Localization of proteins controlling motility and chemotaxis in Escherichia coli.

Flagellar proteins controlling motility and chemotaxis in Escherichia coli were selectively labeled in vivo with [35S]methionine. This distribution of these proteins in subcellular fractions was examined by sodium dodecyl sulfatepolyacrylamide gel electrophoresis and autoradiography. The motA, motB, cheM, and cheD gene products were found to be confined exclusively to the inner cytoplasmic membrane fraction, whereas the cheY, cheW, and cheA (66,000 daltons) polypeptides appeared only in the soluble cytoplasmic fraction. The cheB, cheX, cheZ, and cheA (76,000 daltons) proteins, however, were distributed in both the cytoplasm and the inner membrane fractions. The hag gene product (flagellin) was the only flagellar protein examined that copurified with the outer lipopolysaccharide membrane. Differences in the intracellular locations of the che and mot gene prodcuts presumably reflect the functional attributes of these components.     

Studies on bacterial chemotaxis. II. Effect of cheB and cheZ mutations on the methylation of methyl-accepting chemotaxis protein of Escherichia coli.

Radioactive proteins from chemotactic mutants of Escherichia coli with continuous tumbling phenotype (cheB and cheZ) and their otherwise isogenic parent were compared by two-dimensional gel electrophoresis. The system was capable of separating non-methylated methyl-accepting chemotaxis protein (MCP) from its methylated equivalent. The analysis of proteins from the envelope fraction of the bacteria showed that the cheB mutants contained a larger portion of methylated MCP than did the parent. However, the change of MCP methylation level was small, if any, in cheZ strains. The results suggest that the product of cheB gene and the product of cheZ gene are not functional complementary. The product of cheB gene functions in controlling the level of methylation at the stationary state of the organisms. In addition to known MCP species, a new MCP of about 43,000 daltons was found. This MCP appears to be involved in transducing signals of some sugars.     

Cloning and characterization of chemotaxis genes in Pseudomonas aeruginosa

Two chemotaxis-defective mutants of Pseudomonas aeruginosa, designated PC3 and PC4, were selected by the swarm plate method after N-methyl-N'-nitro-N-nitrosoguanidine mutagenesis. These mutants were not complemented by the P. aeruginosa cheY and cheZ genes, which had been previously cloned (Masduki et al., J. Bacteriol., 177, 948-952, 1995). DNA sequences downstream of the cheY and cheZ genes were able to complement PC3 but not PC4. Sequence analysis of a 9.7-kb region directly downstream of the cheZ gene found three chemotaxis genes, cheA, cheB, and cheW, and seven unknown open reading frames (ORFs). The predicted translation products of the cheA, cheB, and cheW genes showed 33, 36, and 31% amino acid identity with Escherichia coli CheA, CheB, and CheW, respectively. Two of the unknown ORFs, ORF1 and ORF2, encoded putative polypeptides that resembled Bacillus subtilis MotA (40% amino acid identity) and MotB (34% amino acid identity) proteins, respectively. Although P. aeruginosa was found to have proteins similar to the enteric chemotaxis proteins CheA, CheB, CheW, CheY, and CheZ, the gene encoding a CheR homologue did not reside in the chemotaxis gene cluster. The P. aeruginosa cheR gene could be cloned by phenotypic complementation of the PC4 mutant. This gene was located at least 1,800 kb away from the chemotaxis gene cluster and encoded a putative polypeptide that had 32% amino acid identity with E. coli CheR.     

Synthesis of mot and che gene products of Escherichia coli programmed by hybrid ColE1 plasmids in minicells.

Hybrid Escherichia coli ColE1 plasmids carrying the genes for motility (mot) and chemotaxis (che) were transferred to a minicell-producing strain. The mot and che genes on the hybrid plasmid directed protein synthesis in minicells. Polypeptides synthesized in minicells were identical to the products of the motA, motB, cheA, cheW, cheM, cheX, cheB, cheY, and cheZ genes previously identified by using hybrid lambda and ultraviolet-irradiated host cells (Silverman and Simon, J. Bacteriol. 130:1317-1325, 1977), thus confirming these gene product assignments. The products of some che genes (cheA and cheM) appeared as more than one band on polyacrylamide gel electrophoresis, but analysis of partial peptide digests of these polypeptides suggested that the multiple forms were coded for by a single gene. Measurement of the physical length of the hybrid plasmids allowed an estimate of the amount of coding capacity of the cloned deoxyribonucleic acid, which was devoted to the synthesis of the mot and che gene products. These estimates were also consistent with the hypothesis that the multiple polypeptides corresponding to cheA and cheM were the products of single genes.     

