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Specific CheA-short (CheAs) residues, L123 and L126, were identified as critical for CheZ binding. In the Cbe[A.sub.s] 'P1-CheZ nuclear magnetic resonance structure, these residues form an interaction surface on [alpha]-helix E in the 'P1 domain. Both L123 and L126 are buried in CheA-long (Che[A.sub.L]), providing an explanation for why Che[A.sub.L] fails to bind CheZ.

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.

This paper focuses on global gene regulation by FIhD/FIhC in enteric bacteria. Even though Yersinia enterocolitica FIhD/FIhC can complement an Escherichia coli flhDC mutant for motility, it is not known if the Y. enterocolitica FIhD/FIhC complex has an effect on metabolism similar to E. coli. To study metabolic gene regulation, a partial Yersinia enterocolitica 8081c microarray was constructed and the expression patterns of wild-type cells were compared to an flhDC mutant strain at 25 and 37 [degrees]C. The overlap between the E. coli and Y. enterocolitica FIhD/FIhC regulated genes was 25%. Genes that were regulated at least fivefold by FIhD/FIhC in Y. enterocolitica are genes encoding urocanate hydratase (hutU), imidazolone propionase (hutl), carbamoylphosphate synthetase (carAB) and aspartate carbamoyltransferase (pyrBI). These enzymes are part of a pathway that is involved in the degradation of L-histidine to L-glutamate and eventually leads into purine/pyrimidine biosynthesis via carbamoylphosphate and carbamoylaspartate. A number of other genes were regulated at a lower rate. In two additional experiments, the expression of wild-type cells grown at 4 or 25 [degrees]C was compared to the same strain grown at 37 [degrees]C. The expression of the flagella master operon flhD was not affected by temperature, whereas the flagella-specific sigma factor fliA was highly expressed at 25 [degrees]C and reduced at 4 and 37 [degrees]C. Several other flagella genes, all of which are under the control of FliA, exhibited a similar temperature profile. These data are consistent with the hypothesis that temperature regulation of flagella genes might be mediated by the flagella-specific sigma factor FliA and not the flagella master regulator FIhD/FIhC.

The CheZ binding domain of CheA(sub s) is identified by testing the various fragments of CheA(sub s) that contain cysteine residues for CheZ binding activity in an ELISA-based CheA(sub s)-CheZ binding assay. It is shown that both the CheZ binding and activity enhancing functions of CheA(sub s) are located in its truncated P1 domain and the accessibility of the cysteine residue in P1 domain is important for the interaction between P1 domain and CheZ.

Research has been conducted on the transiently phosphorylated signal transduction protein CheY. The structural characterization of the activated site of this protein has been carried out.

The synthesis and biochemical characterization of an analogue of CheY-phosphate, a signal transduction protein in bacterial chemotaxis, were conducted. A combination of site-directed mutagenesis and chemical modification that leads to a stable protein with at least some of the biochemical characteristics of the normally phosphorylated protein was used. The analogue, designated phosphono-CheY, behaves like CheY-P(sub i) but is more chemically stable and is much more amenable to long-term studies of its structure.

The effect of FlhD, a positive regulator of cadA in Escherichia coli, on cell division rate is found to be mediated through the induction of cadA. The cad operon is composed of cadB and cadA, encoding the lysine/cadaverine antiporter CadB and the enzyme lysine decarboxylase CadA. Expression of this operon is dependent on the regulator CadC whose gene is found upstream of cadA. The expression from the p(sub cad) promoter is induced by low pH, low oxygen, rich medium and excess lysine.

The 106th residue of the Escherichia coli CheY response regulator is a conserved tyrosine or phenylalanine aromatic residue and is essential for signal transduction during chemotaxis. The substitution of this residue with different amino acids produces phenotypes with hyperactivity, low activity or no activity. The autodephosphorylation of all the mutants takes place at the same rate. The binding of phosphoryl-CheY to the flagellar motor is necessary though not sufficient for signal transduction.

The role of the Escherichia coli flagellar transcriptional activator, flhD, in cell division was determined. Analysis of the flhD mutant cells grown in tryptone broth by electron microscopy revealed a normal cell division rate and normal cell density. The mutant cultures had biomasses similar to that of wild-type flhD cultures. flhD mutant cells also exhibited normal uptake of serine from the medium. However, wild-type cells reduced their cell division rate at 50 muM serine. There was a strong correlation between levels of serine and cell division rates.

Examination of the distribution of FliG and FliM in Salmonella typhimurium cellular fractions and flagellar basal bodies showed both proteins to be present mainly in the fraction where flagellar structures are found. Large amounts of both proteins were detected in an equimolar ratio in the flagellum. These provide evidence that FliG and FliM function as a component of the extended basal structure in performing their role in flagellar morphogenesis.

Purified FlhC and FlhD proteins of the Escherichia coli flhD operon form a FlhD/FlhC protein molecule complex that exhibits DNA binding to the regions upstream of flhB, fliA, and fliL operons. DNase I footprinting of the binding activity indicates the preservation of a 48-bp region of the fliA operon, a 48-bp region of the fliL operon and a 50-bp region of the flhB operon. The FlhD/FlhC complex functions as a transcriptional activator of class II operon transcription.

The Escherichia coli CheY consists of a (beta/alpha)5 motif of five parallel beta strands in the center, which is enclosed by three alpha-helicases. The regions where the protein structure of the magnesium binds to the bacterial chemotaxis are not well defined. The lack of proper definition is due to the slow chemical exchange recorded on the nuclear magnetic time scale. The salt bridge between the Asp-57 and the Lys-109 is disturbed by the presence of the magnesium ions.

The fliP gene product of Escherichia coli is possibly a precursor protein with a 21 amino acid N-terminal. The fliP protein exhibits homology to the protein product of flaA gene of Rhizobium meliloti and spa24 gene of Shigella flexneri, revealing the protein's possible role in protein translocation. The fliO, fliQ and fliR genes of the fliL operon are identified by complementing with a 2.2-kb PstI restriction fragment.

The second meeting on Bacterial Locomotion and Signal Transduction saw 180 researchers discuss their developments, particularly at the molecular level. Most of the research is summarized, including those regarding chemoreceptors, signaling circuitry, the response regulator CheY, signaling components of other organisms, bacterial flagella, myxobacteria, crosstalk and integrated regulation, and tactic behavior in an archaebacterium. Trends of increasing relevance are also discussed.

The cell cycle regulation of flagellar genes in response to environmental stress was analyzed. Results of the study revealed that the expression of the flagellar master operon, flhDC, is significantly high at the middle of three consecutive cell cycles. On the other hand, it was lowest at the moment of cell division. Furthermore, the speed of individual cells were noticeably higher during the same period.

The amount of wild-type CheAL and CheAS in wild-type chemotactic cells were analyzed to determine the physical interactions of CheW, CheAL, CheZ and CheAS. Analysis of phosphorylating and dephosphorylating protein complexes in chemotactic bacterial cells indicated the ability of CheAS-CheW binding to inhibit CheAS-CheZ interaction. Furthermore, a CheAL-CheAS-CheW complex with a stoichiometric ration of 1:1:1 was detected in situ from chemotactic bacterial cells.

The protein product of the FlhD lhC genes in E. coli binds directly to the C-terminus of the alpha subunit of RNA polymerase and is therefore a class I transcription factor. These genes are part of the flagellar operons.

Research has been conducted on chemotaxis methyl-accepting protein Aer. The regulation of transcriptional activators of this protein and of flagellar expression has been investigated via the glass slide DNA microarrays, and the results are reported.