PROCESSING AND METHYLATION OF PULG, A PILIN-LIKE COMPONENT OF THE GENERAL SECRETORY PATHWAY OF KLEBSIELLA-OXYTOCA

The signal sequence of the Klebsiella oxytoca pulG gene product, which is required for extracellular secretion of the enzyme pullulanase, is similar in many respects to the corresponding segment of the precurso rs of type IV (me-Phe) pilins. The significance of this similarity is confirmed by the observation that the pu10 gene product processes preP ulG at the consensus type IV prepilin peptidase cleavage site at the a mino-terminal end of the PulG signal sequence. Like most type IV pilin s, processed PulG was found to have a methylated amino-terminal phenyl alanine residue. Site-directed mutagenesis was used to replace amino a cids in prePulG that correspond to residues shown by others to be esse ntial for processing, methylation and assembly of type IV pilins. The glycine residue on the amino-terminal side of the prePulG cleavage sit e is absolutely required for processing and for pullulanase secretion. The glutamate residue at position 11(+5) is also required for pullula nase secretion but not for processing or methylation. This result cont rasts with that reported for corresponding variants of Pseudomonas aer uginosa type IV prepilin, which were processed but only inefficiently N-methylated. Cleavage of prePulG and pullulanase secretion were both unaffected by replacement of the phenylalanine residue on the carboxy- terminal side of the cleavage site by leucine, isoleucine or valine, b y a conservative substitution within the hydrophobic core of the prePu lG signal sequence, or by a glutamine to proline substitution within t he processed segment. However, replacement of the same glutamine resid ue by arginine abolished secretion without affecting either processing or methylation.