Analysis of the pilU gene for the prepilin peptidase involved in the biogenesis of type IV pili encoded by plasmid R64.
In many type IV pili, the N-terminal amino acid of the pilin subunit is N-methylated phenylalanine. A prepilin peptidase removes the leader peptide from the precursor and methylates the amino group of the newly formed phenylalanine. PilS, the precursor of the pilin encoded by plasmid R64, is processed by the prepilin peptidase PilU, but the N-terminal amino acid of the mature pilin is a non-methylated tryptophan that is otherwise modified. To study the relationship between the structure and function of PilU, 42 missense pilU mutations were constructed by PCR and site-directed mutagenesis, and the ability of these pilU mutants to complement a pilU null mutant for mating in liquid culture was analyzed. Although practically no conjugation was noted for 21 of the mutants, the remaining 21 supported varying levels of residual plasmid transfer activity. Two mutants with aspartic acid replacements in conserved motifs exhibited no PilU activity, suggesting that the product of the pilU gene is an aspartic acid peptidase, like TcpJ, the prepilin peptidare of Vibrio cholerae. No PilS processing was detected in 21 of the mutants, but the remaining 21 exhibited varying levels of residual PilS processing. A close correlation was noted between residual PilS processing activity and conjugative transfer, suggesting that the pilU gene product possesses prepilin peptidase activity, but is unable to methylate the N-terminal tryptophan. Based on the activity of pilU-phoA and pilU-lacZ fusion genes encoding different segments of PilU, a model for the membrane topology of the protein is also proposed. Furthermore, some amino acid substitutions in the pilU portion of the pilU-phoA and pilU-lacZ fusion genes were found to alter the membrane topology of the product.
Akahane
K
K
Department of Biology, Tokyo Metropolitan University, Minamiohsawa, Hachioji, Tokyo, 192-0397, Japan.
Sakai
D
D
Furuya
N
N
Komano
T
T
D000595:Amino Acid Sequence; D001419:Bacteria / Q000235:genetics*; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D015153:Blotting, Western; D003227:Conjugation, Genetic / Q000235:genetics; D017931:DNA Primers; D010450:Endopeptidases / Q000235:genetics*; D036781:Fimbriae Proteins / Q000235:genetics; D010861:Fimbriae, Bacterial / Q000235:genetics / Q000378:metabolism*; D040461:Gene Components; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000235:genetics; D016133:Polymerase Chain Reaction; D011815:R Factors / Q000235:genetics*; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism
D001426:Bacterial Proteins; D017931:DNA Primers; C091957:PilU protein, Pseudomonas aeruginosa; D014157:Transcription Factors; C080687:pilS protein, Bacteria; D036781:Fimbriae Proteins; D010450:Endopeptidases; C067127:prepilin peptidase protein, Bacteria