Chemotaxis away from uncouplers of oxidative phosphorylation in Bacillus subtilis.

In a capillary assay, uncouplers of oxidative phosphorylation and inhibitors of electron transport are repellents for Bacillus subtilis. They also cause transient tumbling in naturally smooth swimming strains. Tumbling strains can be made to swim smoothly by addition of attractant and then immediately returned to tumbling by subsequent addition of repellent. Arsenate does not cause transient tumbling, suggesting that decrease in concentration of adenosine triphosphate does not cause tumbling and that adenosine triphosphate concentration does not govern tumbling frequency. Instead, the evidence suggests that diminution of the energized state of the membrane, or membrane potential, causes tumbling although the level of the energized state itself does not govern tumbling frequency.

Purification and reconstitution of the methyl-accepting chemotaxis proteins from Bacillus subtilis.

The methyl-accepting chemotaxis proteins (MCPs) from Bacillus subtilis, designated as H1, H2, and H3, have been purified to near homogeneity. These purified MCPs were reconstituted into proteoliposome vesicles using a detergent dilution procedure. The ability of the reconstituted MCPs to be methylated in vitro strongly suggests that they are in a functionally active conformation. The MCPs of B. subtilis are considerably larger than those of Escherichia coli, with molecular weights of the purified proteins being 76, 86, and 97 kDa for H3, H2, and H1, respectively. Two-dimensional electrophoresis demonstrates that the isoelectric point of H1 and H2 is 5.1, while H3 is slightly more basic, having an isoelectric point of 5.3. Immunoblot analysis using the cross reacting E. coli anti-Trg antibody reveals that maximal MCP expression occurs approx. 4 h after the onset of stationary phase, and remains relatively stable thereafter. However, the ability of the MCPs to be methylated in vivo is significantly reduced.

Identification and purification of a calcium-binding protein from Bacillus subtilis.

A Ca(2+)-binding protein was identified in Bacillus subtilis in the log phase of growth. The molecular mass of this protein is about 38 kDa as estimated by polyacrylamide gel electrophoresis in the presence of SDS and by gel filtration. The protein was found to be resistant 10 min at 65 degrees C and was purified about 400 times, starting from heated crude extract, by conventional procedures. This novel protein is able to bind Ca2+ in the presence of an excess of MgCl2 and KCl both in solution and after SDS gel electrophoresis and electrotransfer. Since an impairment of the Ca2+ intake, in Bacillus subtilis, results in an impairment of chemotactic behavior (Matsushita, T. et al (1988) FEBS lett. 236, 437-440), 38 kDa protein may be involved in the regulation of chemotaxis.

The range of attractant concentrations for bacterial chemotaxis and the threshold and size of response over this range. Weber law and related phenomena.

Attractant was added to a suspension of bacteria (the background concentration of attractant) and then these bacteria were exposed to a yet higher concentration of attractant in a capillary. Chemotaxis was measured by determining how many bacteria accumulated in the capillary. The response range for chemotaxis lies between the threshold concentration and the saturating concentration. The breadth of this range is different for attractants detected by different chemoreceptors. Attractants detected by the same chemoreceptor can have their response ranges in widely different places. Over the center of the response range (on a logarithmic scale), bacteria give similar sized responses to similar fractional increases of concentration, i.e. they respond to ratios of attractant concentration, but the response peaks at the center of the range. The size of the response is different for attractants detected by different chemoreceptors. For a detectable response, a smaller increase in attractant concentration is needed for attractants detected by some chemoreceptors than for attractants detected by others. Although the data are inadequate, it appears that the Weber law may be observed over a wide range of concentrations for some attractants but not for others. In the Appendix we aim to explain some of these results in terms of the interaction of an attractant with its chemoreceptor according to the law of mass action.

Chemotaxis in Bacillus subtilis: how bacteria monitor environmental signals.

Virtually all organisms have means of monitoring their environment and making use of information gained to aid their survival. Many organisms, from bacteria to animals, move from place to place and can alter their movements. Chemotaxis is a signal transduction system found in motile bacteria that allows them to sense changes in the concentrations of various extracellular compounds and change their swimming behavior in a way that moves them toward more favorable environments. Chemotaxis is the most ancient sensory-motor process in nature. For years, studies of enteric bacteria, such as Escherichia coli and Salmonella typhimurium, have served as the paradigm for understanding this process on a molecular level. Recent studies on the gram-positive bacterium, Bacillus subtilis, and other bacteria, suggest that a slightly more complex system may be ancestral to that of the more extensively studied enterics. Aspects of chemotaxis that are unique to B. subtilis include a more complex adaptation system, with protein-protein methyl group transfer, chemotaxis proteins having no counterparts in E. coli, and a very extensive repertoire of repellents that are sensed at very low concentrations by receptors.

