Characterization and Use of Catabolite-Repressed Promoters from Gluconate Genes in Corynebacterium glutamicum

Abstract

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.

Effect of carbon source availability and growth phase on expression of Corynebacterium glutamicum genes involved in the tricarboxylic acid cycle and glyoxylate bypass

“…Treating His-tagged GlxR with EnterokinaseMax (Invitrogen) to remove the His tag had no effect on binding (data not shown). In agreement with previous reports (Kim et al, 2004;Letek et al, 2006), the presence of retarded bands was directly dependent on the presence of cAMP in the reaction mixture. No retarded bands were observed in the absence of cAMP or His-tagged GlxR (data not shown).…”

“…The transcriptional regulator GlxR contains a domain with similarity to the cAMP-binding motifs of Crp from E. coli and, in fact, cAMP is essential for binding of GlxR to the aceA/aceB intergenic region in C. glutamicum (Kim et al, 2004;Letek et al, 2006). The regulation of C. glutamicum metabolism in the presence of various carbon sources clearly differs from that of E. coli or B. subtilis (Gerstmeir et al, 2003;Hayashi et al, 2002;Letek et al, 2006;Muffler et al, 2002). No direct evidence has been found of a CCR system in C. glutamicum (Bruckner & Titgemeyer, 2002;Gerstmeir et al, 2003;Han et al, 2007), although a CCR-like phenomenon has been reported from cells grown on medium containing glucose with either glutamate (Gerstmeir et al, 2003;Kramer et al, 1990;Kronemeyer et al, 1995) or ethanol (Arndt & Eikmanns, 2007;Kotrbova-Kozak et al, 2007).…”

“…Expression of Escherichia coli aconitase genes is also subject to CCR mediated by Crp (Cunningham et al, 1997;Gruer & Guest, 1994;Gruer et al, 1997). The transcriptional regulator GlxR contains a domain with similarity to the cAMP-binding motifs of Crp from E. coli and, in fact, cAMP is essential for binding of GlxR to the aceA/aceB intergenic region in C. glutamicum (Kim et al, 2004;Letek et al, 2006). The regulation of C. glutamicum metabolism in the presence of various carbon sources clearly differs from that of E. coli or B. subtilis (Gerstmeir et al, 2003;Hayashi et al, 2002;Letek et al, 2006;Muffler et al, 2002).…”

“…The genes required for the utilization of a specific carbon source are, in most cases, expressed only if that carbon source is present in the medium and if preferred carbon sources, such as acetate and glucose, are absent from the growth medium. These two processes in C. glutamicum are referred to as substrate induction and carbon catabolite repression (CCR), respectively (Letek et al, 2006). CCR is an environment-sensing mechanism used by bacteria to establish priorities in carbon metabolism.…”

Efflux Permease CgAcr3-1 of Corynebacterium glutamicum Is an Arsenite-specific Antiporter

“…Heterologous and homologous complementation analyses of Cgacr3s expressed under their own promoters were performed either in E. coli AW3110 (corynebacterial promoters are frequently functional in E. coli) (26,28), using the pKars1up derivative vectors pKacr3-1, pKacr3-2, and pKacr3-3, or in C. glutamicum 2⌬ars, in the latter case using integrative suicide (pKacr3-1, pKacr3-2, and pKacr3-3) or bifunctional (pECacr3-1, pECacr3-2, and pECacr3-3) vectors. Integrative pKars1up derivatives were incorporated into a 0.5-kb chromosomal region located at the 5Ј-upstream region of the ars1 C. glutamicum operon by means of an identical 0.5-kb fragment present in all of the pKars1up derivatives.…”