Identification of polypeptides necessary for chemotaxis in Escherichia coli.

Molecular cloning techniques were used to construct Escherichia coli-lambda hybrids that contained many of the genes necessary for flagellar rotation and chemotaxis. The properties of specific hybrids that carried the classical "cheA" and "cheB" loci were examined by genetic complementation and by measuring the capacity of the hybrids to direct the synthesis of specific polypeptides. The results of these tests with lambda hybrids and with a series of deletion mutations derived from the hybrids redefined the "cheA" and "cheB" regions. Six genes were resolved: cheA, cheW, cheX, cheB, cheY, and cheZ. They directed the synthesis of specific polypeptides with the following apparent molecular weights: cheA, 76,000 and 66,000; cheW, 12,000; cheX, 28,000; cheB, 38,000; cheY, 8,000; and cheZ, 24,000. The presence of another gene, cheM, was inferred from the protein synthesis experiments. The cheM gene directed the synthesis of polypeptides with apparent molecular weights of 63,000, 61,000, and 60,000. The synthesis of all of these polypeptides is regulated by the same mechanisms that regulate the synthesis of flagellar-related structural components.     

Exploiting genome sequence: predictions for mechanisms of Campylobacter chemotaxis

The genome sequence of Campylobacter jejuni NCTC 11168 reveals the presence of orthologues of the chemotaxis genes cheA, cheW, cheV, cheY, cheR and cheB, ten chemoreceptor genes and two aerotaxis genes. The presence of cheV and a response regulator domain in CheA, combined with the absence of a cheZ gene and the lack of a response regulator domain in CheB, reveals significant differences in the C. jejuni chemotaxis system compared with that found in other bacteria.     

Studies on bacterial chemotaxis. III. Effect of methyl esters on the chemotactic response of Escherichia coli.

The methylation-demethylation reaction of methyl-accepting chemotaxis protein (MCP) is tightly coupled to the appearance of the chemotactic response in Escherichia coli. The bacteria might therefore show a unique response upon the addition of a compound containing a methyl group. We selected methyl N-methyl anthranilate (NMMA) and its analogs for examination. When NMMA was added to a suspension of E. coli (wild type), the bacteria tumbled as it does in the presence of a repellent. NMMA caused tumbling of wild-type bacteria for at least 20 min, while a conventional repellent makes the bacteria tumble for at most one min. The effect of NMMA requires functional MCP, cheA gene product, cheB gene product, and possibly cheX gene product. A positive signal of NMMA (i.e. sudden dilution) was detected by cheZ mutants with much higher sensitivity than that of a conventional repellent, indole, while both signals were rather poorly but equally detected by cheB mutants. These results suggest that the drug is related to the function of cheB gene product, a possible demethylating enzyme of MCP.     

Role of the CheW protein in bacterial chemotaxis: overexpression is equivalent to absence.

The cheW gene from Escherichia coli has been cloned an inducible promoter, and the effects of the overproduction of the CheW protein on chemotactic behavior and receptor covalent modification have been examined. Plasmids that contain the cheW gene behind a regulatable promoter complement a cheW mutation when the CheW protein is produced at low levels. However, when the CheW protein is greatly overproduced in either a wild-type strain or a cheW mutant, chemotaxis is greatly inhibited, cheW null mutant cells swim smoothly as if they were constantly responding to an attractant. Surprisingly, cells in which the CheW protein is overproduced also swim smoothly. The behavioral defect produced by overproduction of the CheW protein does not require the presence of the cheR, cheB, or cheZ gene. Receptor demethylation is also inhibited by overproduction of the CheW protein, as it is by a mutation in the cheW gene or a response to an attractant. In all respects, therefore, overproduction of the CheW protein has the same consequences as does a mutation in the cheW gene or a response to an attractant. A model involving two states of the CheW protein is proposed to explain its role in bacterial chemotaxis.     

Tumbling chemotaxis mutants of Escherichia coli: possible gene-dependent effect of methionine starvation.

Some mutants defective in chemotaxis show incessant tumbling behavior and are called tumbling mutants. Previously described tumbling mutations lie in two genes, cheB and cheZ (41.5 min on Escherichia coli map). Genetic analysis of various tumbling mutants, however, revealed that two more genetic loci, cheC (43 min) and cheE (99.2 min), could also mutate to produce tumbling mutants. The genetic map around cheC was revised: his flaP flaQ flaR flbD flaA (= cheC) flaE. flbD is a new gene. When cells were starved for methionine, the tumbling mutants changed their swimming behavior depending on the che gene mutated. cheZ mutants, like wild-type bacteria, ceased tumbling shortly after removal of methionine. The tumbling of cheB or cheE mutants was depressed after prolonged methionine starvation in the presence of a constant level of an attractant. cheC tumbling mutants appeared unique in that they did not cease tumbling even when cells were deprived of methionine. By contrast, arsenate treatment of the tumbling mutants resulted in smooth swimming of the cells in every case. These results suggest that two different processes are involved in regulation of tumbling; one requiring methionine and the other requiring some phosphorylated compound.     

