The motile behavior of the bacterium Escherichia coli depends on the direction of rotation of its flagellar motors. Binding of the phosphorylated signaling molecule CheY to a motor component FliM is known to enhance clockwise rotation. It is difficult to study this interaction in vivo, because the dynamics of phosphorylation of CheY by its kinase CheA and the hydrolysis of CheY (accelerated by CheZ) are not under direct experimental control. Here, we examine instead the interaction with the flagellar motor of a double mutant CheY 13DK106YW that is active without phosphorylation. The behavioral assays were carried out on tethered cells lacking CheA and CheZ. The effects of variation in intracellular concentration of the mutant protein were highly nonlinear. However, they can be explained by a thermal isomerization model in which the free energies of clockwise and counterclockwise states depend linearly on the amount of CheY bound.Motile bacteria such as Escherichia coli actively respond to a variety of stimuli by modulating the direction of rotation of their flagella. Addition of a chemical attractant (e.g., aspartate) or removal of a chemical repellent (e.g., leucine) enhances counterclockwise (CCW) rotation, causing cells to extend runs that carry them in a favorable direction. This occurs through regulation of a kinase, CheA, which phosphorylates an effector molecule, CheY. Phospho CheY (CheY-P) when bound to a component at the base of the flagellar motor, FliM, promotes clockwise (CW) rotation (for reviews, see refs. 1-4). This interaction has been studied in vivo (5), but interpretation of the results is complicated by the dynamics of phosphorylation and hydrolysis: the intracellular concentration of CheY-P was not measured.One solution to this problem is to use CheY mutants that are active without phosphorylation. Wild-type CheY (CheY wt ) is activated by phosphorylation at Asp-57 (6). Replacement of Asp-13 by Lys (or Arg) results in a CW phenotype (7). CheY 13DK can be phosphorylated to some extent, but phosphorylation is not required for activity. Replacement of Tyr-106 by Trp results in an even stronger CW phenotype (8), but only when CheY 106YW is phosphorylated. The double mutant CheY 13DK106YW -we will call this protein CheY**-is active without phosphorylation (X. Zhu and P. Matsumura, private communication). We chose this mutant to study the dynamics of the interaction of CheY with the flagellar motor.There are two kinds of mechanisms that might explain how CheY controls the direction of the flagellar motor. In one, the switch is thrown when a certain number of CheY molecules are bound. In the other, the number bound only determines the probability of CW or CCW rotation, and the switch is thrown by thermal fluctuations. Our results argue for the latter, stochastic mechanism. MATERIALS AND METHODSBacteria, Phage, and Plasmids. (See Table 1.) The following were gifts: pXYZ202 from Xiangyang Zhu and Phil Matsumura, EC0 and phage f1R408 from Jon Beckwith, pBIP from Steven Slater, MM5008 from Mike...

Halorhodopsin, the chloride pump from Natronobacterium pharaonis (pharaonis hR), was isolated under conditions of low ionic strength. The quotient between the optical densities of pharaonis hR in 4 M NaCl at 280 and 577 nm amounts to 1.1, indicating a high purity of the protein and integrity of the chromophore. Gel filtration chromatography of the purified pharaonos hR at neutral pH and in the absence of inorganic salts leads to a shift to the absorption maximum to 600 nm (pharaonis hRblue). The purple color can be regained by the addition of anions such as chloride, iodide, azide, nitrate, and also fluoride. The absorption maxima are dependent on the nature of the anion and the pH. At pH 7, sulfate does not influence the 600-nm absorption, while at pH 4.5 the absorption maximum is shifted to 581 nm. The blue form of halorhodopsin (pharaonis hRblue) was titrated with different sodium salts, such as chloride, azide, and nitrate. The half-maximal binding is in the millimolar range, with Br < Cl < NO3 < N3 < BrO3. Deprotonation of the Schiff base can be accomplished by treatment of pharaonis hR or pharaonis hRblue with base. The apparent pK of the Schiff base in pharaonis hRblue was determined to be 8.5. The pK shifted to 8.0 in the presence of 150 mM Na2SO4, whereas the pK of pharaonis hR in 150 mM NaCl is 9.6. In the photocycle of the chloride- and nitrate-containing pharaonis hR, a species similar to hR520 could be detected. Pharaonis hR reconstituted with azide shows photochemical reactions similar to the photocycle of bacteriorhodopsin.(ABSTRACT TRUNCATED AT 250 WORDS)

Beneficial plant-microbe associations play critical roles in plant health. Bacterial chemotaxis provides a competitive advantage to motile flagellated bacteria in colonization of plant root surfaces, which is a prerequisite for the establishment of beneficial associations. Chemotaxis signaling enables motile soil bacteria to sense and respond to gradients of chemical compounds released by plant roots. This process allows bacteria to actively swim towards plant roots and is thus critical for competitive root surface colonization. The complete genome sequences of several plant-associated bacterial species indicate the presence of multiple chemotaxis systems and a large number of chemoreceptors. Further, most soil bacteria are motile and capable of chemotaxis, and chemotaxis-encoding genes are enriched in the bacteria found in the rhizosphere compared to the bulk soil. This review compares the architecture and diversity of chemotaxis signaling systems in model beneficial plant-associated bacteria and discusses their relevance to the rhizosphere lifestyle. While it is unclear how controlling chemotaxis via multiple parallel chemotaxis systems provides a competitive advantage to certain bacterial species, the presence of a larger number of chemoreceptors is likely to contribute to the ability of motile bacteria to survive in the soil and to compete for root surface colonization.

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Bacterial motility and chemotaxis are essential qualities for optimum adaptation to different environments. Flagellar synthesis and motility are maximal under nutritional stress and require 2 to 3% of the energy of a cell. Therefore, the expression of some 50 genes involved in this process is strictly regulated by a hierarchy of controls (19). The current paradigm of flagellar gene regulation was derived from studies of Escherichia coli and Salmonella enterica serovar Typhimurium (13,15,18,20,29). The E. coli chemotaxis, flagellar, and motility genes map in four separate clusters often referred to as the flagellar regulon. The genes of this regulon are organized in three classes that are expressed in hierarchial order, although their map positions do not necessarily reflect the assignments to these classes. Class I is represented by a master operon that encodes the transcription activators, FlhC and FlhD, which in turn regulate the expression of class II (18). This includes genes that determine the flagellar basal body and the flagellinspecific export apparatus and fliA, which encodes a 28 (F) transcription factor for class III.Sinorhizobium (Rhizobium) meliloti, a member of the alpha subgroup of proteobacteria, exhibits significant deviations from the enterobacterial (gamma subgroup) paradigm of chemotaxis in its flagellar structure and mode of rotation (23) (20), we refer to the entirety of these genes as the flagellar regulon.We have identified in S. meliloti two related members of the LuxR family, VisN and VisR (for "vital for swimming"), formerly named Orf12 and Orf13, respectively (33), that function as global activators of the flagellar regulon.