Molecular cloning and characterization of a proline iminopeptidase gene from Neisseria gonorrhoeae.
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 promotor. 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.
Albertson
N H
NH
Department of Microbiology and Immunology, University of Michigan Medical School, Ann Arbor 48109-0620.
Koomey
M
M
D000595:Amino Acid Sequence; D000626:Aminopeptidases / Q000096:biosynthesis* / Q000235:genetics* / Q000302:isolation & purification; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009344:Neisseria gonorrhoeae / Q000201:enzymology* / Q000235:genetics*; D011401:Promoter Regions, Genetic; D014176:Protein Biosynthesis; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D015183:Restriction Mapping; D014158:Transcription, Genetic
D017931:DNA Primers; D011994:Recombinant Proteins; D000626:Aminopeptidases; C022450:prolyl aminopeptidase