Methylation of chemotaxis-specific proteins in Escherichia coli cells permeable to S-adenosylmethionine

Using a modification of the EGTA treatment of Oishi and Smith [Oishi, M., & Smith, C. L. (1978) Proc. Natl. Acad. Sci. U.S.A. 75, 3569], Escherichia coli cells have been made permeable to S-adenosylmethionine and other related molecules in order to facilitate the study of methylation in chemotaxis. The permeable cells are nonmotile but respond to chemotactic stimuli by reversible methylation of their methyl-accepting chemotactic proteins (MCP I and MCP II) in a manner similar to that of untreated, motile cells. Addition of S-adenosyl-L-[methyl-3H]methionine to the permeable cells specifically labels two proteins, MCP I and MCP II. Methylation of these MCP's is dependent on the presence of wild-type gene products of flaI, flaA, cheB, cheX, tsr, and tar. The extent of methylation of the MCP's is affected by the presence of attractants or repellents: addition of attractant increases the steady-state level of methylation; addition of repellent causes rapid demethylation to a new steady-state level. Methylation is inhibited by the addition of the transmethylase inhibitors A9145C and Sinefungin, which are S-adenosylmethionine analogues, and by S-adenosylhomocysteine.     

Determinants of chemotactic signal amplification in Escherichia coli

A well-characterized protein phosphorelay mediates Escherichia coli chemotaxis towards the amino acid attractant aspartate. The protein CheY shuttles between flagellar motors and methyl-accepting chemoreceptor (MCP) complexes containing the linker CheW and the kinase CheA. CheA-CheY phosphotransfer generates phospho-CheY, CheY-P. Aspartate triggers smooth swim responses by inactivation of the CheA bound to the target MCP, Tar; but this mechanism alone cannot explain the observed response sensitivity. Here, we used behavioral analysis of mutants deleted for CheZ, a catalyst of CheY-P dephosphorylation, or the methyltransferase CheR and/or the methylesterase CheB to examine the roles of accelerated CheY-P dephosphorylation and MCP methylation in enhancement of the chemotactic response. The extreme motile bias of the mutants was adjusted towards wild-type values, while preserving much of the aspartate response sensitivity by expressing fragments of the MCP, Tsr, that either activate or inhibit CheA. We then measured responses to small jumps of aspartate, generated by flash photolysis of photo-labile precursors. The stimulus-response relation for Delta cheZ mutants overlapped that for the host strains. Delta cheZ excitation response times increased with stimulus size consistent with formation of an occluded CheA state. Thus, neither CheZ-dependent or independent increases in CheY-P dephosphorylation contribute to the excitation response. In Delta cheB Delta cheR or Delta cheR mutants, the dose for a half-maximal response, [Asp](50), was ca 10 microM; but was elevated to 100 microM in Delta cheB mutants. In addition, the stimulus-response relation for these mutants was linear, consistent with stoichiometric inactivation, in contrast to the non-linear relation for wild-type E. coli. These data suggest that response sensitivity is controlled by differential binding of CheR and/or CheB to distinct MCP signaling conformations.     

Cluster II che genes from Pseudomonas aeruginosa are required for an optimal chemotactic response

Pseudomonas aeruginosa, a gamma-proteobacterium, is motile by means of a single polar flagellum and is chemotactic to a variety of organic compounds and phosphate. P. aeruginosa has multiple homologues of Escherichia coli chemotaxis genes that are organized into five gene clusters. Previously, it was demonstrated that genes in cluster I and cluster V are essential for chemotaxis. A third cluster (cluster II) contains a complete set of che genes, as well as two genes, mcpA and mcpB, encoding methyl-accepting chemotaxis proteins. Mutations were constructed in several of the cluster II che genes and in the mcp genes to examine their possible contributions to P. aeruginosa chemotaxis. A cheB2 mutant was partially impaired in chemotaxis in soft-agar swarm plate assays. Providing cheB2 in trans complemented this defect. Further, overexpression of CheB2 restored chemotaxis to a completely nonchemotactic, cluster I, cheB-deficient strain to near wild-type levels. An mcpA mutant was defective in chemotaxis in media that were low in magnesium. The defect could be relieved by the addition of magnesium to the swarm plate medium. An mcpB mutant was defective in chemotaxis when assayed in dilute rich soft-agar swarm medium or in minimal-medium swarm plates containing any 1 of 60 chemoattractants. The mutant phenotype could be complemented by the addition of mcpB in trans. Overexpression of either McpA or McpB in P. aeruginosa or Escherichia coli resulted in impairment of chemotaxis, and these cells had smooth-swimming phenotypes when observed under the microscope. Expression of P. aeruginosa cheA2, cheB2, or cheW2 in E. coli K-12 completely disrupted wild-type chemotaxis, while expression of cheY2 had no effect. These results indicate that che cluster II genes are expressed in P. aeruginosa and are required for an optimal chemotactic response.     

