Che Mutants Research Articles

Butyrylcholinesterase with altered catalytic triad, acting as a promising bioscavenger against organophosphorus agents

Cholinesterases (ChEs) are irreversibly inhibited by organophosphorus compounds (OP) through phosphylation of their active site serine. OPs can be regarded as pseudo-substrates of ChEs in which water-mediated dephosphylation is very slow or impossible. Then, it has been attempted by genetic engineering to convert ChEs into OP hydrolases by speeding up dephosphylation rates. The knowledge of ChEs crystal structure and implementation of potent in silico methods allow rational computer design of new mutants. Such ChE mutants could be used as catalytic bioscavengers in medical counter-measures of OP poisoning. In the present work, using QM/MM MD, we designed a new mutant of human butyrylcholinesterase (BChE). This double mutant, Glu325Gly/Asn322Glu, alters the functioning of the catalytic triad (Ser198.His438.Glu325 in wild-type enzyme), making a new one Ser198.His438.Glu322. Calculations with echothiophate as a model OP showed that the new catalytic triad is stable and capable of hydrolysing the phosphorylated serine. Thus, self-reactivation of the mutated enzyme is possible. Although the calculated dephosphylation rate constant is low, it is expected that the introduction of a third mutation will lead to a much faster turnover.

Identification of a Kinase-Active CheA Conformation in Escherichia coli Chemoreceptor Signaling Complexes.

Escherichia coli chemotaxis relies on control of the autophosphorylation activity of the histidine kinase CheA by transmembrane chemoreceptors. Core signaling units contain two receptor trimers of dimers, one CheA homodimer, and two monomeric CheW proteins that couple CheA activity to receptor control. Core signaling units appear to operate as two-state devices, with distinct kinase-on and kinase-off CheA output states whose structural nature is poorly understood. A recent all-atom molecular dynamic simulation of a receptor core unit revealed two alternative conformations, "dipped" and "undipped," for the ATP-binding CheA.P4 domain that could be related to kinase activity states. To explore possible signaling roles for the dipped CheA.P4 conformation, we created CheA mutants with amino acid replacements at residues (R265, E368, and D372) implicated in promoting the dipped conformation and examined their signaling consequences with in vivo Förster resonance energy transfer (FRET)-based kinase assays. We used cysteine-directed in vivo cross-linking reporters for the dipped and undipped conformations to assess mutant proteins for these distinct CheA.P4 domain configurations. Phenotypic suppression analyses revealed functional interactions among the conformation-controlling residues. We found that structural interactions between R265, located at the N terminus of the CheA.P3 dimerization domain, and E368/D372 in the CheA.P4 domain played a critical role in stabilizing the dipped conformation and in producing kinase-on output. Charge reversal replacements at any of these residues abrogated the dipped cross-linking signal, CheA kinase activity, and chemotactic ability. We conclude that the dipped conformation of the CheA.P4 domain is critical to the kinase-active state in core signaling units.IMPORTANCE Regulation of CheA kinase in chemoreceptor arrays is critical for Escherichia coli chemotaxis. However, to date, little is known about the CheA conformations that lead to the kinase-on or kinase-off states. Here, we explore the signaling roles of a distinct conformation of the ATP-binding CheA.P4 domain identified by all-atom molecular dynamics simulation. Amino acid replacements at residues predicted to stabilize the so-called "dipped" CheA.P4 conformation abolished the kinase activity of CheA and its ability to support chemotaxis. Our findings indicate that the dipped conformation of the CheA.P4 domain is critical for reaching the kinase-active state in chemoreceptor signaling arrays.

Impact of motility and chemotaxis features of the rhizobacterium Pseudomonas chlororaphis PCL1606 on its biocontrol of avocado white root rot.