Gene-protein relationships in the flagellar hook-basal body complex of Bacillus subtilis: sequences of the flgB, flgC, flgG, fliE and fliF genes.

The nucleotide sequence of five genes from the major Bacillus subtilis chemotaxis locus has been determined. Four of these genes encode proteins that are homologous to the Salmonella typhimurium FlgB, FlgC, FlgG and FliF proteins. One gene encodes a protein that is homologous to the Escherichia coli FliE protein. The data from S. typhimurium and E. coli suggest that all of these proteins form part of the hook-basal body (HBB) complex of the bacterial flagella. The FlgB, FlgC and FlgG proteins are components of the proximal and distal rods. The FliF protein forms the M-ring that anchors the rod assembly to the membrane. The role of the FliE protein within the HBB complex has not yet been determined. The similarity between the B. subtilis and S. typhimurium proteins suggests that the structure of the M-ring and the rod may be similar in the two species. However, we observed some differences in size and amino acid composition between some of the corresponding homologues that suggest the basal body proteins may be organized slightly differently within B. subtilis.

Bacillus subtilis flagellar proteins FliP, FliQ, FliR and FlhB are related to Shigella flexneri virulence factors.

The amino acid sequences of the Bacillus subtilis flagellar proteins, FliP, FliQ, FliR and FlhB, as deduced from their respective nucleotide sequences, were found to share significant homology to the Shigella flexneri Spa24, Spa9, Spa29 and Spa40 virulence proteins, respectively. These proteins are required for the presentation of surface plasmid antigens. These results further support the growing hypothesis that a superfamily of proteins exists for the biosynthesis of supramolecular structures that lie in an external to the cell membrane.

A putative ATP-binding protein from the che/fla locus of Bacillus subtilis.

A majority of the chemotaxis and flagellar genes of Bacillus subtilis are found in the major che/fla operon which spans over 26 kilobases of DNA and encodes at least 30 genes. In this operon, a single open reading frame, designated orf298, has been demonstrated to encode a 33,131 Dalton protein which shows no evidence of being involved in chemotaxis or motility. A strain disrupted in orf298 was assayed for motility and chemotaxis and always behaved as the wild-type strain. Inspection of the translated sequence revealed a consensus ATP-binding region. While the role of ORF298 has not yet been determined, ATP-binding and/or hydrolysis is a likely function.

Bacterial chemotaxis: biochemistry of behavior in a single cell.

Bacterial chemotaxis is a primitive behavioral system that shows great promise for being amenable to a description of its molecular mechanism. In Gram-negatives like Escherichia coli, addition of amino acid attractant begins a series of events, starting with binding to certain intrinsic membrane proteins, the MCPs, and ending with a period of smooth swimming. Immediately, methyl-esterification of these MCPs begins and continues during this period. By contrast in the Gram-positive Bacillus subtilis, demethylation of MCPs occurs during the same period. At least two other mechanisms for mediating chemotaxis toward the attractants oxygen and phosphotransferase sugars exist in E. coli, and in these, changes in methylation of MCPs plays no role. Moreover, chemotaxis away from many repellents by B. subtilis appears to involve different mechanisms. Many of the repellents include drugs and toxicants, many of them man-made, so that chemoreceptors could not have specifically evolved; yet the bacteria are often exquisitely sensitive to them. Indeed, the B. subtilis membrane seems to act like a generalized antenna for noxious membrane-active substances.

Recognition sites for chemotactic repellents of Bacillus subtilis.

Repellents of Bacillus subtilis include many membrane-active compounds, such as uncouplers of oxidative phosphorylation, local anesthetics, chlorpromazine (a central nervous system depressant), and tetraphenylboron (a lipophilic anion). Normally, bacteria swim smoothly, and occasionally tumble, but addition of repellent causes all bacteria to tumble, then later resume original frequency of swimming and tumbling (adaptation). Bacteria adapted to repellent can then be tested to determine the minimum concentration (threshold) of the same or different repellents that causes tumbling. The results indicate that repellents act at (saturable) recognition sites, which differ for chemically different species. An implication is that uncouplers of oxidative phosphorylation affect cell properties by interaction at specific locations.

Control of tumbling in bacterial chemotaxis by divalent cation.

