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.     

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.     

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.     

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.     

Chemotaxis in Escherichia coli: associations of protein components

Interactions between protein components of the chemotaxis mechanism in Escherichia coli were investigated by using the cleavable cross-linking reagent, dithiobis(succinimidyl propionate). Two methods were used to allow detection of chemotaxis-specific proteins in intact cells. The first method was to program their synthesis in the presence of [35S]methionine using lambda E. coli hybrid phages which carry the chemotaxis genes. The second method was to label endogenous methyl-accepting chemotaxis proteins (MCP's), with the methyl donor S-adenosyl-L-[methyl-3H]methionine, after permeabilizing the cells with EGTA. Physical associations between proteins were analyzed, after cross-linking, by two dimensional NaDodSO4-polyacrylamide gel electrophoresis. Both labeling methods demonstrate that MCP I and MCP II exist as functional tetramers. Other proteins involved with chemotaxis were found to form dimers and higher polymers. Phage-directed products of cheW, cheX, motA, and cheA formed dimers. CheB and hag products formed multimers. A number of apparent interactions between different gene products were detected as well. Products of cheB, cheW, cheZ, motA, and motB were found to form complexes with other gene products. Included are results consistent with interactions between the products of cheB and cheZ.     

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.     

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.     

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.     

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.     

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.     

Chemosensory and photosensory perception in purple photosynthetic bacteria utilize common signal transduction components.

The chemotaxis gene cluster from the photosynthetic bacterium Rhodospirillum centenum contains five open reading frames (ORFs) that have significant sequence homology to chemotaxis genes from other bacteria. To elucidate the functions of each ORF, we have made various mutations in the gene cluster and analyzed their phenotypic defects. Deletion of the entire che operon (delta che), as well as nonpolar disruptions of cheAY, cheW, and cheR, resulted in a smooth-swimming phenotype, whereas disruption of cheB resulted in a locked tumbly phenotype. Each of these mutants was defective in chemotactic response. Interestingly, disruption of cheY resulted in a slight increase in the frequency of tumbling/reversal with no obvious defects in chemotactic response. In contrast to observations with Escherichia coli and several other bacteria, we found that all of the che mutant cells were capable of differentiating into hyperflagellated swarmer cells when plated on a solid agar surface. When viewed microscopically, the smooth-swimming che mutants exhibited active surface motility but were unable to respond to a step-down in light intensity. Both positive and negative phototactic responses were abolished in all che mutants, including the cheY mutant. These results indicate that eubacterial photosensory perception is mediated by light-generated signals that are transmitted through the chemotaxis signal transduction cascade.     

Isolation and characterization of chemotaxis mutants and genes of Pseudomonas aeruginosa.

Two chemotaxis-defective mutants of Pseudomonas aeruginosa, designated PC1 and PC2, were selected by the swarm plate method after N-methyl-N'-nitro-N-nitrosoguanidine mutagenesis. These mutants were fully motile but incapable of swarming, suggesting that they had a defect in the intracellular signalling pathway. Computer-assisted capillary assays confirmed that they failed to show behavioral responses to chemical stimuli, including peptone, methyl thiocyanate, and phosphate. Two chemotaxis genes were cloned by phenotypic complementation of PC1 and PC2. From nucleotide sequence analysis, one gene was found to encode a putative polypeptide that was homologous to the enteric CheZ protein, while the other gene was cheY, which had been previously reported (M. N. Starnbach and S. Lory, Mol. Microbiol. 6:459-469, 1992). Deletion and complementation analysis showed that PC1 was a cheY mutant, whereas PC2 had a double mutation in the cheY and cheZ genes. A chromosomal cheZ mutant, constructed by inserting a kanamycin resistance gene cassette into the wild-type gene, changed its swimming direction much more frequently than did wild-type strain PAO1. In contrast, cheY mutants were found to rarely reverse their swimming directions.     

Function of a chemotaxis-like signal transduction pathway in modulating motility, cell clumping, and cell length in the alphaproteobacterium Azospirillum brasilense