The biocontrol rhizobacterium Pseudomonas chlororaphis PCL1606 has the ability to protect avocado plants against white root rot produced by the phytopathogenic fungus Rosellinia necatrix. Moreover, PCL1606 displayed direct interactions with avocado roots and the pathogenic fungus. Thus, nonmotile (flgK mutant) and non-chemotactic (cheA mutant) derivatives of PCL1606 were constructed to emphasize the importance of motility and chemotaxis in the biological behaviour of PCL1606 during the biocontrol interaction. Plate chemotaxis assay showed that PCL1606 was attracted to the single compounds tested, such as glucose, glutamate, succinate, aspartate and malate, but no chemotaxis was observed to avocado or R. necatrix exudates. Using the more sensitive capillary assay, it was reported that smaller concentrations (1 mM) of single compounds elicited high chemotactic responses, and strong attraction was confirmed to avocado and R. necatrix exudates. Finally, biocontrol experiments revealed that the cheA and fglK derivative mutants reduced root protection against R. necatrix, suggesting an important role for these biological traits in biocontrol by P. chlororaphis PCL1606. [Int Microbiol 20(2):94-104 (2017)].

More than Motility: Salmonella Flagella Contribute to Overriding Friction and Facilitating Colony Hydration during Swarming

We show in this study that Salmonella cells, which do not upregulate flagellar gene expression during swarming, also do not increase flagellar numbers per μm of cell length as determined by systematic counting of both flagellar filaments and hooks. Instead, doubling of the average length of a swarmer cell by suppression of cell division effectively doubles the number of flagella per cell. The highest agar concentration at which Salmonella cells swarmed increased from the normal 0.5% to 1%, either when flagella were overproduced or when expression of the FliL protein was enhanced in conjunction with stator proteins MotAB. We surmise that bacteria use the resulting increase in motor power to overcome the higher friction associated with harder agar. Higher flagellar numbers also suppress the swarming defect of mutants with changes in the chemotaxis pathway that were previously shown to be defective in hydrating their colonies. Here we show that the swarming defect of these mutants can also be suppressed by application of osmolytes to the surface of swarm agar. The "dry" colony morphology displayed by che mutants was also observed with other mutants that do not actively rotate their flagella. The flagellum/motor thus participates in two functions critical for swarming, enabling hydration and overriding surface friction. We consider some ideas for how the flagellum might help attract water to the agar surface, where there is no free water.

Identification of Chemotaxis Sensory Proteins for Amino Acids in <i>Pseudomonas fluorescens</i> Pf0-1 and Their Involvement in Chemotaxis to Tomato Root Exudate and Root Colonization

Pseudomonas fluorescens Pf0-1 showed positive chemotactic responses toward 20 commonly-occurring l-amino acids. Genomic analysis revealed that P. fluorescens Pf0-1 possesses three genes (Pfl01_0124, Pfl01_0354, and Pfl01_4431) homologous to the Pseudomonas aeruginosa PAO1 pctA gene, which has been identified as a chemotaxis sensory protein for amino acids. When Pf01_4431, Pfl01_0124, and Pfl01_0354 were introduced into the pctA pctB pctC triple mutant of P. aeruginosa PAO1, a mutant defective in chemotaxis to amino acids, its transformants showed chemotactic responses to 18, 16, and one amino acid, respectively. This result suggests that Pf01_4431, Pfl01_0124, and Pfl01_0354 are chemotaxis sensory proteins for amino acids and their genes were designated ctaA, ctaB, and ctaC, respectively. The ctaA ctaB ctaC triple mutant of P. fluorescens Pf0-1 showed only weak responses to Cys and Pro but no responses to the other 18 amino acids, indicating that CtaA, CtaB, and CtaC are major chemotaxis sensory proteins in P. fluorescens Pf0-1. Tomato root colonization by P. fluorescens strains was analyzed by gnotobiotic competitive root colonization assay. It was found that ctaA ctaB ctaC mutant was less competitive than the wild-type strain, suggesting that chemotaxis to amino acids, major components of root exudate, has an important role in root colonization by P. fluorescens Pf0-1. The ctaA ctaB ctaC triple mutant was more competitive than the cheA mutant of P. fluorescens Pf0-1, which is non-chemotactic, but motile. This result suggests that chemoattractants other than amino acids are also involved in root colonization by P. fluorescens Pf0-1.

