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Molecular cloning and characterization of a proline iminopeptidase gene from Neisseria gonorrhoeae
Author(s) -
Albertson N. H.,
Koomey M.
Publication year - 1993
Publication title -
molecular microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.857
H-Index - 247
eISSN - 1365-2958
pISSN - 0950-382X
DOI - 10.1111/j.1365-2958.1993.tb01249.x
Subject(s) - biology , primer extension , microbiology and biotechnology , complementation , neisseria gonorrhoeae , mutant , gene , escherichia coli , genomic library , proline , biochemistry , genetics , peptide sequence , amino acid , nucleotide
Summary Proline iminopeptidase (Pip) is a hydrolase elaborated by virtually all strains of Neisseria gonorrhoeae that selectively removes N ‐terminal proline residues from peptides. Escherichia coli clones expressing the gonococcal gene coding for Pip were identified in a genomic cosmid library using a synthetic colorimetric substrate. Nucleotide sequence determination and analyses of polypeptides detected by coupled in vitro transcription/translation reactions revealed that Pip is a 311‐amino‐acid polypeptide with a M r of 35 kDa and a pi of 5.4. Southern hybridization showed that the pip gene is present in a single copy on the chromosome of N. gonorrhoeae strain MS11 which maps immediately upstream of the previously identified opaA locus. The transcriptional start site of pip in E. coli , determined by primer extension analysis, was characteristic of an NtrA or sigma‐54‐dependent pro‐motor. Complementation of an E. coli mutant deficient in both proline biosynthesis and dipeptide uptake confirmed that Pip is capable of releasing biologically active proline from peptides. Pip expression was found to be non‐essential for in vitro growth of N. gonorrhoeae , based on the viability of a Pip − gonococcal mutant.