Processing of TCP pilin by TcpJ typifies a common step intrinsic to a newly recognized pathway of extracellular protein secretion by gram-negative bacteria.

Abstract

Biogenesis of the Vibrio cholerae toxin-coregulated pilus (TCP) requires the activities of at least seven accessory proteins. We demonstrate that a portion of this pathway involves a novel processing step in which a hydrophilic leader peptide is proteolytically removed from TcpA by the gene product characterized in this report, TcpJ, to yield the mature, export-competent form of the pilin. Cleavage of the pilin leader peptide is independent of known signal peptidases as demonstrated by pilin-processing profiles in Escherichia coli strains conditionally defective for production of leader peptidase or grown in the presence of the antibiotic globomycin. Additionally, pilin cleavage did not rely on the SecA protein, as evidenced by TcpA processing in azide-treated cells. These results suggest that TcpJ is representative of a new class of proteins involved in SecA-independent proteolytic cleavage of a set of atypical leader peptides during extracellular export.

A plasmid-encoded prepilin peptidase gene from enteropathogenic Escherichia coli

“…However, the pilD probe failed to hybridize with the EPEC DNA, even under conditions of reduced stringency. Prepilin peptidase genes have been identified in other species by sequence analysis in the vicinity of pilin or pilin-like genes (11,23,25,35). We therefore suspected that an EPEC prepilin peptidase gene might reside near the bfpA gene.…”

“…Prepilin peptidase genes have been identified in several species including P. aeruginosa (PilD), Klebsiella oxytoca (PulO), V cholerae (TcpJ), and N. gonorrhoeae (PilD) (11,23,25,35,39). One of these (P. aeruginosa PilD) has been shown to be bifunctional, capable of both cleavage and subsequent N PAK(pMMB67HE), PAK-DfQ(pBFP6), and PAKDfl(pMMB67HE) were grown in LB broth to an optical density at 600 nm 0.5, induced with IPTG (isopropyl-p-Dthiogalactopyranoside) (0.5 mM), and incubated for 2 h. EPEC E2348/69 and derivatives 31-6-1(1), 31-6-1(1)(pMSD201), and 31-6-1(1)(pMSD217) were grown overnight in Eagle's minimal essential medium to induce bfpA expression (9).…”

The Type 4 Prepilin Peptidases Comprise a Novel Family of Aspartic Acid Proteases

“…Strain JM290 is JM109 carrying pTrc99A/EpsI (6)His (19). Strain MK90 is K38 carrying p3Z-A, which provides tcpA under the control of a T7 promoter, and pGP1-2, which provides the heat-inducible T7 RNA polymerase that drives transcription of the tcpA on p3Z-A (22 (21), was constructed by the cloning of a 980-bp EcoRI/NsiI fragment containing tcpJ into the EcoRI and PstI sites in the polylinker of pUC18. Plasmid pCL10 was constructed by the additional cloning of a 2.0-kilobase pair HindIII fragment that contains tcpA into the HindIII site of the pCL9 polylinker.…”

“…Primer J-Histag 3Ј codes for 6 His residues, which are added to the C terminus of the TcpJ protein. Plasmid p3Z-A was constructed by the cloning of the 1-kilobase pair DraI tcpA fragment into the SmaI site of the Promega expression vector, pGEM3Z (22). Plasmid pJM294 is a pACYC184 derivative containing vcpD (19).…”

Common components in the assembly of type 4 fimbriae, DNA transfer systems, filamentous phage and protein‐secretion apparatus: a general system for the formation of surface‐associated protein complexes

“…The N. gonorrhoeae type 4 fimbrial proteins PiID , PiIF , PilT and ORF4 were described by Lauer et al (1993)a. TcpE , TcpT and TcpJ are from the V. cholerae fimbrial gene cluster (Ogierm an et al, 1993;Kaufman et al, 1991;. Other proteins in the Table are all referred to in the text.…”