A histidine-kinase cheA gene of Pseudomonas pseudoalcaligens KF707 not only has a key role in chemotaxis but also affects biofilm formation and cell metabolism

A histidine-kinase cheA gene in Pseudomonas pseudoalcaligenes KF707 plays a central role in the regulation of metabolic responses as well as in chemotaxis. Non-chemotactic mutants harboring insertions into the cheA gene were screened for their ability to form biofilms in the Calgary biofilm device. Notably, ≥95% decrease in the number of cells attached to the polystyrene surface was observed in cheA mutants compared to the KF707 wild-type biofilm phenotype. The ability to form mature biofilms was restored to wild-type levels, providing functional copies of the KF707 cheA gene to the mutants. In addition, phenotype micro-arrays and proteomic analyses revealed that several basic metabolic activities and a few periplasmic binding proteins of cheA mutant cells differed compared to those of wild-type cells. These results are interpreted as evidence of a strong integration between chemotactic and metabolic pathways in the process of biofilm development by P. pseudoalcaligenes KF707.

Thermal Domain Motions of CheA Kinase in Solution: Disulfide Trapping Reveals the Motional Constraints Leading to Trans-autophosphorylation

The histidine kinase CheA is a central component of the bacterial chemotaxis signaling cluster, in which transmembrane receptors regulate CheA autokinase activity. CheA is a homodimer, and each of the two identical subunits possesses five different domains with distinct structures and functions. The free enzyme, like the receptor-bound enzyme, catalyzes a trans-autokinase reaction in which the catalytic domain (P4) of one subunit phosphorylates the substrate domain (P1) of the other subunit. Molecular analysis of CheA domain motions has important implications for the mechanism of CheA trans-autophosphorylation, for CheA assembly into the signaling cluster and for receptor regulation of CheA activity. In this initial study of the free CheA dimer, we employ disulfide trapping to analyze collisions between pairs of domains, thereby mapping out the ranges and kinetics of domain motions. A library of 33 functional single-cysteine CheA mutants, all retaining normal autokinase activity, is used to analyze intradimer collisions between symmetric domain pairs. The homodimeric structure of CheA ensures that each mutant contains a pair of symmetric, surface-exposed cysteine residues. Cysteine-cysteine collisions trapped by disulfide bond formation indicate that P1 is the most mobile CheA domain, but large amplitude P2, P4, and P5 domain motions are also detected. The mobility of P1 is further analyzed using a library of 17 functional dicysteine CheA mutants, wherein each mutant subunit possesses one cysteine at a fixed probe position on the P1 domain and a second cysteine on a different domain. The resulting CheA homodimers contain four cysteine residues; thus disulfide trapping yields multiple products that are identified by assignment methods. The findings reveal that the P1 substrate domain collides rapidly with residues on the P4' catalytic domain in the sister subunit, but no intrasubunit collisions are detected. This observation provides a direct, motional explanation for CheA trans-autophosphorylation, explains why the long linkers of the P1-P2 region do not become tangled in the dimer, and has important implications for other aspects of CheA function. Finally, a working model is proposed for the motional constraints that limit the P1 domain to the region of space near the P4' catalytic domain of the sister subunit.

Chemotaxis in Rhodobacter sphaeroides Requires an Atypical Histidine Protein Kinase

