FlgN Is Required for Flagellum-Based Motility by Bacillus subtilis

Abstract

The assembly of the bacterial flagellum is exquisitely controlled. Flagellar biosynthesis is underpinned by a specialized type III secretion system that allows export of proteins from the cytoplasm to the nascent structure. Bacillus subtilis regulates flagellar assembly using both conserved and species-specific mechanisms. Here, we show that YvyG is essential for flagellar filament assembly. We define YvyG as an orthologue of the Salmonella enterica serovar Typhimurium type III secretion system chaperone, FlgN, which is required for the export of the hook-filament junction proteins, FlgK and FlgL. Deletion of flgN (yvyG) results in a nonmotile phenotype that is attributable to a decrease in hag translation and a complete lack of filament polymerization. Analyses indicate that a flgK-flgL double mutant strain phenocopies deletion of flgN and that overexpression of flgK-flgL cannot complement the motility defect of a ΔflgN strain. Furthermore, in contrast to previous work suggesting that phosphorylation of FlgN alters its subcellular localization, we show that mutation of the identified tyrosine and arginine FlgN phosphorylation sites has no effect on motility. These data emphasize that flagellar biosynthesis is differentially regulated in B. subtilis from classically studied Gram-negative flagellar systems and questions the biological relevance of some posttranslational modifications identified by global proteomic approaches.

Evidence for a Functional O-Linked N-Acetylglucosamine (O-GlcNAc) System in the Thermophilic Bacterium Thermobaculum terrenum

“…The gel was blotted onto nitrocellulose membrane using a Novex Semi-Dry Blotter, and the membranes were blocked in 3% milk in TBS, 0.1% Tween 20. The primary purified antibodies against Tt OGT (dilution, 1:100), Tt OGA (dilution, 1:1,000), and B. subtilis RpoD ( 44 ) (dilution, 1:1000) were incubated with the membranes overnight at 4 °C and detected with HRP-conjugated anti-rabbit secondary antibodies.…”

Completed Genomic Sequence ofBacillus thuringiensisHER1410 Reveals aCry-Containing Chromosome, Two Megaplasmids, and an Integrative Plasmidial Prophage

“…This is the case, for example, of phIS3501 that disrupts the production of hlyII toxin when it integrates into the chromosome (Moumen et al, 2012). FlgK is an essential element of the hook-filament junction and its absence causes flagellar misassembly and accumulation in the extracellular milieu of Bacillus subtilis (Cairns et al, 2014). The intermittent disruption of this gene by pLUSID3 integration could have an impact on the motility of B. thuringiensis HER1410 and biofilm formation (Houry et al, 2010).…”

Quantification of Motility in Bacillus subtilis at Temperatures Up to 84°C Using a Submersible Volumetric Microscope and Automated Tracking

“…Bacillus subtilis is a model organism for which regulation of motility genes ( Mukherjee and Kearns, 2014 ) and heat shock responses ( Schumann, 2003 ) have been well studied. There is a strict dependence upon FlgN for motility in this species ( Cairns et al, 2014 ). Flagellar synthesis is affected by a number of the genes involved in the heat shock response.…”