The structure and regulation of flagella in Bacillus subtilis

Abstract

Bacterial flagellar motility is among the most extensively studied physiological systems in biology, but most research has been restricted to using the highly similar Gram-negative species Escherichia coli and Salmonella enterica. Here, we review the recent advances in the study of flagellar structure and regulation of the distantly related and genetically tractable Gram-positive bacterium Bacillus subtilis. B. subtilis has a thicker layer of peptidoglycan and lacks the outer membrane of the Gram-negative bacteria; thus, not only phylogenetic separation but also differences in fundamental cell architecture contribute to deviations in flagellar structure and regulation. We speculate that a large number of flagella and the absence of a periplasm make B. subtilis a premier organism for the study of the earliest events in flagellar morphogenesis and the type III secretion system. Furthermore, B. subtilis has been instrumental in the study of heterogeneous gene transcription in subpopulations and of flagellar regulation at the translational and functional level.

Keywords: Bacillus; bistability; flagella; homeostasis; motility.

Figure 1. Flagellar structure and genetic hierarchy

A) Cartoon depicting the putative structure of the B. subtilis flagellum based on empirical data and similarity to S. enterica. Peptidoglycan is indicated in light gray. Membrane is indicated in dark gray. Flagellar components are colored and labeled. Micrographs are super-resolution fluorescence micrographs of the indicated structures in wild type cells: filament (maleimide-stained Hag^T209C), hook (maleimide-strained FlgE^T123C) and basal body (FliM-GFP fusion protein). B) Cartoon depicting genetic hierarchy of flagellar genes in B. subtilis. Open arrows are genes. Bent arrows are promoters. Closed arrows indicate activation; T-bars indicate inhibition. Genes are color coded to match structures in panel A. Micrograph indicates bistable gene expression found in a swrA mutant of B. subtilis. Membranes colored red (stained with FM 4–64 dye). P_hag expression colored green cytoplasmically (P_hag-YFP). Filaments colored green extracellularly (maleimide-stained Hag^T209C). Only a subpopulation of cells expresses the filament gene hag and assembles filaments. Scale bars are 2μm.

Figure 2. Flagellar regulation

A) FlgM (red) antagonizes σ^D (cyan) but σ^D activates FlgM expression prioritized by DegU-P. The autoinhibitory loop on σ^D is broken by completed hook-basal body structure by an unknown mechanism and hag transcript (green arrow) is made. B) CsrA (red triangle) binds to the RBS (open box) of the hag transcript and inhibits translation. When Hag (green barbell) is secreted aided by FliS (orange pentagon), FliW (blue hexagon) switches partners and antagonizes CsrA allowing high level Hag translation for the duration of filament assembly. C) EpsE (red circle) is relieved from SinR repression during biofilm formation and acts as a clutch by binding to FliG and disengaging the rotor (purple) from MotA (brown). D) DgrA (red octagon) is activated by binding c-di-GMP and interacts with MotA to inhibit flagellar rotation.