Rhodobacter sphaeroides has a complex chemosensory system comprising two classic CheAs, two atypical CheAs, and eight response regulators (six CheYs and two CheBs). The classic CheAs, CheA(1) and CheA(2), have similar domain structures to Escherichia coli CheA, whereas the atypical CheAs, CheA(3) and CheA(4), lack some of the domains found in E. coli CheA. CheA(2), CheA(3), and CheA(4) are all essential for chemotaxis. Here we demonstrate that CheA(3) and CheA(4) are both unable to undergo ATP-dependent autophosphorylation, however, CheA(4) is able to phosphorylate CheA(3). The in vitro kinetics of this phosphorylation reaction were consistent with a reaction mechanism in which CheA(3) associates with a CheA(4) dimer forming a complex, CheA(3)A(4). To the best of our knowledge, CheA(3)A(4) is the first characterized histidine protein kinase where the subunits are encoded by distinct genes. Selective phosphotransfer was observed from CheA(3)-P to the response regulators CheY(1), CheY(6), and CheB(2). Using phosphorylation site and kinase domain mutants of CheA we show that phosphosignaling involving CheA(2), CheA(3), and CheA(4) is essential for chemotaxis in R. sphaeroides. Interestingly, CheA(3) was not phosphorylated in vitro by CheA(1) or CheA(2), although CheA(1) and CheA(2) mutants with defective kinase domains were phosphorylated by CheA(4). Because in vivo CheA(3) and CheA(4) localize to the cytoplasmic chemotaxis cluster, while CheA(2) localizes to the polar chemotaxis cluster, it is likely that the physical separation of CheA(2) and CheA(4) prevents unwanted cross-talk between these CheAs.

Role of Chemotaxis Toward Fusaric Acid in Colonization of Hyphae of Fusarium oxysporum f. sp. radicis-lycopersici by Pseudomonas fluorescens WCS365

Pseudomonas fluorescens WCS365 is an excellent competitive colonizer of tomato root tips after bacterization of seed or seedlings. The strain controls tomato foot and root rot (TFRR) caused by the phytopathogenic fungus Fusarium oxysporum f. sp. radicis-lycopersici. Under biocontrol conditions, fungal hyphae were shown to be colonized by WCS365 bacteria. Because chemotaxis is required for root colonization by WCS365 cells, we studied whether chemotaxis also is required for hyphae colonization. To that end, an in vitro assay was developed to study hyphae colonization by bacteria. The results indicated that cells of the cheA mutant FAJ2060 colonize hyphae less efficiently than cells of wild-type strain WCS365, when single strains were analyzed as well as when both strains were applied together. Cells of WCS365 show a chemotactic response toward the spent growth medium of F. oxysporum f. sp. radicis-lycopersici, but those of its cheA mutant, FAJ2060, did not. Fusaric acid, a secondary metabolite secreted by Fusarium strains, appeared to be an excellent chemo-attractant. Supernatant fluids of a number of Fusarium strains secreting different levels of fusaric acid were tested as chemo-attractants. A positive correlation was found between chemo-attractant activity and fusaric acid level. No chemotactic response was observed toward the low fusaric acid-producer FO242. Nevertheless, the hyphae of FO242 still were colonized by WCS365, suggesting that other metabolites also play a role in this process. The possible function of hyphae colonization for the bacterium is discussed.

Flagella-driven chemotaxis towards exudate components is an important trait for tomato root colonization by Pseudomonas fluorescens.

Motility is a major trait for competitive tomato root-tip colonization by Pseudomonas fluorescens. To test the hypothesis that this role of motility is based on chemotaxis toward exudate components, cheA mutants that were defective in flagella-driven chemotaxis but retained motility were constructed in four P. fluorescens strains. After inoculation of seedlings with a 1:1 mixture of wild-type and nonmotile mutants all mutants had a strongly reduced competitive root colonizing ability after 7 days of plant growth, both in a gnotobiotic sand system as well as in nonsterile potting soil. The differences were significant on all root parts and increased from root base to root tip. Significant differences at the root tip could already be detected after 2 to 3 days. These experiments show that chemotaxis is an important competitive colonization trait. The best competitive root-tip colonizer, strain WCS365, was tested for chemotaxis toward tomato root exudate and its major identified components. A chemotactic response was detected toward root exudate, some organic acids, and some amino acids from this exudate but not toward its sugars. Comparison of the minimal concentrations required for a chemotactic response with concentrations estimated for exudates suggested that malic acid and citric acid are among major chemo-attractants for P. fluorescens WCS365 cells in the tomato rhizosphere.