Chemotaxis is migration of organisms to higher concentrations of attractant or lower concentrations of repellent. Understanding the switch than controls whether the flagella rotate counterclockwise for swimming or clockwise for tumbling (thrashing about without making much forward progress) is central to understanding chemotaxis of peritrichous bacteria, since chemotaxis results from selective suppression of tumbles. Depletion of divalent cation by chelating agents in the presence of A23187, an ionophore that conveys divalent cation across membrane, causes incessant tumbling in Bacillus subtilis. Small additions of MgCl2 prevent this tumbling. In this tumbling condition, the bacteria which normally swim extensively when given attractant, do not respond even to 10 mM alanine, a strong attractant. MnCl2, by contrast to others potentiated by the ionophore. Permanent cations, including tetraphenylarsonium ion and triphenylmethylphosphonium ion, cause permanent swimming, even in the presence of A23187 and chelating agents. We propose that divalent cation, probably Mg2+ ion, binds to the switch to cause swimming and that, in the absence of divalent cation at the switch, the bacterium tumbles.

Effect of methionine on chemotaxis by Bacillus subtilis.

Bacillus subtilis, like Escherichia coli and Salmonella typhimurium, carries out chemotaxis by modulating the relative frequency of smooth swimming and tumbling. Like these enteric bacteria, methionine auxotrophs starved for methionine show an abnormally long-period of smooth swimming after addition of attractant. This "hypersensitive" state requires an hour of starvation for its genesis, which can be hastened by including alanine, a strong attractant, in starvation medium. Susceptibility to repellent, which causes transient tumbling when added, if anything, increases slightly by starvation for methionine. The results are interpreted by postulating the existence of a methionine-derived structure that hastens recovery of attractant-stimulated bacteria back to normal.

Functional homology of chemotactic methylesterases from Bacillus subtilis and Escherichia coli.

The methylesterase enzyme from Bacillus subtilis was compared with that from Escherichia coli. Both enzymes were able to demethylate methyl-accepting chemotaxis proteins (MCPs) from the other organism and were similarly affected by variations in glycerol, magnesium ion, or pH. When attractants were added to a mixture of B. subtilis MCPs and E. coli methylesterase, the rate of demethylation was enhanced. Conversely, when attractants were added to a mixture of E. coli MCPs and B. subtilis methylesterase, the rate of demethylation was diminished. These effects are what would be expected if, in these in vitro systems, the MCPs determined the rate of demethylation. These data suggest that, although the enzymes are from evolutionarily divergent organisms and are different in size, they have considerable functional homology.

Nucleotide sequence and expression of cheF, an essential gene for chemotaxis in Bacillus subtilis.

The cheF gene, which is involved in chemotaxis in Bacillus subtilis, has been cloned, expressed, and sequenced. This gene is contained in a 0.7-kilobase PstI DNA fragment that was isolated from a lambda Charon 4A B. subtilis chromosomal DNA library. This fragment was subcloned into the expression vector pSI-1 and shown to complement the cheF mutation both for chemotaxis and for methanol production in response to the addition of attractants. Plasmid-encoded DNA expression in B. subtilis maxicells indicated that a membrane-associated polypeptide of 20-kilodaltons was expressed from this 0.7-kilobase DNA. The nucleotide sequence of this DNA fragment was determined, and an open reading frame capable of encoding a putative 175-amino-acid protein (Mr 20,002) was identified. In an effort to understand the function of the cheF protein, the dosage of the cheF gene product was varied by altering the concentration of IPTG (isopropyl-beta-D-thiogalactopyranoside) during growth. In the presence of high concentrations of IPTG, chemotaxis was inhibited and methanol production was impaired.

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.

Identification and characterization of FliY, a novel component of the Bacillus subtilis flagellar switch complex.

The Bacillus subtilis gene encoding FliY has been cloned and sequenced. The gene encodes a 379-amino-acid protein with a predicted molecular mass of 41,054 daltons. FliY is partly homologous to the Escherichia coli and Salmonella typhimurium switch proteins FliM and FliN. The N-terminus of FliY has 33% identity with the first 122 amino acids of FliM, whereas the C-terminus of FliY has 52% identity with the last 30 amino acids of FliN. The middle 60% of FliY is not significantly homologous to either of the proteins. A fliY::cat null mutant has no flagella. Motility can be restored to the mutant by expression of fliY from a plasmid, although chemotaxis is still defective since the strain exhibits smooth swimming behaviour. fliY::cat is in the cheD complementation group. One of the cheD point mutants does not switch although the population grown from a single cell has both smooth swimming and tumbling bacteria, implying that the switch is locked. Expression of fliY in wild-type B. subtilis makes the cells more smooth-swimming but does not appear to affect chemotaxis. Expression of fliY in wild-type S. typhimurium severely inhibits chemotaxis and also makes the cells smooth swimming. Expression in a non-motile S. typhimurium fliN mutant restores motility but not chemotaxis, although expression in a non-motile E. coli fliM mutant does not restore motility. The homology, multiple phenotypes, and interspecies complementation suggest that FliY forms part of the B. subtilis switch complex.(ABSTRACT TRUNCATED AT 250 WORDS)