Identification of a chemotaxis operon with two cheY genes in Rhodobacter sphaeroides

A large chemotaxis operon was identified in Rhodobacter sphaeroides WS8-N using a probe based on the 3' terminal portion of the Rhizobium meliloti cheA gene. Two genes homologous to the enteric cheY were identified in an operon also containing cheA , cheW , and cheR homologues. The deduced protein sequences of che gene products were aligned with those from Escherichia coli and shown to be highly conserved. A mutant with an interrupted copy of cheA showed normal patterns of swimming, unlike the equivalent mutants in E. coli which are smooth swimming. Tethered cheA mutant cells showed normal responses to changes in organic acids, but increased, inverted responses to sugars. The unusual behaviour of the cheA mutant and the identification of two homologues of cheY suggests that R. sphaeroides has at least two pathways controlling motor activity. To identify functional similarity between the newly identified R. sphaeroides Che pathway and the methyl-accepting chemotaxis protein (MCP)-dependent pathway in enteric bacteria, the R. sphaeroides cheW gene was expressed in a cheW mutant strain of E. coli and found to complement, causing a partial return to a swarming phenotype. In addition, expression of the R. sphaeroides gene in wild-type E. coli resulted in the same increased tumbling and reduced swarming as seen when the native gene is over-expressed in E. coli . The identification of che homologues in R. sphaeroides and complementation by cheW suggests the presence of MCPs in an organism previously considered to use only MCP-independent sensing. The MCP-dependent pathway, appears conserved. In R. sphaeroides this pathway may mediate responses to sugars, while responses to organic acids may in involve a second system, possibly using the second CheY protein identified in this study.     

Requirement of the cheB function for sensory adaptation in Escherichia coli.

The chemotactic behavior of Escherichia coli mutants defective in cheB function, which is required to remove methyl esters from methyl-accepting chemotaxis proteins, was investigated by subjecting swimming or antibody-tethered cells to various attractant chemicals. Two cheB point mutants, one missense and one nonsense, exhibited stimulus response times much longer than did the wild type, but they eventually returned to the prestimulus swimming pattern, indicating that they were not completely defective in sensory adaptation. In contrast, strains deleted for the cheB function showed no evidence of adaptation ability after stimulation. The crucial difference between these strains appeared to be the residual level of cheB-dependent methylesterase activity they contained. Both point mutants showed detectable levels of methanol evolution due to turnover of methyl groups on methyl-accepting chemotaxis protein molecules, whereas the cheB deletion mutant did not. In addition, it was possible to incorporate the methyl label into the methyl-accepting chemotaxis proteins of the point mutants but not into those of the cheB deletion strain. These findings indicate that cheB function is essential for sensory adaptation in Escherichia coli.     

The roles of the multiple CheW and CheA homologues in chemotaxis and in chemoreceptor localization in Rhodobacter sphaeroides

Rhodobacter sphaeroides has multiple homologues of most of the Escherichia coli chemotaxis genes, organized in two major operons and other, unlinked, loci. These include cheA1 and cheW1 (che Op1) and cheA2, cheW2 and cheW3 (che Op2). We have deleted each of these cheA and cheW homologues in-frame and examined the chemosensory behaviour of these strains on swarm plates and in tethered cell assays. In addition, we have examined the effect of these deletions on the polar localization of the chemoreceptor McpG. In E. coli, deletion of either cheA or cheW results in a non-chemotactic phenotype, and these strains also show no receptor clustering. Here, we demonstrate that CheW2 and CheA2 are required for the normal localization of McpG and for normal chemotactic responses under both aerobic and photoheterotrophic conditions. Under aerobic conditions, deletion of cheW3 has no significant effect on McpG localization and only has an effect on chemotaxis to shallow gradients in swarm plates. Under photoheterotrophic conditions, however, CheW3 is required for McpG localization and also for chemotaxis both on swarm plates and in the tethered cell assay. These phenotypes are not a direct result of delocalization of McpG, as this chemoreceptor does not mediate chemotaxis to any of the compounds tested and can therefore be considered a marker for general methyl-accepting chemotaxis protein (MCP) clustering. Thus, there is a correlation between the normal localization of McpG (and presumably other chemoreceptors) and chemotaxis. We propose a model in which the multiple different MCPs in R. sphaeroides are contained within a polar chemoreceptor cluster. Deletion of cheW2 and cheA2 under both aerobic and photoheterotrophic conditions, and cheW3 under photoheterotrophic conditions, disrupts the cluster and hence reduces chemotaxis to any compound sensed by these MCPs.     