Motility and chemotaxis in tissue penetration of oral epithelial cell layers by Treponema denticola.

The ability to penetrate tissue is an important virulence factor for pathogenic spirochetes. Previous studies have recognized the role of motility in allowing pathogenic spirochetes to invade tissues and migrate to sites favorable for bacterial proliferation. However, the nature of the movements, whether they are random or controlled by chemotaxis systems, has yet to be established. In this study, we addressed the role of motility and chemotaxis in tissue penetration by the periodontal disease-associated oral spirochete Treponema denticola using an oral epithelial cell line-based experimental approach. Wild-type T. denticola ATCC 35405 was found to penetrate the tissue layers effectively, whereas a nonmotile mutant was unable to overcome the tissue barrier. Interestingly, the chemotaxis mutants also showed impaired tissue penetration. A cheA mutant that is motile but lacks the central kinase of the chemotaxis pathway showed only about 2 to 3% of the wild-type penetration rate. The two known chemoreceptors of T. denticola, DmcA and DmcB, also appear to be involved in the invasion process. The dmc mutants were actively motile but exhibited reduced tissue penetration of about 30 and 10% of the wild-type behavior, respectively. These data suggest that not only motility but also chemotaxis is involved in the tissue penetration by T. denticola.

Identification of a fourth cheY gene in Rhodobacter sphaeroides and interspecies interaction within the bacterial chemotaxis signal transduction pathway.

The Escherichia coli chemotaxis signal transduction pathway has: CheA, a histidine protein kinase; CheW, a linker between CheA and sensory proteins; CheY, the effector; and CheZ, a signal terminator. Rhodobacter sphaeroides has multiple copies of these proteins (2 x CheA, 3 x CheW and 3 x CheY, but no CheZ). In this study, we found a fourth cheY and expressed these R. sphaeroides proteins in E. coli. CheA2 (but not CheA1) restored swarming to an E. coli cheA mutant (RP9535). CheW3 (but not CheW2) restored swarming to a cheW mutant of E. coli (RP4606). R. sphaeroides CheYs did not affect E. coli lacking CheY, but restored swarming to a cheZ strain (RP1616), indicating that they can act as signal terminators in E. coli. An E. coli CheY, which is phosphorylated but cannot bind the motor (CheY109KR), was expressed in RP1616 but had no effect. Overexpression of CheA2, CheW2, CheW3, CheY1, CheY3 and CheY4 inhibited chemotaxis of wild-type E. coli (RP437) by increasing its smooth-swimming bias. While some R. sphaeroides proteins restore tumbling to smooth-swimming E. coli mutants, their activity is not controlled by the chemosensory receptors. R. sphaeroides possesses a phosphorelay cascade compatible with that of E. coli, but has additional incompatible homologues.

TNP-ATP and TNP-ADP as probes of the nucleotide binding site of CheA, the histidine protein kinase in the chemotaxis signal transduction pathway of Escherichia coli.

