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

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 Tn10delta16delta17 element in prpB and provide genetic evidence supporting the conclusion that the prpBCDE genes constitute an operon.

[1]  J. Escalante‐Semerena,et al.  cobB function is required for catabolism of propionate in Salmonella typhimurium LT2: evidence for existence of a substitute function for CobB within the 1,2-propanediol utilization (pdu) operon , 1996, Journal of bacteriology.

[2]  D. Rank,et al.  Identification of a new prp locus required for propionate catabolism in Salmonella typhimurium LT2. , 1996, FEMS microbiology letters.

[3]  M. Rondon,et al.  DNA polymerase I function is required for the utilization of ethanolamine, 1,2-propanediol, and propionate by Salmonella typhimurium LT2 , 1995, Journal of bacteriology.

[4]  M. Rondon,et al.  Glutathione is required for maximal transcription of the cobalamin biosynthetic and 1,2-propanediol utilization (cob/pdu) regulon and for the catabolism of ethanolamine, 1,2-propanediol, and propionate in Salmonella typhimurium LT2 , 1995, Journal of bacteriology.

[5]  D. Belin,et al.  Tight regulation, modulation, and high-level expression by vectors containing the arabinose PBAD promoter , 1995, Journal of bacteriology.

[6]  K. Sanderson,et al.  Genetic map of Salmonella typhimurium, edition VIII , 1995, Microbiological reviews.

[7]  S. Suh,et al.  Purification and initial characterization of the ATP:corrinoid adenosyltransferase encoded by the cobA gene of Salmonella typhimurium , 1995, Journal of bacteriology.

[8]  G. O’Toole,et al.  The cobT gene of Salmonella typhimurium encodes the NaMN: 5,6-dimethylbenzimidazole phosphoribosyltransferase responsible for the synthesis of N1-(5-phospho-alpha-D-ribosyl)-5,6-dimethylbenzimidazole, an intermediate in the synthesis of the nucleotide loop of cobalamin , 1994, Journal of bacteriology.

[9]  W. A. Scheffers,et al.  Propionate metabolism in Saccharomyces cerevisiae: implications for the metabolon hypothesis. , 1994, Microbiology.

[10]  M. Merrick,et al.  In a class of its own — the RNA polymerase sigma factor σ;54 (σN) , 1993 .

[11]  G. O’Toole,et al.  cobU-dependent assimilation of nonadenosylated cobinamide in cobA mutants of Salmonella typhimurium , 1993, Journal of bacteriology.

[12]  B. Sumegi,et al.  [13C]propionate oxidation in wild-type and citrate synthase mutant Escherichia coli: evidence for multiple pathways of propionate utilization. , 1993, The Biochemical journal.

[13]  M. Hidaka,et al.  Studies on the biosynthesis of bialaphos (SF-1293). 14. Nucleotide sequence of phosphoenolpyruvate phosphomutase gene isolated from a bialaphos producing organism, Streptomyces hygroscopicus, and its expression in Streptomyces lividans. , 1992, The Journal of antibiotics.

[14]  J. Knowles,et al.  Cloning, overexpression and mechanistic studies of carboxyphosphonoenolpyruvate mutase from Streptomyces hygroscopicus. , 1992, European journal of biochemistry.

[15]  B. Goldman,et al.  Rapid mapping in Salmonella typhimurium with Mud-P22 prophages , 1992, Journal of bacteriology.

[16]  M. G. Johnson,et al.  The CobII and CobIII regions of the cobalamin (vitamin B12) biosynthetic operon of Salmonella typhimurium , 1992, Journal of bacteriology.

[17]  F. de la Cruz,et al.  Construction and properties of a family of pACYC184-derived cloning vectors compatible with pBR322 and its derivatives. , 1991, Gene.

[18]  E. Myers,et al.  Basic local alignment search tool. , 1990, Journal of molecular biology.

[19]  H. Seto,et al.  Carboxyphosphonoenolpyruvate phosphonomutase, a novel enzyme catalyzing C-P bond formation , 1990, Journal of bacteriology.

[20]  E. Wurtele,et al.  Plants contain multiple biotin enzymes: discovery of 3-methylcrotonyl-CoA carboxylase, propionyl-CoA carboxylase and pyruvate carboxylase in the plant kingdom. , 1990, Archives of biochemistry and biophysics.

[21]  R. J. Hartin,et al.  Transformation in restriction-deficient Salmonella typhimurium LT2. , 1989, Journal of general microbiology.

[22]  F. de la Cruz,et al.  pACYC184-derived cloning vectors containing the multiple cloning site and lacZ alpha reporter gene of pUC8/9 and pUC18/19 plasmids. , 1988, Gene.

[23]  A. Fernández-Briera,et al.  A degradation pathway of propionate in Salmonella typhimurium LT-2. , 1988, Biochimie.

[24]  P. Youderian,et al.  Packaging specific segments of the Salmonella chromosome with locked-in Mud-P22 prophages. , 1988, Genetics.

[25]  H. Uchiyama,et al.  Distribution of the Methylcitric Acid Cycle and β-Oxidation Pathway for Propionate Catabolism in Fungi , 1987 .

[26]  G. J. Blomquist,et al.  Metabolism of propionate to acetate in the cockroach Periplaneta americana. , 1985, Archives of biochemistry and biophysics.

[27]  B. Ames,et al.  Localized mutagenesis of any specific small region of the bacterial chromosome. , 1971, Proceedings of the National Academy of Sciences of the United States of America.

[28]  U. K. Laemmli,et al.  Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4 , 1970, Nature.

[29]  H. Reeves,et al.  Alternate pathways of metabolism of short-chain fatty acids. , 1968, Bacteriological reviews.

[30]  Vagelos Pr.  Propionic acid metabolism. IV. Synthesis of malonyl coenzyme A. , 1960 .

[31]  Vagelos Pr,et al.  Propionic acid metabolism. III. beta-Hydroxypropionyl coenzyme A and malonyl semialdehyde coenzyme A, intermediates in propionate oxidation by Clostridium kluyveri. , 1959 .

[32]  G. Ames,et al.  Rapid polymerase chain reaction amplification using intact bacterial cells. , 1991, BioTechniques.

[33]  瀬戸 治男,et al.  Studies on the biosynthesis of bialaphos (SF-1293) 8 purification and characterization of 2-phosphinomethylmalic acid synthase from streptomyces hygroscopicus SF-1293 , 1988 .

[34]  R. Chalmers,et al.  Disorders of propionate and methylmalonate metabolism , 1982 .

[35]  H. Malke.  R. W. Davis, D. Botstein and J. R. Roth, A Manual for Genetic Engineering, Advanced Bacterial Genetics. 251 S., 15 Abb. Cold Spring Harbor 1980. Cold Spring Harbor Laboratory. $ 28.20 , 1981 .

[36]  P. Vagelos.  Propionic acid metabolism. IV. Synthesis of malonyl coenzyme A. , 1960, The Journal of biological chemistry.