pepM is an essential gene in Salmonella typhimurium.

The pepM gene of Salmonella typhimurium codes for a methionine-specific aminopeptidase that removes N-terminal methionine residues from proteins. This gene was inactivated in vitro by the insertion of a DNA fragment coding for kanamycin resistance. The inactivated gene could not replace the wild-type chromosomal pepM gene unless another functional copy was present in the cell. The lethal effect of the pepM insertion was not a result of polarity on any gene downstream, nor was it affected by the presence or absence of other peptidases.

A two-component regulatory system (phoP phoQ) controls Salmonella typhimurium virulence.

We have determined that Salmonella typhimurium strains with mutations in the positive regulatory locus phoP are markedly attenuated in virulence for BALB/c mice. The DNA sequence for the phoP locus indicates that it is composed of two genes present in an operon, termed phoP and phoQ. The deduced amino acid sequence of the phoP and phoQ gene products are highly similar to other members of bacterial two-component transcriptional regulators that respond to environmental stimuli. S. typhimurium strains with transposon insertions that create transcriptional and translational gene fusions that require phoP and phoQ for expression have been isolated and have different chromosomal locations, indicating that this system is a regulon. One of these fusion strains, containing a mutation in a gene termed pagC, has a virulence defect. Other strains, including those containing mutations in the phoN gene, encoding an acid phosphatase, have wild-type virulence. Strains with pagC, phoP, or phoQ mutations have decreased survival in cultured mouse macrophages. When used as live vaccines in mice, strains with phoP or phoQ mutations afford partial protection to subsequent challenge by wild-type S. typhimurium.

Genetic analysis in Salmonella typhimurium with a small collection of randomly spaced insertions of transposon Tn10 delta 16 delta 17.

We report the isolation of a group of 279 Salmonella typhimurium strains carrying randomly spaced insertions of the minitransposon Tn10 delta 16 delta 17 and describe the use of these strains to facilitate genetic analysis. The insertions were isolated initially in individual recombinant lambda clones from a genomic library. Individual insertions were then moved into the S. typhimurium chromosome, where the distribution of insertion sites relative to standard genetic markers was analyzed in a series of transductional crosses. Since a different, randomly chosen clone was used to generate each insertion, the distribution of insertion positions should have been as random as the cloning events leading to the formation of the library. In agreement with this expectation, most S. typhimurium markers tested were cotransducible with one or more of these Tn10 delta 16 delta 17 insertions. We expect that most new mutations will be quickly classified and mapped by determination of the pattern of cotransduction with this set of insertions. This use is illustrated by the analysis of a group of lac operon fusions regulated by anaerobiosis. We also describe several other applications that should make this collection a useful new tool in S. typhimurium genetics.

N-terminal methionine-specific peptidase in Salmonella typhimurium.

Crude extracts of a multiply peptidase-deficient strain of Salmonella typhimurium contain an aminopeptidase that specifically removes N-terminal methionine from peptides. This activity shows pronounced specificity for the peptide's second amino acid. Methionine is removed from peptides with alanine, threonine, or glycine in this position but not when the second amino acid is leucine or methionine. The activity is stimulated by Co2+ and is inhibited by EDTA. Mutations that lead to overproduction (up to 30-fold) of the activity have been obtained by selecting for growth on Met-Gly-Gly as a methionine source. These mutations map at approximately 3 map units, phage P22 cotransducible with leu. The overproducer mutations are dominant to wild type, and duplication of the wild-type allele of the locus leads to a gene dosage effect on peptidase levels. This suggests that the locus of the overproducer mutations may be the structural gene for the peptidase. NaDodSO4/PAGE shows an increased level of a single protein (34 kDa) in the overproducer mutant. This protein is highly enriched in a purified preparation of the peptidase. The specificity of this enzyme suggests that it is involved in the cleavage of methionine from newly synthesized peptide chains. This activity can specifically remove methionine from the N terminus of a completed protein. Treatment of purified, unprocessed (N-terminal methionine) interleukin 1 beta with the purified peptidase results in removal of N-terminal methionine with no additional alterations. N-terminal processing of at least this protein can occur after translation is complete. We propose to call this enzyme peptidase M (methionine-specific aminopeptidase).