The assembly of large and complex organelles, such as the bacterial flagellum, poses the formidable problem of coupling temporal gene expression to specific stages of the organelle-assembly process. The discovery that levels of the bacterial flagellar regulatory protein FlgM are controlled by its secretion from the cell in response to the completion of an intermediate flagellar structure (the hook-basal body) was only the first of several discoveries of unique mechanisms that coordinate flagellar gene expression with assembly. In this Review, we discuss this mechanism, together with others that also coordinate gene regulation and flagellar assembly in Gram-negative bacteria.

SUMMARY How do organisms assess the degree of completion of a large structure, especially an extracellular structure such as a flagellum? Bacteria can do this. Mutants that lack key components needed early in assembly fail to express proteins that would normally be added at later assembly stages. In some cases, the regulatory circuitry is able to sense completion of structures beyond the cell surface, such as completion of the external hook structure. In Salmonella and Escherichia coli, regulation occurs at both transcriptional and posttranscriptional levels. One transcriptional regulatory mechanism involves a regulatory protein, FlgM, that escapes from the cell (and thus can no longer act) through a complete flagellum and is held inside when the structure has not reached a later stage of completion. FlgM prevents late flagellar gene transcription by binding the flagellum-specific transcription factor ς28. FlgM is itself regulated in response to the assembly of an incomplete flagellum known as the hook-basal body intermediate structure. Upon completion of the hook-basal body structure, FlgM is exported through this structure out of the cell. Inhibition of ς28-dependent transcription is relieved, and genes required for the later assembly stages are expressed, allowing completion of the flagellar organelle. Distinct posttranscriptional regulatory mechanisms occur in response to assembly of the flagellar type III secretion apparatus and of ring structures in the peptidoglycan and lipopolysaccharide layers. The entire flagellar regulatory pathway is regulated in response to environmental cues. Cell cycle control and flagellar development are codependent. We discuss how all these levels of regulation ensure efficient assembly of the flagellum in response to environmental stimuli.

The ability of a regulatory protein to sense the integrity of the bacterial flagellar structure was investigated. In response to a defective hook-basal body complex, the anti-sigma 28 FlgM protein inhibits flagellin transcription. In cells with a functional hook-basal body complex, the flagellin genes are transcribed normally and the FlgM protein is expelled into the growth medium. In strains with a defective hook-basal body structure, FlgM is absent from the media. The presence of flagellin protein in the media is substantially reduced in strains carrying a FlgM-LacZ protein fusion, suggesting that the fusion is blocking the flagellar export apparatus. These results suggest that the FlgM protein assesses the integrity of the flagellar hook-basal body complex by itself being a substrate for export by the flagellar-specific export apparatus.

Bacterial flagella contain a specialized secretion apparatus that functions to deliver the protein subunits that form the filament and other structures to outside the membrane 1 . This apparatus is related to the injectisome used by many gram-negative pathogens and symbionts to transfer effector proteins into host cells; in both systems this export mechanism is termed 'type III' secretion 2,3 . The flagellar secretion apparatus comprises a membrane-embedded complex of about five proteins, and soluble factors, which include export-dedicated chaperones and an ATPase, FliI, that was thought to provide the energy for export 1,4 . Here we show that flagellar secretion in Salmonella enterica requires the proton motive force (PMF) and does not require ATP hydrolysis by FliI. The export of several flagellar export substrates was prevented by treatment with the protonophore CCCP, with no accompanying decrease in cellular ATP levels. Weak swarming motility and rare flagella were observed in a mutant deleted for FliI and for the nonflagellar type-III secretion ATPases InvJ and SsaN. These findings show that the flagellar secretion apparatus functions as a protondriven protein exporter and that ATP hydrolysis is not essential for type III secretion.Flagellar assembly begins with structures in the cytoplasmic membrane and proceeds through steps that add the exterior structures in a proximal-to-distal sequence (Fig. 1) 1 . Assembly of the rod, hook and filament requires the action of the secretion apparatus, which transports the needed subunits into a central channel through the structure that conducts them to their site of incorporation at the tip ( Fig. 1). Flagellar export is notably fast: in the early stages of filament growth flagellin is delivered at a rate of several 55 kDa subunits per second 5 .ATP hydrolysis by FliI was thought to provide the energy for export because mutations that delete or reduce the activity of FliI block flagellar synthesis at the stage of rod assembly 1,4,6 (Fig. 1). Homologues of FliI also occur in the type III secretion apparatus of injectisomes and are usually assumed to energize export in those systems as well. Some evidence for a different view has also been reported: it was observed that type III secretion in Yersinia enterocolitica was prevented by the protonophore CCCP 7 , and it was shown that the secretion ATPase InvC of Salmonella functions to dissociate export substrate from the chaperone 8 , a role distinct from transport itself. The energy source for type III secretion thus remains uncertain.To address the energy requirements for type III secretion, we first measured the effect of the uncoupler CCCP on flagellar export in S. enterica, assayed by accumulation of the export substrate FlgM in the medium. FlgM export was prevented by 10 mM or more CCCP (Fig. 2a). Overall cellular energy levels seemed unaffected, because cells grew normally in 10 mM CCCP (growth data not shown) and ATP levels were unchanged ( Supplementary Fig. 1). The effect was reversible: FlgM export was largely re...

A mechanism for regulating gene expression at the level of transcription utilizes an antagonist of the sigma transcription factor known as the anti-sigma (anti-sigma) factor. The cytoplasmic class of anti-sigma factors has been well characterized. The class includes AsiA form bacteriophage T4, which inhibits Escherichia coli sigma 70; FlgM, present in both gram-positive and gram-negative bacteria, which inhibits the flagella sigma factor sigma 28; SpoIIAB, which inhibits the sporulation-specific sigma factor, sigma F and sigma G, of Bacillus subtilis; RbsW of B. subtilis, which inhibits stress response sigma factor sigma B; and DnaK, a general regulator of the heat shock response, which in bacteria inhibits the heat shock sigma factor sigma 32. In addition to this class of well-characterized cytoplasmic anti-sigma factors, a new class of homologous, inner-membrane-bound anti-sigma factors has recently been discovered in a variety of eubacteria. This new class of anti-sigma factors regulates the expression of so-called extracytoplasmic functions, and hence is known as the ECF subfamily of anti-sigma factors. The range of cell processes regulated by anti-sigma factors is highly varied and includes bacteriophage phage growth, sporulation, stress response, flagellar biosynthesis, pigment production, ion transport, and virulence.