The highly conserved domain of the Caulobacter McpA chemoreceptor is required for its polar localization

We have fused GFP to the C-terminus of McpA to study chemoreceptor polar localization in Caulobacter crescentus. The full-length McpA-GFP fusion is polarly localized and methylated. The methylation is dependent on the chemoreceptor methyltransferase (cheR) and chemoreceptor methylesterase (cheB) genes present in the mcpA operon. C-terminal and internal deletions of McpA were constructed and fused to the N-terminus of GFP to identify the domains required for polar localization. When the R1 methylation domain was deleted, the McpA-GFP fusion was still polarly localized, suggesting that this domain is dispensable for polar localization. However, when the highly conserved domain (HCD), which is involved in interacting with CheW, was deleted either by an internal deletion or C-terminal deletion, the resulting McpA-GFP fusions were completely delocalized. When the mcpA operon, which contains the cheW and cheA homologues, was deleted, the full-length McpA-GFP fusion was delocalized. Although additional chemotaxis genes are required for the polar localization of McpA-GFP, the presence of the single polar flagellum is not required. However, in filamentous cells, which are frequently found in C. crescentus fliF mutants, the McpA-GFP fusion was observed at mid-cell positions.     

Degradation of a Caulobacter soluble cytoplasmic chemoreceptor is ClpX dependent

In order to determine whether ClpXP-mediated proteolysis is a common mechanism used to regulate the chemotaxis machinery during the cell cycle of Caulobacter crescentus, we have characterized a soluble cytoplasmic chemoreceptor, McpB. The mcpB gene lies adjacent to the major chemotaxis operon, which encodes 12 chemotaxis proteins, including the membrane chemoreceptor McpA. Like McpA, McpB possesses a C-terminal CheBR docking motif and three potential methylation sites, which we suggest are methylated. The McpB protein is degraded via a ClpX-dependent pathway during the swarmer-to-stalked cell transition, and a motif, which is 3 amino acids N-terminal to the McpB CheBR docking site, is required for proteolysis. Analysis of the degradation signal in McpB and McpA reveals a common motif present in the other four chemoreceptors that possess CheBR docking sites. A green fluorescent protein (GFP) fusion bearing 58 amino acids from the C terminus of McpA, which contains this motif, is degraded, suggesting that the C-terminal sequence is sufficient to confer ClpXP protease susceptibility.     

Proteolysis of the Caulobacter McpA chemoreceptor is cell cycle regulated by a ClpX-dependent pathway

Proteolysis is involved in cell differentiation and the progression through the cell cycle in Caulobacter crescentus. We have constitutively expressed the transmembrane chemoreceptor McpA from a multicopy plasmid to demonstrate that McpA degradation is modulated during the cell cycle. The level of McpA protein starts to decrease only when the swarmer cells differentiate into stalked cells. The reduction in McpA protein levels is maintained until the stalked cells develop into predivisional cells, at which point the level returns to that observed in swarmer cells. The cell-cycle-regulated degradation of McpA does not require the last 12 C-terminal amino acids, but it does require three amino acids (AAL) located 15 residues away from the C terminus. The ClpXP protease is essential in C. crescentus for viability, and thus, we tested McpA degradation in xylose conditional mutants. The effect on McpA degradation occurred within two generations from the start of ClpX depletion. The conditional mutants' growth rate was only slightly affected, suggesting that ClpX is directly involved in McpA proteolysis.     

Identification of a methyl-accepting chemotaxis protein in Rhodobacter sphaeroides

Analysis of the DNA sequence directly upstream of the chemotaxis operon of Rhodobacter sphaeroides identified a single gene whose product has strong similarity to the methyl-accepting chemotaxis proteins (MCPs) found in enteric bacteria. The deduced protein had a highly conserved signalling sequence and only one very hydrophobic region at the N-terminus, in contrast to enteric MCPs. A possible cytoplasmic location of the majority of the protein was supported by Western blotting. The mcpA gene was insertionally inactivated and the resulting phenotype examined using swarm plate assays. The mutant lacking McpA lost chemotaxis to a wide range of attractant stimuli but only under aerobic conditions; it retained almost normal chemotaxis under anaerobic/photosynthetic conditions. The identification of a sensory protein which is active only under one set of growth conditions suggests that R. sphaeroides probably has several MCPs, which co-ordinately respond to changes in environmental conditions. Southern hybridization at relaxed stringency to the conserved sequence of the R. sphaeroides and Caulobacter crescentus mcp genes identified three possible additional mcp genes.     

Two different Pseudomonas aeruginosa chemosensory signal transduction complexes localize to cell poles and form and remould in stationary phase

Pseudomonas aeruginosa has sets of sensory genes designated che and che2. The che genes are required for flagella-mediated chemotaxis. The che2 genes are expressed in the stationary phase of growth and are probably also involved in flagella-mediated behavioural responses. P. aeruginosa also has 26 chemoreceptor genes, six of which are preferentially expressed in stationary phase. Subcellular localization experiments indicated that Che proteins form signal transduction complexes at cell poles throughout growth. Cyan fluorescent protein (CFP)-tagged McpA, a stationary phase-expressed chemoreceptor, appeared and colocalized with yellow fluorescent protein (YFP)-tagged CheA when cells entered stationary phase. This indicates that P. aeruginosa chemotaxis protein complexes are subject to remoulding by chemoreceptor proteins that are expressed when cells stop growing. CheA-CFP and CheY2-YFP tagged proteins that were coexpressed in the same cell had separate subcellular locations, indicating that Che2 proteins do not enter into direct physical interactions with Che proteins. Che2 protein complex formation required McpB, another stationary phase induced chemoreceptor that is predicted to be soluble. This implies that Che2 complexes have a function that depends on just one chemoreceptor. Our results suggest that motile P. aeruginosa cells have signal transduction systems that are adapted to allow non-growing cells to sense and respond to their environment differently from actively growing cells.     

