Propionate catabolism in Salmonella typhimurium LT2: two divergently transcribed units comprise the prp locus at 8.5 centisomes, prpR encodes a member of the sigma-54 family of activators, and the prpBCDE genes constitute an operon

Abstract

We present the initial genetic and biochemical characterization of the propionate (prp) locus at 8.5 centisomes of the Salmonella typhimurium LT2 chromosome (T. A. Hammelman et al., FEMS Microbiol. Lett. 137: 233-239, 1996). In this paper, we report the nucleotide sequences of two divergently transcribed transcriptional units. One unit is comprised of the prpR gene (1,626 bp) encoding a member of the sigma-54 family of transcriptional activators; the second unit contains an operon of four genes designated prpB (888 bp), prpC (1,170 bp), prpD (1,452 bp), and prpE (1,923 bp). The heme biosynthetic gene hemB was shown by DNA sequencing to be located immediately downstream of the prpBCDE operon; hemB is divergently transcribed from prpBCDE and is separated from prpE by a 66-bp gap. In addition, we demonstrate the involvement of PrpB, PrpC, and PrpD in propionate catabolism by complementation analysis of mutants using plasmids carrying a single prp gene under the control of the arabinose-responsive P BAD promoter. Expression of prpB to high levels was deleterious to the growth of a prp ؉ strain on minimal medium supplemented with propionate as a carbon and energy source. We also report the cloning and overexpression of prpB, prpC, prpD, and prpE in the T7 system. PrpB, PrpC, PrpD, and PrpE had molecular masses of ca. 32, ca. 44, ca. 53, and ca. 70 kDa, respectively. PrpB showed homology to carboxyphosphonoenolpyruvate phosphonomutase of Streptomyces hygroscopicus and to its homolog in the carnation Dianthus caryophyllus; PrpC was homologous to both archaeal and bacterial citrate synthases; PrpD showed homology to yeast and Bacillus subtilis proteins of unknown function; PrpE showed homology to acetyl coenzyme A synthetases. We identified a sigma-54 (RpoN)-dependent promoter with a consensus RpoN binding site upstream of the initiating methionine codon of prpB, the promoter-proximal gene of the prp operon. Consistent with this finding, an rpoN prp ؉ mutant failed to use propionate as carbon and energy source. Finally, we report the location of MudI1734 elements inserted in prpC or prpD and of a Tn10⌬16⌬17 element in prpB and provide genetic evidence supporting the conclusion that the prpBCDE genes constitute an operon.

The prpE gene of Salmonella typhimurium LT2 encodes propionyl-CoA synthetase

“…the acetate kinase (AckA) and phosphotransacetylase (Pta) pathway, and the acetyl-CoA synthetase (Acs) pathway. In a previous paper, we proposed that PrpE catalysed the synthesis of propionyl-CoA during propionate breakdown (Horswill & Escalante-Semerena, 1997). However, no evidence to support this hypothesis was presented.…”

“…The activation of propionate to propionyl-CoA is the proposed first step of the methylcitric acid cycle and of all other propionate breakdown pathways in bacteria (Horswill & Escalante-Semerena, 1997 ;Textor et a/., 1997). However, no evidence has been reported identiAbbreviations: Ap, ampicillin; Cm, chloramphenicol; Km, kanamycin; Tc, tetracycline.…”

“…Growth of Salmonella typhimurium on propionate as a sole carbon and energy source requires enzymes encoded by the p r p locus (Hammelman et al, 1996;Horswill & Escalante-Semerena, 1997). Five genes, prpRBCDE, make up this locus and four of these genes show DNA and amino acid sequence similarity to proteins with known biochemical activities (Fig.…”

Biochemical evidence that the pduS gene encodes a bifunctional cobalamin reductase

“…Subsequently, propionaldehyde is converted to propanol and propionate by alcohol dehydrogenase, CoA-dependent propionaldehyde dehydrogenase, phosphotransacylase and propionate kinase (Obradors et al, 1988;Toraya et al, 1979). This pathway generates one ATP, an electron sink for the regeneration of NAD and an intermediate (propionyl-CoA) that can serve as a carbon and energy source via the methylcitrate pathway (Horswill & Escalante-Semerena, 1997).…”

“…Subsequently, propionaldehyde is converted to propanol and propionate by alcohol dehydrogenase, CoA-dependent propionaldehyde dehydrogenase, phosphotransacylase and propionate kinase (Obradors et al, 1988;Toraya et al, 1979). This pathway generates one ATP, an electron sink for the regeneration of NAD and an intermediate (propionyl-CoA) that can serve as a carbon and energy source via the methylcitrate pathway (Horswill & Escalante-Semerena, 1997).Although the pathway of 1,2-PD degradation appears quite straightforward, the catabolism of this small molecule is in fact a very elaborate process (Bobik et al, 1999;Havemann et al, 2002;Havemann & Bobik, 2003). Recent studies have shown that a polyhedral body is involved in AdoCbl-dependent 1,2-PD degradation by S. enterica (Bobik et al, 1999;Havemann et al, 2002;Havemann & Bobik, 2003).…”