The interaction of CheA with ATP has important consequences in the chemotaxis signal transduction pathway of Escherichia coli. This interaction results in autophosphorylation of CheA, a histidine protein kinase. Autophosphorylation of CheA sets in motion a chain of biochemical events that enables the chemotaxis receptor proteins to communicate with the flagellar motors. As a result of this communication, CheA allows the receptors to control the cell swimming pattern in response to gradients of attractant and repellent chemicals. To probe CheA interactions with ATP, we investigated the interaction of CheA with the fluorescent nucleotide analogues TNP-ATP [2'(3')-O-(2,4,6-trinitrophenyl)adenosine 5'-triphosphate] and TNP-ADP. Spectroscopic studies indicated that CheA bound TNP-ATP and TNP-ADP with high affinity (micromolar Kd values) and caused a marked enhancement of the fluorescence of the TNP moiety of these modified nucleotides. Analysis of titration experiments indicated a binding stoichiometry of two molecules of TNP-ATP (TNP-ADP) per CheA dimer and suggested that the two binding sites on the CheA dimer operate independently. Binding of TNP-ATP to CheA was inhibited by ATP, and analysis of this inhibition indicated that the CheA dimer binds 2 molecules of ATP. Competition experiments also indicated that CheA binds TNP-ATP considerably more tightly than it binds unmodified ATP. Binding of TNP-ADP to CheA was inhibited by ADP in a similar manner. TNP-ATP was not a substrate for CheA and served as a potent inhibitor of CheA autophosphorylation (Ki < 1 microM). The glycine-rich regions (G1 and G2) of CheA and other histidine protein kinases have been presumed to play important roles in ATP binding and/or catalysis of CheA autophosphorylation, although few experimental tests of these functional assignments have been made. Here, we demonstrate that a CheA mutant protein with Gly-->Ala substitutions in G1 and G2 has a markedly reduced affinity for ATP and ADP, as measured by Hummel-Dreyer chromatography. This mutant protein also bound TNP-ATP and TNP-ADP very poorly and had no detectable autokinase activity. Surprisingly, a distinct single-site substitution in G2 (Gly470-->Lys) had no observable effect on the affinity of CheA for ATP and ADP, despite the fact that it rendered CheA completely inactive as an autokinase. This mutant protein also bound TNP-ATP and TNP-ADP with affinities and stoichiometries that were indistinguishable from those observed with wild-type CheA. These results provide some preliminary insight into the possible functional roles of G1 and G2, and they suggest that TNP-nucleotides are useful tools for exploring the effects of additional mutations on the active site of CheA.

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.

Chemotactic signaling by the P1 phosphorylation domain liberated from the CheA histidine kinase of Escherichia coli.

CheA is a histidine kinase central to the signal transduction pathway for chemotaxis in Escherichia coli. CheA autophosphorylates at His-48, with ATP as the phosphodonor, and then donates its phosphoryl groups to two aspartate autokinases, CheY and CheB. Phospho-CheY controls the flagellar motors, whereas phospho-CheB participates in sensory adaptation. Polypeptides encompassing the N-terminal P1 domain of CheA can be transphosphorylated in vitro by the CheA catalytic domain and yet have no deleterious effect on chemotactic ability when expressed at high levels in wild-type cells. To find out why, we examined the effects of a purified P1 fragment, CheA[1-149], on CheA-related signaling activities in vitro and devised in vivo assays for those same activities. Although readily phosphorylated by CheA[260-537], the CheA catalytic domain, CheA[1-149], was a poor substrate for transphosphorylation by full-length CheA molecules, implying that the resident P1 domain monopolizes the CheA catalytic center. CheA-H48Q, a nonphosphorylatable mutant, failed to transphosphorylate CheA[1-149], suggesting that phosphorylation of the P1 domain in cis may alleviate the exclusion effect. In agreement with these findings, a 40-fold excess of CheA[1-149] fragments did not impair the CheA autophosphorylation reaction. CheA[1-149] did acquire phosphoryl groups via reversible phosphotransfer reactions with CheB and CheY molecules. An H48Q mutant of CheA[1-149] could not participate in these reactions, indicating that His-48 is probably the substrate site. The low level of efficiency of these phosphotransfer reactions and the inability of CheA[1-149] to interfere with CheA autophosphorylation most likely account for the failure of liberated P1 domains to jam chemotactic signaling in wild-type cells. However, an excess of CheA[1-149] fragments was able to support chemotactic signaling by P1-deficient cheA mutants, demonstrating that CheA[1-149] fragments have both transphosphorylation and phosphotransfer capability in vivo.

CheC and CheD interact to regulate methylation of Bacillus subtilis methyl-accepting chemotaxis proteins.

