Regulation of the central glycolytic genes in Bacillus subtilis: binding of the repressor CggR to its single DNA target sequence is modulated by fructose‐1,6‐bisphosphate

Abstract

SummaryGlycolysis is one of the best and widely conserved general metabolic pathways. Bacillus subtilis enzymes catalysing the central part of glycolysis, gathering the steps of interconversion of the triose phosphates from dihydroxyacetone-phosphate to phosphoenolpyruvate, are encoded by five genes, gapA , pgk , tpi , pgm and eno . They are transcribed in a hexacistronic operon together with cggR , the first cistron, encoding the repressor of this gapA operon. Using deletion analysis, we have localized the CggR operator between the promoter and the first gene of the operon. CggR was purified and used in gel mobility shift assays and DNase I footprinting experiments to delimit its target sequence. Site-directed mutagenesis and in vivo tests demonstrated that it consists of two direct-repeats (CGGGACN 6 TGTCN 4 CGGGACN 6 TG TC). Sequence analysis and transcriptome comparison of a wild-type and a cggR mutant strain strongly suggested that CggR regulates only the gapA operon. The presence of glycolytic carbon sources induces expression of the gapA operon. Genetic experiments allowed us to identify the metabolic steps required for the formation of the CggR effector. In vitro experiments with the suggested candidates allowed us to demonstrate that fructose-1,6-biphosphate (FBP) acts as an inhibitor of CggR DNA-binding activity (10 mM for full inhibition). FBP is thus the major signal for both CcpA-dependent catabolite repression (or activation) and activation of the central glycolytic genes. Genomic sequence comparisons suggest that these results can apply to numerous low-G + + + + C, Grampositive bacterial species.

Carbon Catabolite Control of the Metabolic Network inBacillus subtilis

“…The in vivo concentration of fructose-1,6-bisphosphate (FBP) increases upon active transport of glucose through the PTS, which diminishes CggR binding ability as to its cis-sequence, CggR being unable to function as a repressor. 126) In contrast, an increase in gluconeogenesis is achieved by relief from CcpN repression of gapB and pckA, specific to gluconeogenesis, in the interaction of CcpN with active YqfL (indicated in blue). 127) The CcpC protein represses the expression of the citB and citZCH operons.…”

“…Transcription of the entire gapA operon from the gapA promoter is repressed by CggR, resulting in the gapA-pgk-tpi-pgm-eno transcript due to endonucleolytic cleavage between cggR and gapA, although the pgk-tpi-pgm-eno transcript is constitutively formed from another promoter in the intergenic region between gapA and pgk. 126,136) FBP is an inhibitor of CggR activity. 126) FBP, in the millimolar range, reduces CggR binding to its cis-sequence which is located downstream of the gapA promoter.…”

“…126,136) FBP is an inhibitor of CggR activity. 126) FBP, in the millimolar range, reduces CggR binding to its cis-sequence which is located downstream of the gapA promoter. 137) Hence FBP is a very suitable signal for the regulation of the genes for glycolysis, since its concentration is much higher during the utilization of glycolytic carbon sources (more than 10 mM) than under gluconeogenic conditions.…”

Glyceraldehyde-3-phosphate dehydrogenase of Xanthomonas campestris pv. campestris is required for extracellular polysaccharide production and full virulence

“…Like many bacterial counterparts, the Xcc GAPDH acquits its enzymic activity by using NAD + specifically as a cofactor. However, differing from the observation that the expression of the gap gene is induced by glucose in Streptomyces aureofaciens, E. coli and B. subtilis (Kormanec et al, 1997;Charpentier et al, 1998;Tobisch et al, 1999;Doan & Aymerich, 2003), the Xcc gapA gene seems to be constitutively expressed. We have also demonstrated that inactivation of the Xcc GAPDH results in impairment of bacterial growth and virulence in the host plant, deficiency of the capability to utilize various carbohydrates, and reduction of ATP and EPS.…”

pBaSysBioII: an integrative plasmid generating gfp transcriptional fusions for high-throughput analysis of gene expression in Bacillus subtilis

“…The cggR gene encodes the repressor of the hexacistronic gapA operon, which includes the cggR, gapA, pgk, tpi, pgm and eno genes. As documented by Doan & Aymerich (2003), binding of the CggR repressor to its single DNA target sequence is modulated by fructose 1,6-bisphosphate. Importantly, the cggR and ptsG expression profiles are known to be very similar (Doan & Aymerich, 2003).…”