Amino acid efflux in response to chemotactic and osmotic signals in Bacillus subtilis.

We observed a large efflux of nonvolatile radioactivity from Bacillus subtilis in response to the addition of 31 mM butyrate or the withdrawal of 0.1 M aspartate in a flow assay. The major nonvolatile components effluxed were methionine, proline, histidine, and lysine. In studies of the release of volatile radioactivity in chemotaxis by B. subtilis cells that had been labeled with [3H]methionine, the breakdown of methionine to methanethiol can contribute substantially to the volatile radioactivity in fractions following addition of 0.1 M aspartate. However, methanol was confirmed to be released after aspartate addition and, in lesser quantities, after aspartate withdrawal. Methanol and methanethiol were positively identified by derivitization with 3,5-dinitro-benzoylchloride. Amino acid efflux but not methanol release was observed in response to 0.1 M aspartate stimulation of a cheR mutant of B. subtilis that lacks the chemotaxis methylesterase. The amino acid efflux could be reproduced by withdrawal of 0.1 M NaCl, 0.2 M sucrose, or 0.2 M xylitol and is probably the result of changes in osmolarity. Chemotaxis to 10 mM alanine or 10 mM proline resulted in methanol release but not efflux of amino acids. In behavioral studies, B. subtilis tumbled for 16 to 18 s in response to a 200 mosM upshift and for 14 s after a 20 mosM downshift in osmolarity when the bacteria were in perfusion buffer (40 mosM). The pattern of methanol release was similar to that observed in chemotaxis. This is consistent with osmotaxis in B. subtilis away from an increase or decrease in the osmolarity of the incubation medium. The release of methanol suggests that osmotaxis is correlated with methylation of a methyl-accepting chemotaxis protein.     

CheB is required for behavioural responses to negative stimuli during chemotaxis in Bacillus subtilis

The methyl-accepting chemotaxis protein, McpB, is the sole receptor mediating asparagine chemotaxis in Bacillus subtilis. In this study, we show that wild-type B. subtilis cells contain approximately 2,000 copies of McpB per cell, that these receptors are localized polarly, and that titration of only a few receptors is sufficient to generate a detectable behavioural response. In contrast to the wild type, a cheB mutant was incapable of tumbling in response to decreasing concentrations of asparagine, but the cheB mutant was able to accumulate to low concentrations of asparagine in the capillary assay, as observed previously in response to azetidine-2-carboxylate. Furthermore, net demethylation of McpB is logarithmically dependent on asparagine concentration, with half-maximal demethylation of McpB occurring when only 3% of the receptors are titrated. Because the corresponding methanol production is exponentially dependent on attractant concentration, net methylation changes and increased turnover of methyl groups must occur on McpB at high concentrations of asparagine. Together, the data support the hypothesis that methylation changes occur on asparagine-bound McpB to enhance the dynamic range of the receptor complex and to enable the cell to respond to a negative stimulus, such as removal of asparagine.     

Aerotaxis in Salmonella typhimurium: role of electron transport

Sensory transduction in aerotaxis required electron transport, in contrast to chemotaxis, which is independent of electron transport. Assays for aerotaxis were developed by employing spatial and temporal oxygen gradients imposed independently of respiration. By varying the step increase in oxygen concentration in the temporal assay, the dose-response relationship was obtained for aerotaxis in Salmonella typhimurium. A half-maximal response at 0.4 microM oxygen and inhibition by 5 mM KCN suggested that the "receptor" for aerotaxis is cytochrome o. The response was independent of adenosine triphosphate formation via oxidative phosphorylation but did correlate with changes in membrane potential monitored with the fluorescent cyanine dye diS-C3-(5). Nitrate and fumarate, which are alternative electron acceptors for the respiratory chain in S. typhimurium, inhibited aerotaxis when nitrate reductase and fumarate reductase were induced. These results support the hypothesis that taxis to oxygen, nitrate, and fumarate is mediated by the electron transport system and by changes in the proton motive force. Aerotaxis was normal in Escherichia coli mutants that were defective in the tsr, tar, or trg genes; in S. typhimurium, oxygen did not stimulate methylation of the products of these genes. A cheC mutant which shows an inverse response to chemoattractants also gave an inverse response to oxygen. Therefore, aerotaxis is transduced by a distinct and unidentified signally protein but is focused into the common chemosensory pathway before the step involving the cheC product. When S. typhimurium became anaerobic, the decreased proton motive force from glycolysis supported slow swimming but not tumbling, indicating that a minimum proton motive force was required for tumbling. The bacteria rapidly adapted to the anaerobic condition and resumed tumbling after about 3 min. The adaptation period was much shorter when the bacteria had been previously grown anaerobically.     

