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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The motB gene product of Escherichia coli is an integral membrane protein required for rotation of the flagellar motor. We have determined the nucleotide sequence of the motB region and find that it contains an open reading frame of 924 nucleotides which we ascribe to the motB gene. The predicted amino acid sequence of the gene product is 308 residues long and indicates an amphipathic protein with one major hydrophobic region, about 22 residues long, near the N terminus. There is no consensus signal sequence. We postulate that the protein has a short N-terminal region in the cytoplasm, an anchoring region in the membrane consisting of two spanning segments, and a large cytoplasmic C-terminal domain. By placing motB under control of the tryptophan operon promoter of Serratia marcescens, we have succeeded in overproducing the MotB protein.Under these conditions, the majority of MotB was found in the cytoplasm, indicating that the membrane has a limited capacity to incorpor.1te the protein. We conclude that insertion of MotB into the membrane requires the presence of other more hydrophobic components, possibly including the MotA protein or other components of the flagellar motor. The results further reinforce the concept that the total flagellar motor consists of more than just the basal body. At least five proteins are essential for motor rotation (41, 47), but none of these have been found within the flagellar basal body (1,. 20), even though the basal body is considered a major part of the motor. Two of these proteins, MotA and MotB, are integral to the cell membrane (6, 37, 41). They are not necessary for assembly of the flagellum and do not copurify with it (20). They can be synthesized after flagellar assembly and used to activate the motor; paralyzed motA motB mutants acquire the ability to rotate their flagella after MotA and MotB synthesis under the direction of lambda-E. coli hybrid bacteriophage (41). Recently, it was shown (6) that, at least in the case of MotB, this acquisition of motility proceeds by quantum increases in flagellar rotation rate, presumably as a result of successive incorporation of subunits of MotB protein. The fact that motA and motB null mutants are nonetheless flagellated makes these genes different from all other flagellum-associated genes and will have * Corresponding author.

The regulation of the expression of the operons in the flagella-chemotaxis regulon in Escherichia coli has been shown to be a highly ordered cascade which closely parallels the assembly of the flagellar structure and the chemotaxis machinery (T. Iino, Annu. Rev. Genet. 11:161-182, 1977; Y. Komeda, J. Bacteriol. 168: 1315-1318). The master operon, flbB, has been sequenced, and one of its gene products (Flal) has been identified. On the basis of the deduced amino acid sequence, the FIbB protein has similarity to an alternate sigma factor which is responsible for expression of flagella in Bacillus subtilis. In addition, we have sequenced the 5' regions of a number of flagellar operons and compared these sequences with the 5' region of flagellar operons directly and indirectly under FIbB and FlaI control.-We found both a consensus sequence which has been shown to be in all other flagellar operons (J. D. Helmano and M. J. Chamberlin, Proc. Natl. Acad. Sci. USA 84:6422-6424) and a derivative consensus sequence, which is found only in -the 5' region of operons directly under FlbB and FlaI control.Over 3% of the Escherichia coli K-12 genome is concerned with the synthesis, assembly, and function of its flagella (21). It is therefore not surprising that the regulation of the assembly of the flagellar apparatus is highly coordinated and controlled at least in part by the sequential expression of known flagellar operons. fla-lacZ fusion studies have revealed a hierarchy of transcription beginning with genes whose products are required for the formation of the basal body apparatus and hook, followed by the flagellar filament itself, and ending with the motility and chemotaxis machinery (21).Expression of the flagellar gene cascade begins with the flbB operon, consisting of theflbB andflaI genes (23, 35, 36).These' genes are required for the expression of all the remaining flagellar genes. Hence, theflbB operon is believed to serve a master regulatory role (37). There is evidence that other genes may be important for flagellar gene expression farther down the cascade (19,22). The production of the flagellum is under positive control from the pleiotropic regulatoiry protein cyclic AMP (cAMP) receptor protein (CRP) (also called catabolite-activated protein) (1,41). Therefore, similar to other operons which are catabolite repressed, flagellar genes are not expressed in the presence of glucose (37). Constitutive flagellar synthesis mutations which are insensitive to catabolite repression have been mapped to thefbB locus in E. coli and to the analogous locus in Salmonella typhimurium (24,36). Presumably, in the presence of sufficient cAMP, a CRP-cAMP complex binds to a region near the flbB operon, thereby inducing its transcription. In addition, the flagellar genes are expressed optimally at 34°C and not at 42°C (30). Mutations which result in the synthesis of flage}la at high temperatures map to two loci: the flaD gene and the flbB operon (37). The regulation of groups of operons in an ordered cascade often involves the part...

CheY is the response regulator in the signal transduction pathway of bacterial chemotaxis. Position 106 of CheY is occupied by a conserved aromatic residue (tyrosine or phenylalanine) in the response regulator superfamily. A number of substitutions at position 106 have been made and characterized by both behavioral and biochemical studies. On the basis of the behavioral studies, the phenotypes of the mutants at position 106 can be divided into three categories: (i) hyperactivity, with a tyrosine-to-tryptophan mutation (Y106W) causing increased tumble signaling but impairing chemotaxis; (ii) low-level activity, with a tyrosine-to-phenylalanine change (Y106F) resulting in decreased tumble signaling and chemotaxis; and (iii) no activity, with substitutions such as Y106L, Y106I, Y106V, Y106G, and Y106C resulting in no chemotaxis and a smooth-swimming phenotype. All three types of mutants can be phosphorylated by CheA-phosphate in vitro to a level similar to that of wild-type CheY. Autodephosphorylation rates are similar for all categories of mutants. All mutant proteins displayed less than twofold increased rates compared with wild-type CheY. Binding of the mutant proteins to FliM was similar to that of the wild-type CheY in the CheY-FliM binding assays. The combined results from in vivo behavioral and in vitro biochemical studies suggest that the diverse phenotypes of the Y106 mutants are not due to a variation in phosphorylation or dephosphorylation ability nor in affinity for the switch. With reference to the structures of wild-type CheY and the T87I CheY mutant, our results suggest that rearrangements of the orientation of the tyrosine side chain at position 106 are involved in the signal transduction of CheY. These data also suggest that the binding of phosphoryl-CheY to the flagellar motor is a necessary, but not sufficient, event for signal transduction.

Motility and chemotaxis allow cells to move away from stressful microenvironments. Motility of Escherichia coli in batch cultures, as measured by cell swimming speed, was low in early-exponential-phase cells, peaked as the cells entered post-exponential phase, and declined into early stationary phase. Transcription from the flhB operon and synthesis of flagellin protein similarly peaked in late exponential and early post-exponential phases, respectively. The increase in swimming speed between early-exponential and post-exponential phases was correlated with twofold increases in both flagellar length and flagellar density per cell volume. This increased investment in flagella probably reflects the increased adaptive value of motility in less favorable environments. The decrease in speed between post-exponential and stationary phases was correlated with a threefold decrease in torque produced by the flagellar motors and presumably reflects decreased proton motive force available to stationary-phase cells.