Propanediol utilization genes (pdu) of Salmonella typhimurium: three genes for the propanediol dehydratase.

The propanediol utilization (pdu) operon of Salmonella typhimurium encodes proteins required for the catabolism of propanediol, including a coenzyme B12-dependent propanediol dehydratase. A clone that expresses propanediol dehydratase activity was isolated from a Salmonella genomic library. DNA sequence analysis showed that the clone included part of the pduF gene, the pduABCDE genes, and a long partial open reading frame (ORF1). The clone included 3.9 kbp of pdu DNA which had not been previously sequenced. Complementation and expression studies with subclones constructed via PCR showed that three genes (pduCDE) are necessary and sufficient for propanediol dehydratase activity. The function of ORF1 was not determined. Analyses showed that the S. typhimurium propanediol dehydratase was related to coenzyme B12-dependent glycerol dehydratases from Citrobacter freundii and Klebsiella pneumoniae. Unexpectedly, the S. typhimurium propanediol dehydratase was found to be 98% identical in amino acid sequence to the Klebsiella oxytoca propanediol dehydratase; this is a much higher identity than expected, given the relationship between these organisms. DNA sequence analyses also supported previous studies indicating that the pdu operon was inherited along with the adjacent cobalamin biosynthesis operon by a single horizontal gene transfer.

Characterization of lip expression in Salmonella typhimurium: analysis of lip::lac operon fusions.

Strains of Salmonella typhimurium which have an auxotrophic requirement for lipoic acid were isolated by mutagenesis with the transposable element Mu dJ. The chromosomal location of these insertion mutations was determined to be at 14 map units by bacteriophage P22-mediated cotransduction. The lip gene is transcribed in the clockwise direction relative to the S. typhimurium genetic map. Strains with lip::lac operon fusions were used to characterize the transcriptional activity of the lip promoter. Transcription of the lip gene is not regulated by catabolite repression or lipoic acid concentration. The data indicate that the lip gene product is expressed constitutively at a low level.

Cobalamin-dependent 1,2-propanediol utilization by Salmonella typhimurium.

The enteric bacterium Salmonella typhimurium utilizes 1,2-propanediol as a sole carbon and energy source during aerobic growth, but only when the cells are also provided with cobalamin as a nutritional supplement. This metabolism is mediated by the cobalamin-dependent propanediol dehydratase enzyme pathway. Thirty-three insertion mutants were isolated that lacked the ability to utilize propanediol, but retained the ability to degrade propionate. This phenotype is consistent with specific blocks in one or more steps of the propanediol dehydratase pathway. Enzyme assays confirmed that propanediol dehydratase activity was absent in some of the mutants. Thus, the affected genes were designated pdu (for defects in propanediol utilization). Seventeen mutants carried pdu::lac operon fusions, and these fusions were induced by propanediol in the culture medium. All of the pdu mutations were located in a single region (41 map units) on the S. typhimurium chromosome between the his (histidine biosynthesis) and branch I cob (cobalamin biosynthesis) operons. They were shown to be P22-cotransducible with a branch I cob marker at a mean frequency of 12%. Mutants that carried deletions of the genetic material between his and cob also failed to utilize propanediol as a sole carbon source. Based upon the formation of duplications and deletions between different pairs of his::MudA and pdu::MudA insertions, the pdu genes were transcribed in a clockwise direction relative to the S. typhimurium genetic map.

Characterization of phosphoenolpyruvate synthase mutants in Salmonella typhimurium.

The enteric bacteria are able to grow by utilizing three-carbon compounds (pyruvate, lactate, and alanine) as sole carbon sources only if they have a functional phosphoenolpyruvate synthase (PEP synthase). PEP synthase catalyzes the phosphorylation of pyruvate to PEP with the hydrolysis of ATP to AMP. This anaplerotic reaction is needed for the synthesis of carbohydrates and citric acid cycle intermediates that are essential for continued cell growth. Insertion mutants were isolated in Salmonella typhimurium that specifically lack the ability to grow on three-carbon compounds. These mutants also fail to utilize acetate as a sole carbon source. Enzyme assays were performed and the results showed that these mutants contain no PEP synthase activity. By using bacteriophage P22, the pps mutations isolated in this study were found to be contransducible with genetic markers in both the aroD and btuC genes. Three-factor crosses pinpointed the order of these genes and their distances with respect to each other. One of the mutants carries a pps::lac operon fusion. This fusion was used to explore the transcriptional regulation of the pps gene. A functional copy of the pps gene is required for its own induction. The pps gene is also under catabolite repression, but the addition of adenosine 3',5'-cyclic monophosphate (cyclic AMP) to cells grown in the presence of glucose does not relieve this repression. These results indicate that the synthesis of PEP synthase is regulated in a more complex manner than has been previously recognized.

Cobalamin (vitamin B12) biosynthetic genes of Salmonella typhimurium.