In vivo and in vitro chemotactic methylation in Bacillus subtilis

Two doublets of Bacillus subtilis membrane proteins with molecular weights of 69,000 and 71,000 and of 30,000 and 30,800, were labeled by C3H3 transfer in the absence of protein synthesis. In addition, there was intense methylation of several low-molecular-weight substances. Both doublets were missing in a chemotaxis mutant. The equivalent proteins in Escherichia coli and Salmonella typhimurium are believed to be the methyl-accepting chemotaxis proteins. The higher-molecular-weight doublet bands were increased in degree of methylation upon addition of attractant to the bacteria. A methyltransferase from B. subtilis that methylates the wild-type membrane significantly better than the mutant membrane, using S-adenosylmethionine, has been partly purified. The methylated product was alkali labile and is probably a gamma-glutamyl methyl ester, as in E. coli and S. typhimurium. Ca2+ ion inhibited the methyltransferase, with a Ki of about 80 nM. Analysis of the in vitro methylation product showed labeling of the 69,000-dalton methyl-accepting chemotaxis protein and a low-molecular-weight protein, using wild-type membrane. Labeling of the low-molecular-weight protein but not of the 69,000 dalton protein was observed when the mutant membrane was used. The chemotaxis mutant tumbled much longer than the wild type when diluted away from attractant.     

In vitro methylation and demethylation of methyl-accepting chemotaxis proteins in Bacillus subtilis

Bacillus subtilis responds to attractants by demethylating a group of integral membrane proteins referred to as methyl-accepting chemotaxis proteins (MCPs). We have studied the methylation and demethylation of these proteins in an in vitro system, consisting of membrane vesicles, and purified methyltransferase and methylesterase. The chemoattractant aspartate was found to inhibit methylation and stimulate demethylation of MCPs. Escherichia coli radiolabeled membranes in the presence of B. subtilis enzyme do not respond to aspartate by an increase demethylation rate. We also report that B. subtilis MCPs are multiply methylated, demethylation resulting in slower migrating proteins on sodium dodecyl sulfate-polyacrylamide gels.     

A methyl-accepting protein involved in multiple-sugar chemotaxis by Cellulomonas gelida.

Tethered-cell and capillary assays indicated that L-methionine is required by Cellulomonas gelida for its normal cell motility pattern and chemotaxis and that S-adenosylmethionine is involved in sugar chemotaxis by this cellulolytic bacterium. In addition, in vivo methylation assays showed that several proteins were methylated in the absence of protein synthesis. The incorporated methyl groups were alkali sensitive. Of special interest was the observation that the methylation level of a 51,000-Mr protein increased two- to fivefold upon addition of various sugar attractants and decreased after the removal of the attractants. The increase was less pronounced in mutants defective in sugar chemotaxis and appeared to be specifically involved with sugar chemotaxis. Furthermore, cell fractionation and in vitro methylation assays demonstrated that the 51,000-Mr protein is located in the cytoplasmic membrane. These results suggest that a specific methyl-accepting chemotaxis protein is involved in multiple-sugar chemotaxis by C gelida. During chemotaxis, the changes of methylesterase activity in C gelida cells were similar to those in Escherichia coli RP437 cells, as determined by a continuous-flow assay for methanol evolution. Thus, the mechanism of methyl-accepting chemotaxis protein-mediated chemotaxis of the gram-positive C. gelida appears to be similar to that of the gram-negative E. coli rather than to that of other gram-positive bacteria, such as Bacillus subtilis.     

