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In this paper, we describe the regulatory roles of VisN, VisR and Rem in the expression of flagellar, motility and chemotaxis genes in Rhizobium leguminosarum biovar viciae strains VF39SM and 3841. Individual mutations in the genes encoding these proteins resulted in a loss of motility and an absence of flagella, indicating that these regulatory genes are essential for flagellar synthesis and function. Transcriptional experiments involving gusA--gene fusions in wild-type and mutant backgrounds were performed to identify the genes under VisN/R and Rem regulation. Results showed that the chemotaxis and motility genes of R. leguminosarum could be separated into two groups: one group under VisN/R-Rem regulation and another group that is independent of this regulation. VisN and VisR regulate the expression of rem, while Rem positively regulates the expression of flaA, flaB, flaC, flaD, motA, motB, che1 and mcpD. All of these genes except mcpD are located within the main motility and chemotaxis gene cluster of R. leguminosarum. Other chemotaxis and motility genes, which are found outside of the main motility gene cluster (che2 operon, flaH for VF39SM, and flaG) or are plasmid-borne (flaE and mcpC), are not part of the VisN/R-Rem regulatory cascade. In addition, all genes exhibited the same regulation pattern in 3841 and in VF39SM, except flaE and flail, flaE is not regulated by VisN/R-Rem in 3841 but it is repressed by Rem in VF39SM. flail is under VisN/R-Rem regulation in 3841, but not in VF39SM. A kinetics experiment demonstrated that a subset of the flagellar genes is continuously expressed in all growth phases, indicating the importance of continuous motility for R. leguminosarum under free-living conditions. On the other hand, motility is repressed under symbiotic conditions. Nodulation experiments showed that the transcriptional activators VisN and Rem are dramatically downregulated in the nodules, suggesting that the symbiotic downregulation of motility-related genes could be mediated by repressing the expression of VisN/R and Rem. DOI 10.1099/mic.0.035386-0

Transmembrane chemoreceptors are central components in bacterial chemotaxis. Receptors couple ligand binding and adaptational modification to receptor conformation in processes that create transmembrane signaling. Homodimers, the fundamental receptor structural units, associate in trimers and localize in patches of thousands. To what degree do conformational coupling and transmembrane signaling require higher-order interactions among dimers? To what degree are they altered by such interactions? To what degree are they inherent features of homodimers? We addressed these questions using nanodiscs to create membrane environments in which receptor dimers had few or no potential interaction partners. Receptors with many, few, or no interaction partners were tested for conformational changes and transmembrane signaling in response to ligand occupancy and adaptational modification. Conformation was assayed by measuring initial rates of receptor methylation, a parameter independent of receptor-receptor interactions. Coupling of ligand occupancy and adaptational modification to receptor conformation and thus to transmembrane signaling occurred with essentially the same sensitivity and magnitude in isolated dimers as for dimers with many neighbors. Thus, we conclude that the chemoreceptor dimer is the fundamental unit of conformational coupling and transmembrane signaling. This implies that in signaling complexes, coupling and transmembrane signaling occur through individual dimers and that changes between dimers in a receptor trimer or among trimer-based signaling complexes are subsequent steps in signaling. doi: 10.1128/JB.01391-09

Regular environmental conditions allow for the evolution of specifically adapted responses, whereas complex environments usually lead to conflicting requirements upon the organism's response. A relevant instance of these issues is bacterial chemotaxis, where the evolutionary and functional reasons for the experimentally observed response to chemoattractants remain a riddle. Sensing and motility requirements are in fact optimized by different responses, which strongly depend on the chemoattractant environmental profiles. It is not clear then how those conflicting requirements quantitatively combine and compromise in shaping the chemotaxis response. Here we show that the experimental bacterial response corresponds to the maximin strategy that ensures the highest minimum uptake of chemoattractants for any profile of concentration. We show that the maximin response is the unique one that always outcompetes motile but nonchemotactic bacteria. The maximin strategy is adapted to the variable environments experienced by bacteria, and we explicitly show its emergence in simulations of bacterial populations in a chemostat. Finally, we recast the contrast of evolution in regular vs. complex environments in terms of minimax vs. maximin game-theoretical strategies. Our results are generally relevant to biological optimization principles and provide a systematic possibility to get around the need to know precisely the statistics of environmental fluctuations. biological optimization | E. coli | evolution | game theory | strategy doi/ 10.1073/pnas.0909673107

