Many bacteria swim in liquid or swarm over solid surfaces by synthesizing rotary flagella. The same bacteria that are motile also commonly form non-motile multicellular aggregates held together by an extracellular matrix called biofilms. Biofilms are an important part of the lifestyle of pathogenic bacteria and it is assumed that there is a motility-to-biofilm transition wherein the inhibition of motility promotes biofilm formation. The transition is largely inferred from regulatory mutants that reveal the opposite regulation of the two phenotypes. Here we review the regulation of motility during biofilm formation in Bacillus, Pseudomonas, Vibrio, and Escherichia, and we conclude that the motility-to-biofilm transition, if necessary, likely involves two steps. In the short term, flagella are functionally regulated to either inhibit rotation or modulate the basal flagellar reversal frequency. Over the long term, flagellar gene transcription is inhibited and in the absence of de novo synthesis, flagella are likely diluted to extinction through growth. Both short term and long term control is likely important to the motility-to-biofilm transition to stabilize aggregates and optimize resource investment. We emphasize the newly discovered classes of flagellar functional regulators and speculate that others await discovery in the context of biofilm formation.

Over the course of more than a century of laboratory experimentation, Bacillus subtilis has become "domesticated," losing its ability to carry out many behaviors characteristic of its wild ancestors. One such characteristic is the ability to form architecturally complex communities, referred to as biofilms. Previous work has shown that the laboratory strain 168 forms markedly attenuated biofilms compared with the wild strain NCIB3610 (3610), even after repair of a mutation in sfp (a gene involved in surfactin production) previously known to impair biofilm formation. Here, we show that in addition to the sfp mutation, mutations in epsC, swrA, and degQ are necessary and sufficient to explain the inability of the laboratory strain to produce robust biofilms. Finally, we show that the architecture of the biofilm is markedly influenced by a large plasmid present in 3610 but not 168 and that the effect of the plasmid can be attributed to a gene we designate rapP. When rapP is introduced into 168 together with wild-type alleles of sfp, epsC, swrA, and degQ, the resulting repaired laboratory strain forms biofilms that are as robust as and essentially indistinguishable in architecture from those of the wild strain, 3610. Thus, domestication of B. subtilis involved the accumulation of four mutations and the loss of a plasmid-borne gene.

SUMMARY Bacterial flagella are highly conserved molecular machines that have been extensively studied for assembly, function, and gene regulation. Less studied is how and why bacteria differ based on the number and arrangement of the flagella they synthesize. Here we explore the cell biology of peritrichous flagella in the model bacterium Bacillus subtilis by fluorescently labeling flagellar basal bodies, hooks, and filaments. We find that the average B. subtilis cell assembles approximately 26 flagellar basal bodies and we show that basal body number is controlled by the protein of unknown function SwrA. Basal bodies are assembled rapidly (< 5 minutes) but the assembly of flagella capable of supporting motility (> 40 minutes) is rate limited by filament polymerization. We find that basal bodies are not positioned randomly on the cell surface. Rather, basal bodies occupy a grid-like pattern organized symmetrically around the midcell and that flagella are discouraged at the poles. Basal body position is genetically determined by FlhF and FlhG homologs to control spatial patterning differently from what is seen in bacteria with polar flagella. Finally, spatial control of flagella in B. subtilis seems more relevant to the inheritance of flagella in individual cells than the motile behavior of populations.

Many bacteria inhibit motility concomitant with the synthesis of an extracellular polysaccharide matrix and the formation of biofilm aggregates. In Bacillus subtilis biofilms, motility is inhibited by EpsE, which acts as a clutch on the flagella rotor to inhibit motility, and which is encoded within the 15 gene eps operon required for EPS production. EpsE shows sequence similarity to the glycosyltransferase family of enzymes, and we demonstrate that the conserved active site motif is required for EPS biosynthesis. We also screen for residues specifically required for either clutch or enzymatic activity and demonstrate that the two functions are genetically separable. Finally, we show that, whereas EPS synthesis activity is dominant for biofilm formation, both functions of EpsE synergize to stabilize cell aggregates and relieve selective pressure to abolish motility by genetic mutation. Thus, the transition from motility to biofilm formation may be governed by a single bifunctional enzyme.

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