Rapid attractant-induced changes in methylation of methyl-accepting chemotaxis proteins in Bacillus subtilis

In Bacillus subtilis, addition of chemotactic attractant causes an immediate change in distribution of methyl groups on methyl-accepting chemotaxis proteins (MCPs), whereas in Escherichia coli, it causes changes that occur throughout the adaptation period. Thus, methylation changes in B. subtilis are probably related to excitation, not adaptation. If labeled cells are exposed to excess nonradioactive methionine, then attractant causes immediate 50% delabeling of the MCPs, suggesting that a flux of methyl groups through the MCPs occurs. Methanol is given off at a high rate during the adaptation period and probably reflects demethylation of some substance to bring about adaptation. The fact that many radioactive methyl groups are lost immediately from the MCPs but only slowly arise as methanol is consistent with the hypothesis that they are transferred from the MCPs to a carrier from which methanol arises. Demethylation of this carrier may cause adaptation.     

Methyl transfer in chemotaxis toward sugars by Bacillus subtilis.

Like amino acids, the sugars glucose and the nonmetabolizable 2-deoxyglucose caused a turnover of methyl groups on the methyl-accepting chemotaxis proteins. These sugars also caused methanol formation on addition. Thus, in contrast to chemotaxis in Escherichia coli, taxis to phosphotransferase sugars by Bacillus subtilis utilizes the methyl-accepting chemotaxis proteins.     

An Archaeal Aerotaxis Transducer Combines Subunit I Core Structures of Eukaryotic Cytochrome c Oxidase and Eubacterial Methyl-Accepting Chemotaxis Proteins

Signal transduction in the archaeon Halobacterium salinarum is mediated by three distinct subfamilies of transducer proteins. Here we report the complete htrVIII gene sequence and present analysis of the encoded primary structure and its functional features. HtrVIII is a 642-amino-acid protein and belongs to halobacterial transducer subfamily B. At the N terminus, the protein contains six transmembrane segments that exhibit homology to the heme-binding sites of the eukaryotic cytochrome c oxidase. The C-terminal domain has high homology with the eubacterial methyl-accepting chemotaxis protein. The HtrVIII protein mediates aerotaxis: a strain with a deletion of the htrVIII gene loses aerotaxis, while an overproducing strain exhibits stronger aerotaxis. We also demonstrate that HtrVIII is a methyl-accepting protein and demethylates during the aerotaxis response.     

Evidence for an intermediate methyl-acceptor for chemotaxis in Bacillus subtilis

Bacillus subtilis responds to chemotactic attractants by demethylating certain membrane-bound proteins, termed methyl-accepting chemotaxis proteins (MCPs) and by augmenting the evolution of methanol. We propose that the methanol comes from a methylated intermediate rather than directly from the MCPs themselves. First, repellent blocks attractant-induced smooth swimming and methanol formation, but not MCP demethylation. Second, prior treatment of cells with much attractant to reduce radiolabeling of MCPs and increase that of the putative intermediate caused increased, rather than decreased, production of methanol upon addition and then removal of the repellent. Third, such cells also produced much, rather than little, methanol upon addition of less attractant than during the pretreatment. We speculate that unmethylated intermediate causes tumbling; attractant causes its methylation and hence absence of tumbling (smooth swimming). Its demethylation during the period of smooth swimming affords adaptation.     

Methyl esterification of glutamic acid residues of methyl-accepting chemotaxis proteins in Bacillus subtilis.

