Signal Recognition Particle-Dependent Inner Membrane Targeting of the PulG Pseudopilin Component of a Type II Secretion System

Abstract

The pseudopilin PulG is an essential component of the pullulanase-specific type II secretion system from Klebsiella oxytoca. PulG is the major subunit of a short, thin-filament pseudopilus, which presumably elongates and retracts in the periplasm, acting as a dynamic piston to promote pullulanase secretion. It has a signal sequence-like N-terminal segment that, according to studies with green and red fluorescent protein chimeras, anchors unassembled PulG in the inner membrane. We analyzed the early steps of PulG inner membrane targeting and insertion in Escherichia coli derivatives defective in different protein targeting and export factors. The ␤-galactosidase activity in strains producing a PulG-LacZ hybrid protein increased substantially when the dsbA, dsbB, or all sec genes tested except secB were compromised by mutations. To facilitate analysis of native PulG membrane insertion, a leader peptidase cleavage site was engineered downstream from the N-terminal transmembrane segment (PrePulG*). Unprocessed PrePulG* was detected in strains carrying mutations in secA, secY, secE, and secD genes, including some novel alleles of secY and secD. Furthermore, depletion of the Ffh component of the signal recognition particle (SRP) completely abolished PrePulG* processing, without affecting the Sec-dependent export of periplasmic MalE and RbsB proteins. Thus, PulG is cotranslationally targeted to the inner membrane Sec translocase by SRP.

Export of the Pseudopilin XcpT of the Pseudomonas aeruginosa Type II Secretion System via the Signal Recognition Particle-Sec Pathway

“…Given the strong conservation of their N-terminal sequences, it is likely that other pilins and pseudopilins also use the SRP/Sec pathway. Indeed, similar conclusions were reached by Francetic et al in the accompanying paper on the export of the pseudopilin PulG of the type II secretion system of Klebsiella oxytoca (21). We propose that, after translocation, the (pseudo)pilins leave the Sec translocon laterally, after which they become recruited by their cognate machinery.…”

Membrane Proteases in the Bacterial Protein Secretion and Quality Control Pathway

“…In addition to signal peptide cleavage, SPIV is also responsible for N-methylation of the phenylalanine at position ϩ1 relative to the cleavage site (116). Pseudopilin signal peptides show clear structural similarities to other types of bacterial signal peptides, and the pseudopilin precursors are most likely targeted to the membrane by signal recognition particle (SRP) and inserted by Sec pathways (9,43). The subsequent assembly of the pseudopilins into a type IV pilus is facilitated by a dedicated pilin assembly pathway.…”

Two Isoforms of Geobacter sulfurreducens PilA Have Distinct Roles in Pilus Biogenesis, Cytochrome Localization, Extracellular Electron Transfer, and Biofilm Formation

“…Type IV pilins in Gram-negative bacteria are synthesized as prepilins, with a leader sequence that is cleaved after a conserved glycine (defined as position Ϫ1) by a specific leader peptidase, PilD, in the inner membrane (3,16,61). Mature pilin subunits have a hydrophobic amino-terminal segment with a consensus sequence that includes a conserved phenylalanine (position 1) and glutamic acid (position 5), which forms the core of the pilus fiber (14).…”