Genes Required for Plasmid R64 Thin-Pilus Biogenesis: Identification and Localization of Products of the pilK, pilM, pilO, pilP, pilR, and pilT Genes

AUTOR(ES)

Sakai, Daisuke

FONTE

American Society for Microbiology

RESUMO

We have previously shown that the pilL, pilN, pilQ, pilS, pilU, and pilV genes of plasmid R64 encode outer membrane lipoprotein, secretin, cytoplasmic ATPase, major pilin, prepilin peptidase, and minor pilin, respectively, which are required for thin-pilus formation. In this work, we characterized the products of the remaining essential genes, pilK, pilM, pilO, pilP, pilR, and pilT, with regard to their localization and processing. Overexpression systems containing pilM, pilO, and pilP genes fused with N-terminal glutathione S-transferase (GST) or a His tag were constructed. Overproduced proteins were purified and used to raise specific antibodies. Localization of PilM, PilO, and PilP proteins was performed by Western blot analysis with anti-GST-PilM, anti-PilO, and anti-PilP antibodies, respectively. The pilK, pilR, and pilT products were produced with a C-terminal His tag and then detected by anti-His tag antibody. Subcellular fractionation experiments with Escherichia coli cells producing R64 thin pili revealed that PilK, PilM, and PilR are inner membrane proteins, and PilP and PilT are periplasmic proteins. PilO protein was localized to the outer membrane in the presence of other Pil proteins, whereas it was localized to the cytoplasm in the absence of these proteins. Furthermore, the cleavage site of PilP protein was determined by N-terminal amino acid sequencing of purified mature PilP protein. We predict that PilK, PilM, PilO, PilP, and PilT proteins function as the components of the pilin transport apparatus and thin-pilus basal body.

Documentos Relacionados

• Twelve pil Genes Are Required for Biogenesis of the R64 Thin Pilus
• The plasmid R64 thin pilus identified as a type IV pilus.
• Thin Pilus PilV Adhesins of Plasmid R64 Recognize Specific Structures of the Lipopolysaccharide Molecules of Recipient Cells
• Identification of pilR, which encodes a transcriptional activator of the Pseudomonas aeruginosa pilin gene.
• Mutational Analysis of Plasmid R64 Thin Pilus Prepilin: the Entire Prepilin Sequence Is Required for Processing by Type IV Prepilin Peptidase