Peptidase Mutants of Salmonella typhimurium

Abstract

Six peptidase activities have been distinguished electrophoretically in cell extracts of Salmonella typhimurium with the aid of a histochemical stain. The activities can also be partially separated by chromatography on diethylaminoethyl-cellulose. These peptidases show overlapping substrate specificities. Mutants (pepN) of the parent strain leu-485 lacking one of these enzymes (peptidase N) were obtained by screening for colonies that do not hydrolyze the chromogenic substrate L-alanyl-,B-naphthylamide. The absence of this broad-specificity peptidase in leu-485 pepNmutants allowed the selection of mutants unable to use L-leucyl-L-alaninamide as a leucine source. These mutants (leu-485 pepN-pepA-) lack a broad-specificity peptidase (peptidase A) similar to aminopeptidase I previously described in Escherichia coli. Mutants (pepD) lacking a dipeptidase (peptidase D) have been isolated from a leu-485 pepN-pepAparent by penicillin selection for mutants unable to use L-leucyl-L-glycine as a leucine source. Mutants (pepB) lacking a fourth peptidase (peptidase B) have been isolated from a leu-485 pepN-pepA-pepDstrain by penicillin selection for failure to utilize L-leucyl-L-leucine as a source of leucine. Single recombinants were obtained by transduction for each of the peptidases missing in a leu-485 pepN-pepA-pepD-pepB-strain. The growth response of these recombinants to leucine peptides shows that all of these peptidases can function in the catabolism of peptides and that they display overlapping substrate specificities in vivo.

The arginine repressor is essential for plasmid-stabilizing site-specific recombination at the ColE1 cer locus.

“…Since xerAlargR protein is unlikely to be the recombinase acting at cer, is the other chromosomal gene that we have isolated and characterized (xerB) the recombinase gene? Our experiments to date suggest not (unpublished); indeed it is likely that the E. coli xerB gene is equivalent to the pepA gene of Salmonella typhimurium which encodes aminopeptidase A (Miller and MacKinnon, 1974). Recently we have identified a third chromosomal locus (xerC) by a TnS mutation which reduces but does not abolish monomerizing recombination at cer.…”

Molecular cloning and amplification of the gene for aminopeptidase N of Escherichia coli

“…Aminopeptidase N is one of numerous peptidases found in Escherichia coli and Salmonella typhimurium [1][2][3]. In these organisms, it is the only enzyme able to hydrolyse substrates like Lalanine-p-nitroanilide or L-alanine-/~-naphtyl amide, and it accounts for less than 1% of total cell proteins [2].…”

“…L-alanine-fl-naphtylamide and Fast Garnet C, BC were employed as indicated by Miller [1], except that tlae products were dissolved in soft agar.…”

Peptidases and proteases of Escherichia coli and Salmonella typhimurium

“…These studies were rather fragmentary, however, and dealt mainly with the enzymatic specificity of these proteins. In 1974, the situation was improved considerably primarily due to the genetic approach used by Miller et al [66,67,69]. Peptidase-deficient mutants were isolated by means of systematic screening.…”