The amino acid residue modified in the reversible methylation of Bacillus subtilis methyl-accepting chemotaxis proteins was identified as glutamic acid; methylation results in the formation of glutamate 5-methyl ester. Identification was made by comparing the behaviour of a 3H-labelled compound isolated from proteolytically hydrolysed methyl-accepting chemotaxis proteins labelled in vivo with that of authentic methylated amino acids by chromatographic and electrophoretic techniques. Also, the isolated compound on mild alkaline hydrolysis shows behaviour identical with that of authentic glutamate 5-methyl ester. [3H]Methanol released by mild alkaline hydrolysis was made to react with 3,5-dinitrobenzyl chloride to form [3H]methyl 3,5-dinitrobenzoate, which was identified by reverse-phase high-pressure liquid chromatography.     

Transposon Tn917lacZ mutagenesis of Bacillus subtilis: identification of two new loci required for motility and chemotaxis.

We have used Tn917lacZ to mutagenize the Bacillus subtilis chromosome and have isolated mutants that are defective in chemotaxis and motility. Mapping of the transposon inserts identified two new loci. Mutations in one of these loci generated mutants that had paralyzed flagella. Accordingly, we designate this a mot locus. The other locus is closely linked to the first and encodes proteins specifying chemotaxis functions. This locus is designated the cheX locus. Both the mot and cheX loci map close to ptsI. An additional transposon insert that maps in the hag locus was obtained. The pattern of beta-galactosidase expression from some of the transposons suggested that the mot locus is regulated by sigD, a minor sigma factor of B. subtilis. The cheX locus appeared to be under the control of vegetative sigA. Four transposon inserts were mapped to a previously characterized che locus near spcB. These mutants did not produce flagellin and were defective in the methylation of the methyl-accepting chemotaxis proteins. This locus probably encodes proteins required for flagellum biosynthesis and other proteins that are required for the methylation response.     

Oxygen as attractant and repellent in bacterial chemotaxis.

Studies of bacterial chemotaxis to oxygen (aerotaxis) over a broad range of oxygen concentrations showed that at high concentrations, oxygen was a repellent of Salmonella typhimurium, Escherichia coli, and some bacilli, whereas it is known that at lower concentrations (less than or equal to 0.25 mM dissolved oxygen), oxygen is an attractant. In a temporal assay of aerotaxis, S. typhimurium in medium equilibrated with air (0.25 mM dissolved oxygen) and then exposed to pure oxygen (1.2 mM) tumbled continuously for approximately 20 s. The oxygen concentration that elicited a half-maximal negative (repellent) response was 1.0 mM for both S. typhimurium and E. coli. The receptor for the negative chemoresponse to high concentrations of oxygen is apparently different from the receptor for the positive chemoresponse to low concentrations of oxygen, since the oxygen concentration that elicits a half-maximal positive (attractant) response in S. typhimurium and E. coli is reported to be 0.7 microM. Adaptation to high concentrations of oxygen, like adaptation to low concentrations of oxygen, was independent of methylation of a transducer protein. Only the response to low oxygen concentrations, however, was altered by interaction with the amidated Tsr transducer in cheB mutants.     

Role of CheB and CheR in the complex chemotactic and aerotactic pathway of Azospirillum brasilense

It has previously been reported that the alpha-proteobacterium Azospirillum brasilense undergoes methylation-independent chemotaxis; however, a recent study revealed cheB and cheR genes in this organism. We have constructed cheB, cheR, and cheBR mutants of A. brasilense and determined that the CheB and CheR proteins under study significantly influence chemotaxis and aerotaxis but are not essential for these behaviors to occur. First, we found that although cells lacking CheB, CheR, or both were no longer capable of responding to the addition of most chemoattractants in a temporal gradient assay, they did show a chemotactic response (albeit reduced) in a spatial gradient assay. Second, in comparison to the wild type, cheB and cheR mutants under steady-state conditions exhibited an altered swimming bias, whereas the cheBR mutant and the che operon mutant did not. Third, cheB and cheR mutants were null for aerotaxis, whereas the cheBR mutant showed reduced aerotaxis. In contrast to the swimming bias for the model organism Escherichia coli, the swimming bias in A. brasilense cells was dependent on the carbon source present and cells released methanol upon addition of some attractants and upon removal of other attractants. In comparison to the wild type, the cheB, cheR, and cheBR mutants showed various altered patterns of methanol release upon exposure to attractants. This study reveals a significant difference between the chemotaxis adaptation system of A. brasilense and that of the model organism E. coli and suggests that multiple chemotaxis systems are present and contribute to chemotaxis and aerotaxis in A. brasilense.     