In this study, we have demonstrated that two unique proteins in Bacillus subtilis chemotaxis, CheC and CheD, interact. We have shown this interaction both by using the yeast two-hybrid system and by precipitation of in vitro translated products using glutathione-S-transferase fusions and glutathione agarose beads. We have also shown that CheC inhibits B. subtilis CheR-mediated methylation of B. subtilis methyl-accepting chemotaxis proteins (MCPs) but not of Escherichia coli MCPs. It was previously reported that cheC mutants tend to swim smoothly and do not adapt to addition of attractant; cheD mutants have very poorly methylated MCPs and are very tumbly, similar to cheA mutants. We hypothesize that CheC exerts its effect on MCP methylation in B. subtilis by controlling the binding of CheD to the MCPs. In absence of CheD, the MCPs are poor substrates for CheR and appear to tie up, rather than activate, CheA. The regulation of CheD by CheC may be part of a unique adaptation system for chemotaxis in B. subtilis, whereby high levels of CheY-P brought about by attractant addition would allow CheC to interact with CheD and consequently leave the MCPs, reducing CheA activity and hence the levels of CheY-P.

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 overexpressed 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.(ABSTRACT TRUNCATED AT 250 WORDS)

Bacillus subtilis CheN, a homolog of CheA, the central regulator of chemotaxis in Escherichia coli

The Bacillus subtilis cheN gene was isolated, sequenced, and expressed. It encodes a large negatively charged protein with a molecular weight of approximately 74,000. The predicted protein sequence has 33 to 34% identity with the Escherichia coli and Salmonella typhimurium CheA and Myxococcus xanthus FrzE sequences. These proteins are found to autophosphorylate and are members of the same histidine kinase signal modulating family. CheN has several conserved regions (including the histidine that is phosphorylated in CheA) that coincide with other autophosphorylated signal transducers. A null mutant is defective in attractant-induced methanol formation and shows no behavioral response to chemoeffectors. These results imply that in B. subtilis the mechanism of chemotaxis involves phosphoryl transfer similar to that in E. coli. However, the CheN null mutant mostly tumbles, whereas CheA mutants swim smoothly, and only in B. subtilis does excitation lead to methyl transfer and methanol formation. Thus, the overall mechanism of chemotaxis is different in the two organisms.

Crosstalk between bacterial chemotaxis signal transduction proteins and regulators of transcription of the Ntr regulon: evidence that nitrogen assimilation and chemotaxis are controlled by a common phosphotransfer mechanism.

We demonstrate by using purified bacterial components that the protein kinases that regulate chemotaxis and transcription of nitrogen-regulated genes, CheA and NRII, respectively, have cross-specificities: CheA can phosphorylate the Ntr transcription factor NRI and thereby activate transcription from the nitrogen-regulated glnA promoter, and NRII can phosphorylate CheY. In addition, we find that a high intracellular concentration of a highly active mutant form of NRII can suppress the smooth-swimming phenotype of a cheA mutant. These results argue strongly that sensory transduction in the Ntr and Che systems involves a common protein phosphotransfer mechanism.

Refined genetic analysis of the region II che mutants in Salmonella typhimurium.

From a detailed complementation analysis of the region II che mutants of Salmonella typhimurium, we have located five che genes, cheA, cheW, cheR, cheB, and cheY. We have shown that corrections are required in the previous assignment of the mutations in four strains: both SL2514 and SL2515 which have been reported to be cheY mutants are cheR mutants, SL2539 is not a cheA but a cheW mutant, and ST171 which has been reported to be a cheZ mutant is a double mutant with defects in both cheA and cheB. Since ST171 is the only "cheZ mutant" so far isolated, the idea that the cheZ gene might play an essential role in chemotaxis in S. typhimurium as in Escherichia coli has lost its experimental basis. Furthermore, a number of deletion mutants in region II resulting from the excision of Tn10 have been isolated and analysed. From these experiments, we propose that the gene order in region II is flaK-flaE-motA-motB-cheA-cheW-cheR-cheB-++ +cheY-flaM-flaC, which is identical with that in E. coli.