Analysis of the pilU gene for the prepilin peptidase involved in the biogenesis of type IV pili encoded by plasmid R64

Abstract

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.

The TadV Protein ofActinobacillus actinomycetemcomitansIs a Novel Aspartic Acid Prepilin Peptidase Required for Maturation of the Flp1 Pilin and TadE and TadF Pseudopilins

“…TadV is predicted to lack a cleavable signal sequence and to have five transmembrane segments (TM1 to TM5) (Fig. 6A), which is consistent with its function as a prepilin peptidase as well as with the determined or inferred localization patterns of other known prepilin peptidases (1,48,72). The catalytic domains of several T4P and T2S peptidases have been characterized in other systems.…”

“…A study of the Vibrio cholerae TcpJ protein, which functions as a T4P prepilin peptidase, was the first to identify two conserved aspartic acid residues as the active sites for proteolysis (48). The corresponding aspartic acid residues have also been shown to be critical for function of other T4P prepilin peptidases (1,48), preflagellin peptidases in Archea (5,76), and the P. aeruginosa TadV homolog, FppA (15).…”

ThePseudomonas aeruginosaPathogenicity Island PAPI-1 Is Transferred via a Novel Type IV Pilus

“…Besides PilS2 and PilV2, which are homologs of the major and minor subunits of R64 thin pilus, PilS and PilV, they are PilL2, a type IV lipoprotein; PilN2, a type IV pilus secretin protein; PilO2, a pilus accessory protein; PilP2, a pilus assembly protein; PilQ2, a pilus retraction ATPase; PilR2, an integral membrane protein and PilM2, an inner membrane protein ( Table 2). The R64 PilU is a prepilin peptidase, which catalyzes the cleavage of the R64 PilS prepilin protein prior to it assembling into the mature pilus (1). No homolog of R64 PilU was identified on the PAPI-1 island; however, R64 PilU is distantly related to PA14 PilD (PA14_58770, 27% identity), which is involved in processing of prepilin protein PilA, the major subunit of P. aeruginosa polar pili (36).…”

Active-Site Residues in the Type IV Prepilin Peptidase Homologue PibD from the Archaeon Sulfolobus solfataricus

“…In conjunction with the positive-inside rule for membrane proteins (50), our results are consistent with a model of six transmembrane segments and extracellular amino and carboxyl termini. Membrane topology data based on reporter gene fusions are available for three bacterial TFPPs, PilD from P. aeruginosa, OutO from Erwinia carotovora, and PilU encoded by plasmid R64 (1,29,36,40). OutO contains eight TMS, and PilU, which belongs to a distinct group of smaller prepilin peptidases, contains six TMS.…”