The enteric bacterium Salmonella typhimurium synthesizes cobalamin (vitamin B12) de novo only under anaerobic growth conditions. We initiated a genetic analysis of the cobalamin biosynthetic (cob) gene cluster, Stable cob::lac operon fusions were generated by insertions of a transposition-defective derivative of bacteriophage Mu d1 (Ap lac) into the cob genes. beta-Galactosidase synthesis was repressed in the presence of exogenously supplied cyanocobalamin, demonstrating that the cobalamin biosynthetic pathway was regulated by end-product repression. Transcriptional polarity studies showed that the cob genes responsible for synthesis of the corrinoid intermediate cobinamide (branch I of the pathway) were organized into a single operon. Genes for the synthesis of 5,6-dimethylbenzimidazole (branch II) and the final assembly of the complete cobalamin molecule (branch III) were organized into two or more additional operons. All of the known cob genes (in branches I, II, and III) were transcribed in a counterclockwise direction relative to the S. typhimurium genetic map. These genes are located at 41 map units and near the his operon. No essential genes lie between the his and cob operons. Mutants that carried deletions extending from the his genes into the cob region were isolated and characterized. By using these mutants, a deletion map of the branch I cob operon was constructed and the order of genes (his-cobI-cobIII-cobII) was inferred.

Inability of Pseudomonas stutzeri denitrification mutants with the phenotype of Pseudomonas aeruginosa to grow in nitrous oxide.

Pseudomonas aeruginosa PAO1 reduced nitrous oxide to dinitrogen but did not grow anaerobically in nitrous oxide. Two transposon insertion Nos- mutants of Pseudomonas stutzeri exhibited the P. aeruginosa phenotype. Growth yield studies demonstrated that nitrous oxide produced in vivo was productively respired, but nitrous oxide supplied exogenously was not. The defect may be in electron transport or in nitrous oxide uptake.

Salmonella typhimurium synthesizes cobalamin (vitamin B12) de novo under anaerobic growth conditions.

In this paper, we report that the enteric bacterium Salmonella typhimurium synthesized cobalamin de novo under anaerobic culture conditions. Aerobically, metE mutants of S. typhimurium need either methionine or cobalamin as a nutritional supplement for growth. The growth response to cobalamin depends upon a cobalamin-requiring enzyme, encoded by the gene metH, that catalyzes the same reaction as the metE enzyme. Anaerobically, metE mutants grew without any nutritional supplements; the metH enzyme functioned under these conditions due to the endogenous biosynthesis of cobalamin. This conclusion was confirmed by using a radiochemical assay to measure cobalamin production. Insertion mutants defective in cobalamin biosynthesis (designated cob) were isolated in the three major branches of the cobalamin biosynthetic pathway. Type I mutations blocked the synthesis of cobinamide, type II mutations blocked the synthesis of 5,6-dimethylbenzimidazole, and type III mutations blocked the synthesis of cobalamin from cobinamide and 5,6-dimethylbanzimidazole. Mutants that did not synthesize siroheme (cysG) were blocked in cobalamin synthesis. Genetic mapping experiments showed that the cob mutations are clustered in the region of the S. typhimurium chromosome between supD (40 map units) and his (42 map units). The discovery that S. typhimurium synthesizes cobalamin de novo only under anaerobic conditions raises the possibility that anaerobically grown cells possess a variety of enzymes which are dependent upon cobalamin as a cofactor.

Isolation and characterization of mutant Pseudomonas aeruginosa strains unable to assimilate nitrate.

Single-site mutants of Pseudomonas aeruginosa that lack the ability aerobically to assimilate nitrate and nitrite as sole sources of nitrogen have been isolated. Twenty-one of these have been subdivided into four groups by transductional analysis. Mutants in only one group, designated nis, lost assimilatory nitrite reductase activity. Mutants in the other three transductional groups, designated ntmA, ntmB, ntmC, display a pleiotropic phenotype: utilization of a number of nitrogen-containing compounds including nitrite as sole nitrogen sources is impaired. Assimilatory nitrite reductase was shown to be the major route by which hydroxylamine is reduced in aerobically-grown cells.

Chromosomal location and function of genes affecting Pseudomonas aeruginosa nitrate assimilation.

Seven known genes control Pseudomonas aeruginosa nitrate assimilation. Three of the genes, designated nas, are required for the synthesis of assimilatory nitrate reductase: nasC encodes a structural component of the enzyme; nasA and nasB encode products that participate in the biosynthesis of the molybdenum cofactor of the enzyme. A fourth gene (nis) is required for the synthesis of assimilatory nitrite reductase. The remaining three genes (ntmA, ntmB, and ntmC) control the assimilation of a number of nitrogen sources. The nas genes and two ntm genes have been located on the chromosome and are well separated from the known nar genes which encode synthesis of dissimilatory nitrate reductase. Our data support the previous conclusion that P. aeruginosa has two distinct nitrate reductase systems, one for the assimilation of nitrate and one for its dissimilation.