Phosphorylation-based signaling pathways employ dephosphorylation mechanisms for signal termination. Histidine to aspartate phosphosignaling in the two-component system that controls bacterial chemotaxis has been studied extensively. Rhodobacter sphaeroides has a complex chemosensory pathway with multiple homologues of the Escherichia coil chemosensory proteins, although it lacks homologues of known signal-terminating CheY-P phosphatases, such as CheZ, CheC, FliY or CheX. Here, we demonstrate that an unusual CheA homologue, Che[A.sub.3], is not only a phosphodonor for the principal CheY protein, Che[Y.sub.6], but is also is a specific phosphatase for Che[Y.sub.6]-P. This phosphatase activity accelerates Che[Y.sub.6]-P dephosphorylation to a rate that is comparable with the measured stimulus response time of approximately 1 s. Che[A.sub.3] possesses only two of the five domains found in classical CheAs, the Hpt (P1) and regulatory (PS) domains, which are joined by a 794-amino acid sequence that is required for phosphatase activity. The P1 domain of Che[A.sub.3] is phosphorylated by Che[A.sub.4], and it subsequently acts as a phosphodonor for the response regulators. A Che[A.sub.3] mutant protein without the 794-amino acid region lacked phosphatase activity, retained phosphotransfer function, but did not support chemotaxis, suggesting that the phosphatase activity may be required for chemotaxis. Using a nested deletion approach, we showed that a 200-amino acid segment of Che[A.sub.3] is required for phosphatase activity. The phosphatase activity of previously identified nonhybrid histidine protein kinases depends on the dimerization and histidine phosphorylation (DHp) domains. However, Che[A.sub.3] lacks a DHp domain, suggesting that its phosphatase mechanism is different from that of other histidine protein kinases. response regulator | signal termination | two-component | Rhodobacter sphaeroides | histidine protein kinase

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 [[DELTA](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.

Research has been conducted on Rhizobium meliloti flagellar, chemotaxis and motility genes. The probing of the gene expression on Western blots and with lacZ fusions has been carried out.

Myxococcus xanthus cells exhibit directed motility up phosphatidylethanolamine (PE) gradients, and we suggest that PE behaves as a chemoattractant. Computer-assisted stop-motion digital microscopy was used to record cell movements in slide culture. PE decreased cellular reversal frequency with molecular specificity that was correlated with the fatty acid composition. Synthetic dilauroyl (di C12:0) PE and dioleoyl (di C18:1 [Omega]9c) PE suppressed direction reversals and stimulated movement up the gradient. Sensory adaptation occurred about 1 hr after the onset of stimulation. Null mutants in a methylated chemotaxis protein homolog (FrzCD) and a CheA/CheY homolog (FrzE) moved up a PE gradient at a reduced rate. The mutants displayed normal excitation but were defective in adaptation. A dominant, hyper-reversal mutant in the M. xanthus methyl accepting chemotaxis protein homolog, frzCD224, failed to respond to PE stimulation, which argued that PE was a transduced stimulus. Neither dilauroyl PE nor dioleoyl PE is present at high enough concentrations in vegetative or developmental PE to account for all of the chemotactic activity. It appears then that there are additional, as yet unknown, PE species that serve as autoattractants. We report on a discrete phospholipid chemoattractant in a gliding bacterium

Motility of Borrelia borgdorferi, the causative microorganism in Lyme disease, requires an environment similar to interstitial fluid. Rabbit serum is an attractant for B burgdorferi while butanol and ethanol were repellants. B burgdorferi shows no observable chemotaxis to common sugars or amino acids, unlike some free-living spirochetes. Furthermore, the frequency and duration for pausing of flagella are essential for chemotaxis of B burgdorferi.

