An enzyme with type IV prepilin peptidase activity is required to process components of the general extracellular protein secretion pathway of Klebsiella oxytoca.
The last gene (pulO) of the pulC-O pullulanase secretion gene operon of Klebsiella oxytoca codes for a protein that is 52% identical to the product of the pilD/xcpA gene required for extracellular protein secretion and type IV pilus biogenesis in Pseudomonas aeruginosa. The PilD/XcpA protein is known to remove the first six amino acids of the signal sequence of the type IV pilin precursor by cleaving after the glycine residue in the conserved sequence GF(M)XXXE (where X represents hydrophobic amino acids). This prepilin peptidase cleavage site is present in the products of four genes in the pulC-O operon (PulG, PulH, Pull and PulJ proteins). It is shown here that PulO processes the pulG gene product in vivo. Processing was maximal within 15 seconds, but experiments in which the expression of pulO was uncoupled from that of the other genes in the secretion operon suggest that processing can also occur post-translationally. The products of two pulG derivatives with internal inframe deletions were also processed by PulO, but the three PulG-PhoA hybrids, two PulJ-PhoA hybrids and the single PulH-PhoA hybrid tested did not appear to be processed. Sucrose gradient fraction experiments showed that both precursor and mature forms of PulG appear to be associated with low-density, outer membrane vesicles prepared by osmotic lysis of sphaeroplasts. Neither the xcpA gene nor the Bacillus subtilis gene comC, which is also homologous to pulO and codes for a protein with type IV prepilin peptidase activity, can correct the pullulanase secretion defect in an Escherichia coli strain carrying all of the genes required for secretion except pulO. Furthermore, neither XcpA nor ComC is able to process prePulG protein in vivo.
Pugsley
A P
AP
Unité de Génétique Moléculaire, CNRS UA 1149, Institut Pasteur, Paris, France.
Dupuy
B
B
D000595:Amino Acid Sequence; D001425:Bacterial Outer Membrane Proteins / Q000378:metabolism*; D001426:Bacterial Proteins / Q000378:metabolism; D010450:Endopeptidases / Q000235:genetics / Q000378:metabolism*; D036781:Fimbriae Proteins; D005798:Genes, Bacterial; D005816:Genetic Complementation Test; D006026:Glycoside Hydrolases / Q000235:genetics / Q000378:metabolism*; D007709:Klebsiella / Q000201:enzymology*; D008565:Membrane Proteins / Q000378:metabolism; D008969:Molecular Sequence Data; D011498:Protein Precursors / Q000378:metabolism*; D011499:Protein Processing, Post-Translational
D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D008565:Membrane Proteins; D011498:Protein Precursors; D036781:Fimbriae Proteins; D006026:Glycoside Hydrolases; C100181:pullulanase; D010450:Endopeptidases; C074273:type IV prepilin peptidase