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Cloning, genetic characterization, and nucleotide sequence of the hemA-prfA operon of Salmonella typhimurium.

ABSTRACT: The first step in heme biosynthesis is the formation of 5-aminolevulinic acid (ALA). Mutations in two genes, hemA and hemL, result in auxotrophy for ALA in Salmonella typhimurium, but the roles played by these genes and the mechanism of ALA synthesis are not understood. I have cloned and sequenced the S. typhimurium hemA gene. The predicted polypeptide sequence for the HemA protein shows no similarity to known ALA synthases, and no ALA synthase activity was detected in extracts prepared from strains carrying the cloned hemA gene. Genetic analysis, DNA sequencing of amber mutations, and maxicell studies proved that the open reading frame identified in the DNA sequence encodes HemA. Another surprising finding of this study is that hemA lies directly upstream of prfA, which encodes peptide chain release factor 1 (RF-1). A hemA::Kan insertion mutation, constructed in vitro, was transferred to the chromosome and used to show that these two genes form an operon. The hemA gene ends with an amber codon, recognized by RF-1. I suggest a model for autogenous control of prfA expression by translation reinitiation.

SUBMITTER: Elliott T

PROVIDER: S-EPMC210147 | biostudies-other | 1989 Jul

REPOSITORIES: biostudies-other

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Cloning, genetic characterization, and nucleotide sequence of the hemA-prfA operon of Salmonella typhimurium.

Elliott T T

Journal of bacteriology 19890701 7

The first step in heme biosynthesis is the formation of 5-aminolevulinic acid (ALA). Mutations in two genes, hemA and hemL, result in auxotrophy for ALA in Salmonella typhimurium, but the roles played by these genes and the mechanism of ALA synthesis are not understood. I have cloned and sequenced the S. typhimurium hemA gene. The predicted polypeptide sequence for the HemA protein shows no similarity to known ALA synthases, and no ALA synthase activity was detected in extracts prepared from str ...[more]

PMID: 2544564

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Project description:In Escherichia coli, efficient mutagenesis by UV requires the umuDC operon. A deficiency in umuDC activity is believed to be responsible for the relatively weak UV mutability of Salmonella typhimurium LT2 compared with that of E. coli. To begin evaluating this hypothesis and the evolutionary relationships among umuDC-related sequences, we cloned and sequenced the S. typhimurium umuDC operon. S. typhimurium umuDC restored mutability to umuD and umuC mutants of E. coli. DNA sequence analysis of 2,497 base pairs (bp) identified two nonoverlapping open reading frames spanning 1,691 bp that were were 67 and 72% identical at the nucleotide sequence level to the umuD and umuC sequences, respectively, from E. coli. The sequences encoded proteins whose deduced primary structures were 73 and 84% identical to the E. coli umuD and umuC gene products, respectively. The two bacterial umuDC sequences were more similar to each other than to mucAB, a plasmid-borne umuDC homolog. The umuD product retained the Cys-24--Gly-25, Ser-60, and Lys-97 amino acid residues believed to be critical for RecA-mediated proteolytic activation of UmuD. The presence of a LexA box 17 bp upstream from the UmuD initiation codon suggests that this operon is a member of an SOS regulon. Mu d-P22 inserts were used to locate the S. typhimurium umuDC operon to a region between 35.9 and 40 min on the S. typhimurium chromosome. In E. coli, umuDC is located at 26 min. The umuDC locus in S. typhimurium thus appears to be near one end of a chromosomal inversion that distinguishes gene order in the 25- to 35-min regions of the E. coli and S. typhimurium chromosomes. It is likely, therefore, that the umuDC operon was present in a common ancestor before S. typhimurium and E. coli diverged approximately 150 million years ago. These results provide new information for investigating the structure, function, and evolutionary origins of umuDC and for exploring the genetic basis for the mutability differences between S. typhimurium and E. coli.

Dataset Information

Cloning, genetic characterization, and nucleotide sequence of the hemA-prfA operon of Salmonella typhimurium.

Publications

Cloning, genetic characterization, and nucleotide sequence of the hemA-prfA operon of Salmonella typhimurium.

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