A 2ND PREPILIN PEPTIDASE GENE IN ESCHERICHIA-COLI K-12

Escherichia coli K-12 strains grown at 37 degrees C or 42 degrees C, b ut not at 30 degrees C, process the precursors of the Neisseria gonorr hoeae type IV pilin PilE and the Klebsiella oxytoca type IV pseudopili n PulG in a manner reminiscent of the prepilin peptidase-dependent pro cessing of these proteins that occurs in these bacteria. Processing ng of prePulG in Escherichia coli requires a glycine at position -1, as does processing by the cognate prepilin peptidase (PulO), and is unaff ected by mutations that inactivate several non-specific proteases. The se data suggested that Ei coli K-12 has a functional prepilin peptidas e, despite the fact that it does not itself appear to express either t ype IV pilin or pseudopilin genes under the conditions that allow preP ilE and prePulG processing. The E. coli K-12 genome contains two genes encoding proteins with significant sequence similarity to prepilin pe ptidases: gspO at minute 74.5 and pppA (f310c) at minute 67 on the gen etic map. We have previously obtained evidence that gspO encodes an ac tive enzyme but is not transcribed. pppA was cloned and shown to code for a functional prepilin peptidase capable of processing typical prep ilin peptidase substrates. Inactivation of pppA eliminated the endogen ous, thermoinducible prepilin peptidase activity. PppA was able to rep lace PulO prepilin peptidase in a pullulanase secretion system reconst ituted in E. coli when expressed from high-copy-number plasmids but no t 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.