Nucleotide sequence and characterization of a Bacillus subtilis gene encoding a flagellar switch protein.

The nucleotide sequence of the Bacillus subtilis fliM gene has been determined. This gene encodes a 38-kDa protein that is homologous to the FliM flagellar switch proteins of Escherichia coli and Salmonella typhimurium. Expression of this gene in Che+ cells of E. coli and B. subtilis interferes with normal chemotaxis. The nature of the chemotaxis defect is dependent upon the host used. In B. subtilis, overproduction of FliM generates mostly nonmotile cells. Those cells that are motile switch less frequently. Expression of B. subtilis FliM in E. coli also generates nonmotile cells. However, those cells that are motile have a tumble bias. The B. subtilis fliM gene cannot complement an E. coli fliM mutant. A frameshift mutation was constructed in the fliM gene, and the mutation was transferred onto the B. subtilis chromosome. The mutant has a Fla- phenotype. This phenotype is consistent with the hypothesis that the FliM protein encodes a component of the flagellar switch in B. subtilis. Additional characterization of the fliM mutant suggests that the hag and mot loci are not expressed. These loci are regulated by the SigD form of RNA polymerase. We also did not observe any methyl-accepting chemotaxis proteins in an in vivo methylation experiment. The expression of these proteins is also dependent upon SigD. It is possible that a functional basal body-hook complex may be required for the expression of SigD-regulated chemotaxis and motility genes.     

Identification of methylation sites and effects of phototaxis stimuli on transducer methylation in Halobacterium salinarum.

The two transducers in the phototaxis system of the archaeon Halobacterium salinarum, HtrI and HtrII, are methyl-accepting proteins homologous to the chemotaxis transducers in eubacteria. Consensus sequences predict three glutamate pairs containing potential methylation sites in HtrI and one in HtrII. Mutagenic substitution of an alanine pair for one of these, Glu265-Glu266, in HtrI and for the homologous Glu513-Glu514 in HtrII eliminated methylation of these two transducers, as demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis autofluorography. Photostimulation of the repellent receptor sensory rhodopsin II (SRII) induced reversible demethylation of HtrII, while no detectable change in the extent of methylation of HtrI was observed in response to stimulation of its cognate sensory rhodopsin, the attractant receptor SRI. Cells containing HtrI or HtrII with all consensus sites replaced by alanine still exhibited phototaxis responses and behavioral adaptation, and methanol release assays showed that methyl group turnover was still induced in response to photostimulation of SRI or SRII. By pulse-chase experiments with in vivo L-[methyl-(3)H]methionine-labeled cells, we found that repetitive photostimulation of SRI complexed with wild-type (or nonmethylatable) HtrI induced methyl group turnover in transducers other than HtrI to the same extent as in wild-type HtrI. Both attractant and repellent stimuli cause a transient increase in the turnover rate of methyl groups in wild-type H. salinarum cells. This result is unlike that obtained with Escherichia coli, in which attractant stimuli decrease and repellent stimuli increase turnover rate, and is similar to that obtained with Bacillus subtilis, which also shows turnover rate increases regardless of the nature of the stimulus. We found that a CheY deletion mutant of H. salinarum exhibited the E. coli-like asymmetric pattern, as has recently also been observed in B. subtilis. Further, we demonstrate that the CheY-dependent feedback effect does not require the stimulated transducer to be methylatable and operates globally on other transducers present in the cell.     