Chemotaxis in Rhodobacter sphaeroides and Sinorhizobium meliloti has a direct link to the environment in which a cell finds itself. The presence of at least two CheY homologues and the discovery that the swimming speed of these bacteria can vary indicate a different control mechanism at the flagellar motor. However, it remains to be understood why R sphaeroides should have at least two functional sets of genes encoding homologues of the enteric chemosensory pathway.

The role of histidine protein kinase PleC in Caulobacter crescentus motility and chemotaxis was analyzed. The study utilized a pseudoreversion analysis of the dwJ332 allele which confers a temperature-sensitive motility phenotype. Results revealed that signal transduction pathways are mediated by PleC-DivK and DivJ-PleD in the regulation of chemotaxis and motility. Furthermore, A temperature-sensitive allele of pleC can significantly reduce viability under certain growth conditions.

A simple method to analyze chemotaxis in Myxococcus xanthus is discussed. It involves the construction of steep and stable chemical gradients in slide cultures of M. xanthus. Application of this method has shown that not all M. xanthus species in the form or single cells and microscopic swarms respond chemotacticaly to chemotaxis.

The requirements for motility and flagellation of Xanthobacter sp. JW-KR2 are discussed. Motility was induced by lack of a utilizable carbon source. Synthesis of bacterial flagella was induced upon transfer of cells froma succinate or fumarate medium to a medium which lacked carbon. Chemotaxis was observed in the species toward 1-propanol, 2-propanol and butanol between 15mM and 500mM and no peak responses were observed. Positive response to butanol wasinhibited by the presence of TCA cycle intermediates.

The swimming behavior of Azospirillum brasilense was characterized. The polar flagella of free-swimming and tethered cells could rotate in both clockwise and counterclockwise directions. A. brasilense shows both chemotaxis and chemokinesis in response to temporal gradients of chemoattractants. The results suggest that A. brasilense does not use a methyl-accepting chemotaxis protein-based chemosensing system and that the main chemosensory pathway in this bacterium is methylation independent.

'Caged' calcium compounds are photolabile calcium chelators which change their affinity for calcium in response to certain wavelengths of light. Caged compounds were electroporated into Escherichia coli cells to investigate the effects of intracellular calcium levels on chemotaxis. The results showed increasing intracellular calcium levels led to cell tumbling, while decreases in intracellular calcium led to smooth swimming. Mutational analysis showed that CheA, CheW and CheY are required for the calcim-induced tumbling.

The Palleroni microcapillary assay was evaluated with regard to the chemotactic behavior of Methylosinus trichosporium OB3b, an obligate methanotroph which degrades a number of chlorinated hydrocarbons. The results showed that the Palleroni microcapillary assay is subject to abiotic hydrodynamic effects arising from a density-induced convection. The source of this convection was most probably the fluid density difference between thee cell suspension and the microcapillary buffer. This procedure is more useful when evaluating the behavior of highly motile strains of bacteria.

Escherichia coli is unable to synthesize pyrroquinoline quinone (PQQ) but compensates by chemotaxis toward PQQ via the phosphoenolpyruvate-sugar-phosphotransferase system. Utilization of swarm plates and capillary assay revealed that the sensory mechanisms in E. coli show chemotaxis toward a mixture of glucose and PQQ. E. coli strains lacking the methylated chemotaxis receptor protein Tar showed no chemotaxis toward PQQ.

The effect of fumarate on the rotational direction of flagellar movement was determined. Tests were conducted on partially lysed Escherichia coli cells with counter clockwise rotating flagella showed that fumarate caused an increase in clockwise rotation, although the proportion of switching cells remained unchanged. These results suggest that fumarate or a fumarate metabolite may be a cytoplasmic switch factor.

Barry L. Taylor [1] Igor B. Zhulin [2] Mark S. Johnson [3] Department of Microbiology and Molecular Genetics, School of Medicine, Loma Linda University, Loma Linda, California 92350; e-mail: [1] [...]