified GlxR protein was used in gel shift mobility assays, and a specific interaction of GlxR with sequences present on PgntP and PgntK fragments was detected only in the presence of cAMP.
Letek
Michal
M
Area de Microbiología, Dpto. Ecología, Genética y Microbiología, Universidad de León, 24071 León, Spain.
Valbuena
Noelia
N
Ramos
Angelina
A
Ordóñez
Efrén
E
Gil
José A
JA
Mateos
Luís M
LM
D015152:Blotting, Northern; D018797:Cell Cycle Proteins; D002455:Cell Division / Q000235:genetics; D048230:Corynebacterium glutamicum / Q000201:enzymology / Q000235:genetics*; D002373:Cyclic AMP Receptor Protein / Q000235:genetics; D029968:Escherichia coli Proteins / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial / Q000235:genetics*; D005942:Gluconates / Q000378:metabolism*; D026901:Membrane Transport Proteins / Q000235:genetics; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000235:genetics; D011401:Promoter Regions, Genetic; D012097:Repressor Proteins / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013379:Substrate Specificity
D018797:Cell Cycle Proteins; D002373:Cyclic AMP Receptor Protein; D029968:Escherichia coli Proteins; D005942:Gluconates; D026901:Membrane Transport Proteins; D012097:Repressor Proteins; C104633:gluconate permease, E coli; D017853:Phosphotransferases (Alcohol Group Acceptor); C020578:gluconokinase; C030691:gluconic acid