The Escherichia coli flhD operon encodes two genes, flhD and flhC. Both gene products were overproduced and purified. The purified proteins formed a complex consisting of two FlhD and two FlhC molecules. Mobility shift assays showed that the FlhD/FlhC complex had a DNA-binding activity and bound to the upstream regions offli4,flhB, andfliL operons (class II), which are under direct control of theflhD operon. DNase I footprinting analyses of FlhD/FlhC binding to the three class II promoter regions revealed protection of a 48-bp region of thefli,4 operon between positions -41 to -88, a 50-bp region of theflhB operon between positions -28 to -77, and a 48-bp region of the fliL operon between positions -29 to -76. In vitro transcription experiments demonstrated that the FlhD/FlhC complex is a transcriptional activator required for the transcription of the three class II operons examined in vitro.The Escherichia coli flagellar regulon consists of at least 14 operons which encode more than 40 genes. Studies of the regulation of the flagellar regulon showed that the genes in the regulon constituted their own transcriptional hierarchy and allowed for coordinate expression (13) (Fig. 1). At the top of the hierarchy is the flhD master operon, which is composed of the flhD and flhC genes. Expression of this operon is required for the expression of all of the remaining operons. Class II consists of operons which are under direct control of the master operon. The genes contained in these operons encode a flagellum-specific sigma factor ((u28 or FliA), which is required for the transcription of class III operons, and many structural components assembled in the early and middle stages of flagellar synthesis as well as some proteins of unknown functions. Class I11a operons are under the dual control of FlhD/FlhC and FliA, whereas class IIIb operons are under the control of FliA (for reviews, see references 18 and 19).Through genetic techniques, it is known that FlhD and FlhC are the master regulatory proteins of the flagellar transcriptional regulon. However, little is known about the molecular mechanism by which gene expression in the regulon is controlled. It was proposed that FlhD and FlhC may function together as a u2 homolog in E. coli (2, 10). This proposal was based on the observation of a Bacillus subtilis ur28 promoter consensus sequence similarity, found in the upstream region of seven E. coli flagellar operons which lacked the promoter consensus sequence recognized by u70 (9), and on the observation that both flhD and flhC gene products functioned as trans-acting positive regulators of the flagellar regulon. Consistent with this idea, an amino acid sequence similarity between c28 of B. subtilis and both F1hD and FlhC has been reported (10). Later, a 28-kDa flagellum-specific sigma factor was isolated (2). However, the sigma factor activity was shown to reside in the class II FliA protein, not in F1hD or FlhC (23).Further analysis at the DNA sequence level indicated that a derivative of the flagellum-specific consensu...

The flhD operon is the master operon of the flagellar regulon and a global regulator of metabolism. The genome sequence of the Escherichia coli K-12 strain MG1655 contained an IS1 insertion sequence element in the regulatory region of the flhD promoter. Another stock of MG1655 was obtained from the E. coli Genetic Stock Center. This stock contained isolates which were poorly motile and had no IS1 element upstream of the flhD promoter. From these isolates, motile subpopulations were identified after extended incubation in motility agar. Purified motile derivatives contained an IS5 element insertion upstream of the flhD promoter, and swarm rates were sevenfold higher than that of the original isolate. For a motile derivative, levels of flhD transcript had increased 2.7-fold, leading to a 32-fold increase in fliA transcript and a 65-fold increase in flhB::luxCDABE expression from a promoter probe vector. A collection of commonly used lab strains was screened for IS element insertion and motility. Five strains (RP437, YK410, MC1000, W3110, and W2637) contained IS5 elements upstream of the flhD promoter at either of two locations. This correlated with high swarm rates. Four other strains (W1485, FB8, MM294, and RB791) did not contain IS elements in the flhD regulatory region and were poorly motile. Primer extension determined that the transcriptional start site of flhD was unaltered by the IS element insertions. We suggest that IS element insertion may activate transcription of the flhD operon by reducing transcriptional repression.An important source of genome plasticity is derived from transpositional events of insertion sequence (IS) elements (34, 35). They generally encode no functions other than those involved in their mobility (for a review, see reference 30) and display a nonrandom distribution in the chromosome of Escherichia coli (9, 16). Many IS elements have been shown to activate the expression of neighboring genes, for example, through the formation of hybrid promoters or disruption of transcriptional repression. This has also been seen with cryptic operons, which depend upon mutations for activation. Two examples in E. coli are the bgl and ade operons, which can be activated by IS element insertion upstream or downstream of the promoter (18,42,49,50,53). The chitobiose operon, chb (formerly cel), was thought to be cryptic but can be induced by chitobiose, as well as being activated by IS element insertion upstream of the structural genes under noninducing conditions (40, 44).Flagellar motility enables bacteria to escape from detrimental conditions and to reach more favorable environments. In E. coli, the flagellar regulon involves the expression of at least 14 operons in a regulated cascade to produce functional flagellar and chemotaxis machinery (for a review, see reference 14). The flhD operon at the apex of the flagellar regulon has been identified as the primary target of regulation by many environmental factors (for a review, see reference 61). It consists of two genes, flhD and flhC, whose product...

The nature of the biochemical signal that is involved in the excitation response in bacterial chemotaxis is not known. However, ATP is required for chemotaxis. We have purified all of the proteins involved in signal transduction and show that the product of the cheA gene is rapidly autophosphorylated, while some mutant CheA proteins cannot be phosphorylated. The presence of stoichiometric levels of two other purified components in the chemotaxis system, the CheY and CheZ proteins, induces dephosphorylation. We suggest that the phosphorylation of CheA by ATP plays a central role in signal transduction in chemotaxis.Bacteria can respond to chemical changes in their environment by altering their pattern of motility, resulting in swimming toward higher concentrations of attractants and away from repellents. The chemotaxis response is mediated by a series of transmembrane receptor-transducer proteins that bind specific ligands and transmit information about changes in ligand concentration as a function of time to the bacterial flagellar apparatus (for reviews, see refs. 1, 2, and 3). The components involved in the intracellular signal transduction pathway for chemotaxis in Escherichia coli and Salmonella have been identified by genetic techniques. Four genes, cheA, cheW, cheY, and cheZ elaborate products that are required for the integration and transduction of information. Two other gene products encoded by cheR and cheB are responsible for adaptation to wide ranges of ligand concentration. They reversibly methylate specific glutamic acid residues on the cytoplasmic portion of the receptor-transducer, modulating its function.While a great deal is known about the components of the information-processing system, little is known about the biochemical nature of the chemotactic signal. A number of laboratories have found that ATP is required for signal transduction (4-7). However, the exact nature of its involvement was not clear. Indirect experiments have led to the formulation of models for the function of the chemotaxis proteins and their interaction with ATP (2, 8). To measure these interactions directly, we purified all of the proteins known to be involved in the central pathway for information transduction. In this paper we show that the cheA gene product can autophosphorylate with ATP. We can isolate a phosphorylated CheA intermediate and show that the CheY and CheZ proteins can influence the course of CheA phosphorylation. Furthermore, mutations that eliminate chemotaxis and map within the cheA gene result in proteins that are defective in the phosphorylation reaction.MATERIALS AND METHODS Protein Purifications. CheA and CheW were overexpressed from a plasmid, pDV4 (P.M., unpublished data), containing the cheA and cheW genes. The plasmid was maintained in an E. coli W3110 derivative SVS402 AtrpE-A, recAl, tna-2, bglR, obtained from R. Bauerle (University of Virginia, Charlottesville, VA). CheA was purified by a protocol including the use of dye-ligand chromatography and gel filtration and will be describe...

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