Large increases in attractant concentration disrupt the polar localization of bacterial chemoreceptors

In bacterial chemotaxis, the chemoreceptors [methyl-accepting chemotaxis proteins (MCPs)] transduce chemotactic signals through the two-component histidine kinase CheA. At low but not high attractant concentrations, chemotactic signals must be amplified. The MCPs are organized into a polar lattice, and this organization has been proposed to be critical for signal amplification. Although evidence in support of this model has emerged, an understanding of how signals are amplified and modulated is lacking. We probed the role of MCP localization under conditions wherein signal amplification must be inhibited. We tested whether a large increase in attractant concentration (a change that should alter receptor occupancy from c. 0% to > 95%) would elicit changes in the chemoreceptor localization. We treated Escherichia coli or Bacillus subtilis with a high level of attractant, exposed cells to the cross-linking agent paraformaldehyde and visualized chemoreceptor location with an anti-MCP antibody. A marked increase in the percentage of cells displaying a diffuse staining pattern was obtained. In contrast, no increase in diffuse MCP staining is observed when cells are treated with a repellent or a low concentration of attractant. For B. subtilis mutants that do not undergo chemotaxis, the addition of a high concentration of attractant has no effect on MCP localization. Our data suggest that interactions between chemoreceptors are decreased when signal amplification is unnecessary.     

Methanol production during chemotaxis to amino acids in Bacillus subtilis

The 20 common amino acids act as attractants during chemotaxis by the Gram-positive organism Bacillus subtilis . In this study, we report that all amino acids induce B. subtilis to produce methanol both upon addition and removal of the chemoeffector. Asparagine-induced methanol production is specific to the McpB receptor and aspartate-induced methanol production correlates with receptor occupancy. These findings suggest that addition and removal of all amino acids cause demethylation of specific receptors which results in methanol production. We also demonstrate that certain attractants cause greater production of methanol after multiple stimulations. CheC and CheD, while affecting the levels of receptor methylation, are not absolutely required for either methylation or demethylation. In contrast, CheY is necessary for methanol formation upon removal of attractant but not upon addition of attractant. We conclude that methanol formation due to negative stimuli indicates the existence of a unique adaptational mechanism in B. subtilis involving the response regulator, CheY.     

Chemotaxis proteins and transducers for aerotaxis in Pseudomonas aeruginosa

It was previously shown that the chemotaxis gene cluster 1 (cheYZABW) was required for chemotaxis. In this study, the involvement of the same cluster in aerotaxis is described and two transducer genes for aerotaxis are identified. Aerotaxis assays of a number of deletion-insertion mutants of Pseudomonas aeruginosa PAO1 revealed that the chemotaxis gene cluster 1 and cheR are required for aerotaxis. Mutant strains which contained deletions in the methyl-accepting chemotaxis protein-like genes tlpC and tlpG showed decreased aerotaxis. A double mutant deficient in tlpC and tlpG was negative for aerotaxis. TlpC has 45% amino acid identity with the Escherichia coli aerotactic transducer Aer. The TlpG protein has a predicted C-terminal segment with 89% identity to the highly conserved domain of the E. coli serine chemoreceptor Tsr. A hydropathy plot of TlpG indicated that hydrophobic membrane-spanning regions are missing in TlpG. A PAS motif was found in the N-terminal domains of TlpC and TlpG. On this basis, the tlpC and tlpG genes were renamed aer and aer-2, respectively. No significant homology other than the PAS motif was detected in the N-terminal domains between Aer and Aer-2.     

Methylation-independent and methylation-dependent chemotaxis in Rhodobacter sphaeroides and Rhodospirillum rubrum.

In vivo and in vitro methylation, methanol production assays, and the use of specific antibodies raised against the sensory transducing protein Tar in Escherichia coli all failed to demonstrate the presence of methyl-accepting chemotaxis proteins (MCPs) in the photosynthetic bacterium Rhodobacter sphaeroides, although such proteins did exist in another photosynthetic bacterium, Rhodospirillum rubrum. The range of chemicals to which Rhodobacter sphaeroides responds, the lack of an all-or-none response, and the lack of true repellents indicate an alternative chemosensory pathway. The existence of MCPs in Rhodospirillum rubrum means that the lack of MCPs is not the result of a phototrophic metabolism, but may be connected to the unidirectional flagellar motor of Rhodobacter sphaeroides.