Genetics of methyl-accepting chemotaxis proteins in Escherichia coli: organization of the tar region.

The tar locus of Escherichia coli specifies one of the major species of methyl-accepting proteins involved in the chemotactic behavior of this organism. The physical and genetic organization of the tar region was investigated with a series of specialized lambda transducing phages and plasmid clones. The tar gene was mapped at the promoter-proximal end of an operon containing five other chemotaxis-related loci. Four of those genes (cheR, cheB, cheY and cheZ) are required for all chemotactic responses; consequently, polar mutations in the tar gene resulted in a generally nonchemotactic phenotype. The fifth gene, tap, was mapped between the tar and cheR loci and specified the production of a 65-kilodalton methyl-accepting protein. Unlike the tar locus, which is required for chemotaxis to aspartate and maltose, mutants lacking only the tap function had no obvious defects in chemotactic ability. Genetic and physical maps of the tar-tap region were constructed with Mu d1 (Apr lac) insertion mutations, whose polar properties conferred a phenotype suitable for deletion mapping studies. Restriction endonuclease analyses of phage and plasmid clones indicated that all of the genetic coding capacity in the tar region is now accounted for.     

Fine tuning bacterial chemotaxis: analysis of Rhodobacter sphaeroides behaviour under aerobic and anaerobic conditions by mutation of the major chemotaxis operons and cheY genes

Rhodobacter sphaeroides chemotaxis is significantly more complex than that of enteric bacteria. Rhodobacter sphaeroides has multiple copies of chemotaxis genes (two cheA, one cheB, two cheR, three cheW, five cheY but no cheZ), controlling a single 'stop-start' flagellum. The growth environment controls the level of expression of different groups of genes. Tethered cell analysis of mutants suggests that CheY(4) and CheY(5) are the motor-binding response regulators. The histidine protein kinase CheA(2) mediates an attractant ('normal') response via CheY(4), while CheA(1) and CheY(5) appear to mediate a repellent ('inverted') response. CheY(3) facilitates signal termination, possibly acting as a phosphate sink, although CheY(1) and CheY(2) can substitute. The normal and inverted responses may be initiated by separate sets of chemoreceptors with their relative strength dependent on growth conditions. Rhodobacter sphaeroides may use antagonistic responses through two chemosensory pathways, expressed at different levels in different environments, to maintain their position in a currently optimum environment. Complex chemotaxis systems are increasingly being identified and the strategy adopted by R.sphaeroides may be common in the bacterial kingdom.     

Identification of a fourth cheY gene in Rhodobacter sphaeroides and interspecies interaction within the bacterial chemotaxis signal transduction pathway

The Escherichia coli chemotaxis signal transduction pathway has: CheA, a histidine protein kinase; CheW, a linker between CheA and sensory proteins; CheY, the effector; and CheZ, a signal terminator. Rhodobacter sphaeroides has multiple copies of these proteins (2 x CheA, 3 x CheW and 3 x CheY, but no CheZ). In this study, we found a fourth cheY and expressed these R. sphaeroides proteins in E. coli. CheA2 (but not CheA1) restored swarming to an E. coli cheA mutant (RP9535). CheW3 (but not CheW2) restored swarming to a cheW mutant of E. coli (RP4606). R. sphaeroides CheYs did not affect E. coli lacking CheY, but restored swarming to a cheZ strain (RP1616), indicating that they can act as signal terminators in E. coli. An E. coli CheY, which is phosphorylated but cannot bind the motor (CheY109KR), was expressed in RP1616 but had no effect. Overexpression of CheA2, CheW2, CheW3, CheY1, CheY3 and CheY4 inhibited chemotaxis of wild-type E. coli (RP437) by increasing its smooth-swimming bias. While some R. sphaeroides proteins restore tumbling to smooth-swimming E. coli mutants, their activity is not controlled by the chemosensory receptors. R. sphaeroides possesses a phosphorelay cascade compatible with that of E. coli, but has additional incompatible homologues.