Catabolite repression of the propionate catabolic genes in Escherichia coli and Salmonella enterica: evidence for involvement of the cyclic AMP receptor protein

Abstract

Previous studies with Salmonella enterica serovar Typhimurium LT2 demonstrated that transcriptional activation of the prpBCDE operon requires the function of transcription factor PrpR, sigma-54, and IHF. In this study, we found that transcription from the prpBCDE and prpR promoters was down-regulated by the addition of glucose or glycerol, indicating that these genes may be regulated by the cyclic AMP (cAMP)-cAMP receptor protein (CRP) complex. Targeted mutagenesis of a putative CRP-binding site in the promoter region between prpR and prpBCDE suggested that these genes are under the control of CRP. Furthermore, cells with defects in cya or crp exhibited reduced transcriptional activation of prpR and prpBCDE in Escherichia coli. These results demonstrate that propionate metabolism is subject to catabolite repression by the global transcriptional regulator CRP and that this regulation is effected through control of both the regulator gene prpR and the prpBCDE operon itself. The unique properties of the regulation of these two divergent promoters may have important implications for mechanisms of CRP-dependent catabolite repression acting in conjunction with a member of the sigma-54 family of transcriptional activators.

FIG. 1.

Nucleotide sequence of the prpR-prpBCDE bidirectional promoter region in E. coli and S. enterica (A). On the basis of previous work (32), a σ⁷⁰ promoter for prpR, a consensus σ⁵⁴ binding region 5′ to prpBCDE, and two ribosome-binding sites (RBS) are underlined and labeled in the promoter region between the two transcriptional units. The proposed ATG start sites for PrpR and PrpB are boxed. Putative CRP-binding sequences are identified, shaded, and labeled. An inverted repeat (GTTTCAT-10 nt-ATGAAAC), which may be a PrpR-binding site for activation of the prpBCDE promoter, is in italics. Nucleotides in the region between the two genes are numbered 5′ to 3′ on the basis of the E. coli sequence. Putative binding sites for regulator proteins are shown (B). The inferred −10 and −35 region and −12 and −24 region of each promoter are indicated. Reporter plasmids were constructed by fusion of the prpBCDE promoter to the gene encoding RFP and/or the prpR promoter to the gene encoding LacZ.

FIG. 2.

Regulation of the prpBCDE and prpR promoters by glucose (Glc) or glycerol (Gly) in E. coli JSW1 and S. enterica TR6583. Strains JSW1 (A, C) and TR6583 (B, D) harboring the dual P_prpBCDE-rfp/P_prpR-lacZ reporter plasmid pAPLPR were grown in LB medium plus the indicated carbon sources at 0.2 or 0.4% and in the absence of propionate (black bars) or in the presence of 10 mM propionate (hatched bars). RFP fluorescence per unit of OD₆₀₀ (A and B; prpBCDE promoter) and β-galactosidase activity (C and D; prpR promoter) were measured.

FIG. 3.

Regulation of the prpBCDE promoter by glucose or glycerol in cells expressing PrpR. E. coli JSW1 strains harboring the P_prpBCDE-rfp reporter with no extra copy of prpR (pZBR, black bars) or the P_prpBCDE-rfp reporter and prpR under the control of Pzt1 (pZBRR, hatched bars) were grown in LB medium plus 0.4% glucose (Glc) or glycerol (Gly) with 10 mM propionate (Prop) or 5 ng of TC per ml.

FIG. 4.

Effects of crp and cya mutations on the prpBCDE and prpR promoters. Strains harboring pAPF8 (P_prpBCDE-rfp) (A) or pAPR8 (P_prpR-lacZ) (B, C) were grown in LB medium with or without 10 mM propionate. RFP fluorescence per unit of OD₆₀₀ (A) and β-galactosidase activity (B, C) were measured.

FIG. 5.

Effects of crp and cya mutations on the prpBCDE promoter in cells expressing PrpR in trans. E. coli strains JSW1, JSW2, and JSW3 harboring P_prpBCDE-rfp with prpR under the control of Pzt1 (pZBRR) were grown in LB medium with or without 10 mM propionate and without TC. RFP fluorescence per unit of OD₆₀₀ was measured.

FIG. 6.

Effects of mutations in the putative CRP-binding site on P_prpBCDE-rfp (A) and P_prpR-lacZ (B) expression in strain JSW1. Strains harboring pAPF8/8# (P_prpBCDE-rfp) or pAPR8/8# (P_prpR-lacZ) were grown in LB medium with or without 10 mM propionate. The site-directed mutations within the putative cAMP-CRP binding site are shown in panel C. The mutation sites are underlined below the wild-type binding sequences. RFP fluorescence per unit of OD₆₀₀ (A) and β-galactosidase activity (B) were measured.

FIG. 7.

Effects of upstream deletions on prpBCDE expression. E. coli W3110 strains harboring one of the plasmids ranging from pAPF1 to pAPF8 were grown in LB medium with 10 mM propionate. After 30 h of incubation, RFP fluorescence per unit of OD₆₀₀ was measured. pAP is an empty vector. The results are averages from two independent experiments.