Analysis of the roles of FlgP and FlgQ in flagellar motility of Campylobacter jejuni

Abstract

Flagellar motility is an important determinant of Campylobacter jejuni that is required for promoting interactions with various hosts to promote gastroenteritis in humans or commensal colonization of many animals. In a previous study, we identified a nonmotile mutant of C. jejuni 81-176 with a transposon insertion in Cj1026c, but verification of the role of the encoded protein in motility was not determined. In this study, we have determined that Cj1026c and the gene immediately downstream, Cj1025c (here annotated as flgP and flgQ, respectively), are both required for motility of C. jejuni but not for flagellar biosynthesis. FlgP and FlgQ are not components of the transcriptional regulatory cascades to activate sigma(28)- or sigma(54)-dependent expression of flagellar genes. In addition, expression of flgP and flgQ is not largely dependent on sigma(28) or sigma(54). Immunblot analyses revealed that the majority of FlgP in C. jejuni is associated with the outer membrane. However, in the absence of FlgQ, the amounts of FlgP in the outer membrane of C. jejuni are greatly reduced, suggesting that FlgQ may be required for localization or stability of FlgP at this location. This study provides insight into features of FlgP and FlgQ, two proteins with previously undefined functions that are required for the larger, multicomponent flagellar system of C. jejuni that is necessary for motility.

FIG. 1.

Deletion of flgP or flgQ does not affect FlgR production in C. jejuni. (A) Organization of the flgPQ locus and surrounding genes. flgP and flgQ are immediately upstream of flgR, and their coding sequences overlap by 27 nucleotides. The arrowhead indicates the location of the solo transposon in strain 81-176 flgP::solo (DRH241). The annotations of flgP and flgQ in the genomic sequences of C. jejuni 11168 and 81-176, respectively, are Cj1026c and Cjj81176_1045 for flgP and Cj1025c and Cjj81176_1044 for flgQ. (B) Immunoblot analysis of FlgR production in C. jejuni flgP and flgQ mutants. Whole-cell lysates of wild-type (wt) 81-176 Sm^r (DRH212), 81-176 Sm^r ΔflgR (DRH737), 81-176 flgP::solo (DRH241), 81-176 Sm^r ΔflgP (DRH2070), and 81-176 Sm^r ΔflgQ (DRH2071) were used for analysis.

FIG. 2.

FlgP and FlgQ are required for flagellar motility of C. jejuni. Strains were inoculated into MH motility agar. Motility was assessed 32 h after inoculation. Strains are (A) wild-type (wt) 81-176 Sm^r (DRH212), 81-176 Sm^r ΔflgR (DRH737), 81-176 flgP::solo (DRH241), 81-176 Sm^r ΔflgP (DRH2070), and 81-176 Sm^r ΔflgQ (DRH2071) and (B) wild-type 81-176 Sm^r/pECO102 (DRH837), 81-176 Sm^r ΔflgP/pECO102 (SMS304), 81-176 Sm^r ΔflgQ/pECO102 (SMS306), 81-176 Sm^r ΔflgP/pSMS265 (SMS308), and 81-176 Sm^r ΔflgQ/pSMS269 (SMS312). pECO102 is the empty vector control, and pSMS265 and pSMS269 are pECO102 derivatives that express flgP and flgQ, respectively.

FIG. 3.

FlgP and FlgQ are not required for flagellar biosynthesis. Bacteria were stained with 1% uranyl acetate. All electron micrographs are at a magnification of ×20,000. The bar for each micrograph represents 0.5 μm. Strains shown are wild-type (wt) 81-176 Sm^r (DRH212), 81-176 Sm^r ΔflgR (DRH737), 81-176 Sm^r ΔflgP (DRH2070), and 81-176 Sm^r ΔflgQ (DRH2071).

FIG. 4.

FlgP requires FlgQ for localization to or stability in the outer membrane. Proteins from whole-cell lysates representing 200 μl of bacterial culture or proteins from outer membrane preparations or cytoplasmic preparations representing 750 μl of bacterial culture were used for immunoblotting. Blots were developed with α-FlgP mouse antiserum M1, α-RpoA mouse antiserum M1, or α-MOMP rabbit antiserum (30). Blots on the left are detecting FlgP. The top and middle blots on the right are detecting RpoA, whereas the bottom blot is detecting MOMP. Strains are wild-type (wt) 81-176 Sm^r (DRH212), 81-176 Sm^r ΔflgP (DRH2070), 81-176 Sm^r ΔflgP/pECO102 (SMS304), 81-176 Sm^r ΔflgP/pSMS265 (SMS308), 81-176 Sm^r ΔflgQ (DRH2071), 81-176 Sm^r ΔflgQ/pECO102 (SMS306), and 81-176 Sm^r ΔflgQ/pSMS269 (SMS312). pECO102 is the empty vector control, and pSMS265 and pSMS269 are pECO102 derivatives that express flgP and flgQ, respectively. WCL, whole-cell lysates.