title: A second prepilin peptidase gene in Escherichia coli K-12.
abstract: Escherichia coli K-12 strains grown at 37 degrees C or 42 degrees C, but not at 30 degrees C, process the precursors of the Neisseria gonorrhoeae type IV pilin PilE and the Klebsiella oxytoca type IV pseudopilin PulG in a manner reminiscent of the prepilin peptidase-dependent processing of these proteins that occurs in these bacteria. Processing of prePulG in Escherichia coli requires a glycine at position -1, as does processing by the cognate prepilin peptidase (PulO), and is unaffected by mutations that inactivate several non-specific proteases. These data suggested that E. coli K-12 has a functional prepilin peptidase, despite the fact that it does not itself appear to express either type IV pilin or pseudopilin genes under the conditions that allow prePilE and prePulG processing. The E. coli K-12 genome contains two genes encoding proteins with significant sequence similarity to prepilin peptidases: gspO at minute 74.5 and pppA (f310c) at minute 67 on the genetic map. We have previously obtained evidence that gspO encodes an active enzyme but is not transcribed. pppA was cloned and shown to code for a functional prepilin peptidase capable of processing typical prepilin peptidase substrates. Inactivation of pppA eliminated the endogenous, thermoinducible prepilin peptidase activity. PppA was able to replace PulO prepilin peptidase in a pullulanase secretion system reconstituted in E. coli when expressed from high-copy-number plasmids but not when present in a single chromosomal copy. The analysis of pppA-lacZ fusions indicated that pppA expression was very low and regulated by the growth temperature at the level of translation, in agreement with the observed temperature dependence of PppA activity. Polymerase chain reaction and Southern hybridization analyses revealed the presence of the pppA gene in 12 out of 15 E. coli isolates.
authors.affiliation: Unité de Génétique Moléculaire, Institut Pasteur, Paris, France.
authors.forename: O
authors.initials: O
authors.lastname: Francetić
authors.forename: S
authors.initials: S
authors.lastname: Lory
authors.forename: A P
authors.initials: AP
authors.lastname: Pugsley
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism*; D001483:Base Sequence; D010450:Endopeptidases; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics* / Q000378:metabolism; D010861:Fimbriae, Bacterial; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D005816:Genetic Complementation Test; D008969:Molecular Sequence Data; D009154:Mutation; D011550:Pseudomonas aeruginosa / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
chemical_list: D001426:Bacterial Proteins; D011994:Recombinant Proteins; D010450:Endopeptidases; C067127:prepilin peptidase protein, Bacteria