A chemotaxis signal transduction pathway (hereafter called Che1) has been previously identified in the alphaproteobacterium Azospirillum brasilense. Previous experiments have demonstrated that although mutants lacking CheB and/or CheR homologs from this pathway are defective in chemotaxis, a mutant in which the entire chemotaxis pathway has been mutated displayed a chemotaxis phenotype mostly similar to that of the parent strain, suggesting that the primary function of this Che1 pathway is not the control of motility behavior. Here, we report that mutants carrying defined mutations in the cheA1 (strain AB101) and the cheY1 (strain AB102) genes and a newly constructed mutant lacking the entire operon [Δ(cheA1-cheR1)::Cm] (strain AB103) were defective, but not null, for chemotaxis and aerotaxis and had a minor defect in swimming pattern. We found that mutations in genes of the Che1 pathway affected the cell length of actively growing cells but not their growth rate. Cells of a mutant lacking functional cheB1 and cheR1 genes (strain BS104) were significantly longer than wild-type cells, whereas cells of mutants impaired in the cheA1 or cheY1 genes, as well as a mutant lacking a functional Che1 pathway, were significantly shorter than wild-type cells. Both the modest chemotaxis defects and the observed differences in cell length could be complemented by expressing the wild-type genes from a plasmid. In addition, under conditions of high aeration, cells of mutants lacking functional cheA1 or cheY1 genes or the Che1 operon formed clumps due to cell-to-cell aggregation, whereas the mutant lacking functional CheB1 and CheR1 (BS104) clumped poorly, if at all. Further analysis suggested that the nature of the exopolysaccharide produced, rather than the amount, may be involved in this behavior. Interestingly, mutants that displayed clumping behavior (lacking cheA1 or cheY1 genes or the Che1 operon) also flocculated earlier and quantitatively more than the wild-type cells, whereas the mutant lacking both CheB1 and CheR1 was delayed in flocculation. We propose that the Che1 chemotaxis-like pathway modulates the cell length as well as clumping behavior, suggesting a link between these two processes. Our data are consistent with a model in which the function of the Che1 pathway in regulating these cellular functions directly affects flocculation, a cellular differentiation process initiated under conditions of nutritional imbalance.     

A che-like signal transduction cascade involved in controlling flagella biosynthesis in Rhodospirillum centenum

Rhodospirillum centenum is a photosynthetic bacterium capable of undergoing swim cell to swarm cell differentiation that allows this species to be motile on both liquid and solid media. Previous experiments have demonstrated that the che1 operon is required for the control of chemotactic and phototactic behaviour of both swim and swarm cells. In this report, we analyse the function of a second che-like gene cluster in R. centenum, the che2 gene cluster. In-frame deletion mutants of cheW2, cheB2, cheR2, cheY2, and of the entire che2 operon, exhibit defects in swim and swarm cell motility. Analysis of these strains demonstrates that they are non-motile, and that the non-motile phenotype is resulting from reduced polar and lateral flagella synthesis. Additionally, mutations in mcp2, ORF204, cheA2 and ORF74 remain chemotacticly and phototacticly competent at both high and low growth temperatures. Mutations in these che2 genes result in elevated levels of flagellin proteins giving rise to a hyperflagellate phenotype. We propose a model in which R. centenum utilizes a che-like signal transduction pathway (che2) for regulating flagellum synthesis in order to optimize swim cell-swarm cell differentiation in response to changing environmental conditions.     

CheA, CheW, and CheY are required for chemotaxis to oxygen and sugars of the phosphotransferase system in Escherichia coli.

We carried out studies with Escherichia coli to determine the site at which the methylation-independent pathways for taxis to oxygen and to sugars of the phosphoenolpyruvate:sugar phosphotransferase transport system converge with the methylation-dependent chemotaxis pathways. Using genetic reconstitution of the pathways in a null strain, we determined that all pathways examined required the products of the genes cheA, cheW, and cheY. Thus, we conclude that both the methylation-independent and methylation-dependent pathways converge at CheA, the histidine kinase product of cheA.     

Interactions between chemotaxis genes and flagellar genes in Escherichia coli

Escherichia coli mutants defective in cheY and cheZ function are motile but generally nonchemotactic; cheY mutants have an extreme counterclockwise bias in flagellar rotation, whereas cheZ mutants have a clockwise rotational bias. Chemotactic pseudorevertants of cheY and cheZ mutants were isolated on semisolid agar and examined for second-site suppressors in other chemotaxis-related loci. Approximately 15% of the cheZ revertants and over 95% of the cheY revertants contained compensatory mutations in the flaA or flaB locus. When transferred to an otherwise wild-type background, most of these suppressor mutations resulted in a generally nonchemotactic phenotype: suppressors of cheY caused a clockwise rotational bias; suppressors of cheZ produced a counterclockwise rotational bias. Chemotactic double mutants containing a che and a fla mutation invariably exhibited flagellar rotation patterns in between the opposing extremes characteristic of the component mutations. This additive effect on flagellar rotation resulted in essentially wild-type swimming behavior and is probably the major basis of suppressor action. However, suppression effects were also allele specific, suggesting that the cheY and cheZ gene products interact directly with the flaA and flaB products. These interactions may be instrumental in establishing the unstimulated swimming pattern of E. coli.