Effects of lipoprotein biogenesis mutations on flagellar assembly in Salmonella

Abstract

Flagellar assembly requires the expression of a large number of flagellum-specific genes. However, mutations in a number of other genes in Salmonella and Escherichia coli have been shown to have pleiotropic effects that affect flagellar assembly. FlgH (the L-ring subunit of the flagellar basal body) is a lipoprotein whose modification is important for L-ring assembly. We therefore tested whether the lack of motility of Salmonella mutants defective in lipoprotein biogenesis is a result of inability to modify FlgH. Our results show that temperature-sensitive apolipoprotein N-acyltransferase [lnt(Ts)] mutants are nonflagellate at 42 degrees C. However, the flagellar assembly defect occurs at a much earlier step in the pathway than L-ring assembly. These mutants failed to assemble even an MS ring, presumably because of the observed decrease in transcription of fliF. In contrast, temperature-sensitive diacylglycerol transferase [lgt(Ts)] mutants were motile at 42 degrees C, provided the strains carried an lpp (Braun lipoprotein) mutation to permit growth. We have isolated second-site mutants from an lgt(Ts) lpp(+) strain that grow but are nonflagellate at 42 degrees C. Thus, lipoprotein biogenesis is a factor that is important for flagellar assembly.

FIG. 1.

Cartoon of the flagellar basal body. The outer-membrane L-ring protein, FlgH, is lipoylated, while the periplasmic P-ring protein, FlgI, undergoes intramolecular disulfide bridge formation.

FIG. 2.

Schematic representation of the pathway leading to formation of a lipoprotein (see the text). LXGC (or LXAC) is the sequence known as a lipobox. UPLP, unmodified prolipoprotein; MPLP, modified prolipoprotein; ALP, apolipoprotein; LP, mature lipoprotein; Lgt, prolipoprotein diacylglyceryltransferase; SPII, lipoprotein-specific signal peptidase; Lnt, apolipoprotein N-acyl transferase; scissors, potential site of cleavage by SPII; -S-DAG, diacylglycerol in thioether linkage to cysteine; AcHN-, N-acylation of terminal amino group.

FIG. 3.

Swarming of FDY40 [lnt(Ts) lpp] and FDY50 (lpp) on soft agar plates after 11 h at 30 and 42°C.

FIG. 4.

Immunoblots, using monoclonal anti-FliF antibody, of strains SJW1684 (fliF), SJW1469 (flgH), FDY50 (lpp), and FDY40 [lnt(Ts) lpp], all grown at 30°C, and of strains FDY50 (lpp) and FDY40 [lnt(Ts) lpp] grown at 42°C.

FIG. 5.

Swarming of FDY126 and FDY127 [both lgt(Ts) lpp⁺ lpr)] on soft agar plates after 8 h at 30 and 42°C.