Identification and characterization of the chemotactic transducer in Pseudomonas aeruginosa PAO1 for positive chemotaxis to trichloroethylene

Pseudomonas aeruginosa PAO1 is repelled by trichloroethylene (TCE), and the methyl-accepting chemotaxis proteins PctA, PctB, and PctC serve as the major chemoreceptors for negative chemotaxis to TCE. In this study, we found that the pctABC triple mutant of P. aeruginosa PAO1 was attracted by TCE. Chemotaxis assays of a set of mutants containing deletions in 26 potential mcp genes revealed that mcpA (PA0180) is the chemoreceptor for positive chemotaxis to TCE. McpA also detects tetrachloroethylene and dichloroethylene isomers as attractants.     

Cluster II che genes from Pseudomonas aeruginosa are required for an optimal chemotactic response

Pseudomonas aeruginosa, a gamma-proteobacterium, is motile by means of a single polar flagellum and is chemotactic to a variety of organic compounds and phosphate. P. aeruginosa has multiple homologues of Escherichia coli chemotaxis genes that are organized into five gene clusters. Previously, it was demonstrated that genes in cluster I and cluster V are essential for chemotaxis. A third cluster (cluster II) contains a complete set of che genes, as well as two genes, mcpA and mcpB, encoding methyl-accepting chemotaxis proteins. Mutations were constructed in several of the cluster II che genes and in the mcp genes to examine their possible contributions to P. aeruginosa chemotaxis. A cheB2 mutant was partially impaired in chemotaxis in soft-agar swarm plate assays. Providing cheB2 in trans complemented this defect. Further, overexpression of CheB2 restored chemotaxis to a completely nonchemotactic, cluster I, cheB-deficient strain to near wild-type levels. An mcpA mutant was defective in chemotaxis in media that were low in magnesium. The defect could be relieved by the addition of magnesium to the swarm plate medium. An mcpB mutant was defective in chemotaxis when assayed in dilute rich soft-agar swarm medium or in minimal-medium swarm plates containing any 1 of 60 chemoattractants. The mutant phenotype could be complemented by the addition of mcpB in trans. Overexpression of either McpA or McpB in P. aeruginosa or Escherichia coli resulted in impairment of chemotaxis, and these cells had smooth-swimming phenotypes when observed under the microscope. Expression of P. aeruginosa cheA2, cheB2, or cheW2 in E. coli K-12 completely disrupted wild-type chemotaxis, while expression of cheY2 had no effect. These results indicate that che cluster II genes are expressed in P. aeruginosa and are required for an optimal chemotactic response.     

The carboxyl-terminal linker is important for chemoreceptor function

Sensory adaptation in bacterial chemotaxis is mediated by chemoreceptor methylation and demethylation. In Escherichia coli, methyltransferase CheR and methylesterase CheB bind both substrate sites and a carboxyl-terminal pentapeptide sequence carried by certain receptors. Pentapeptide binding enhances enzyme action, an enhancement required for effective adaptation and chemotaxis. Pentapeptides are linked to the conserved body of chemoreceptors through a notably variable sequence of 30-35 residues. We created nested deletions from the distal end of this linker in chemoreceptor Tar. Chemotaxis was eliminated by deletion of 20-40 residues and reduced by shorter deletions. This did not reflect generalized disruption, because all but the most extremely truncated receptors activated kinase, were substrates for adaptational modification and performed transmembrane signalling. In contrast, linker truncations reduced rates of adaptational modification in parallel with chemotaxis. We concluded the linker is important for chemotaxis because of its role in adaptational modification. Effects of linker truncations on CheR binding to receptor-borne pentapeptide implied linker (i) makes pentapeptide available to modification enzymes by separation from the helical receptor body, and (ii) is a flexible arm allowing dual binding of enzyme to pentapeptide and modification site. The data suggest linker and the helix from which it emerges are structurally dynamic.     

CheR- and CheB-Dependent Chemosensory Adaptation System of Rhodobacter sphaeroides

Rhodobacter sphaeroides has multiple homologues of most of the Escherichia coli chemotaxis genes, organized in three major operons and other, unlinked, loci. These include cheA(1) and cheR(1) (che Op(1)) and cheA(2), cheR(2), and cheB(1) (che Op(2)). In-frame deletions of these cheR and cheB homologues were constructed and the chemosensory behaviour of the resultant mutants examined on swarm plates and in tethered cell assays. Under the conditions tested, CheR(2) and CheB(1) were essential for normal chemotaxis, whereas CheR(1) was not. cheR(2) and cheB(1), but not cheR(1), were also able to complement the equivalent E. coli mutants. However, none of the proteins were required for the correct polar localization of the chemoreceptor McpG in R. sphaeroides. In E. coli, CheR binds to the NWETF motif on the high-abundance receptors, allowing methylation of both high- and low-abundance receptors. This motif is not contained on any R. sphaeroides chemoreceptors thus far identified, although 2 of the 13 putative chemoreceptors, McpA and TlpT, do have similar sequences. This suggests that CheR(2) either interacts with the NWETF motif of E. coli methyl-accepting chemotaxis proteins (MCPs), even though its native motif may be slightly different, or with another conserved region of the MCPs. Methanol release measurements show that R. sphaeroides has an adaptation system that is different from that of Bacillus subtilis and E. coli, with methanol release measurable on the addition of attractant but not on its removal. Intriguingly, CheA(2), but not CheA(1), is able to phosphorylate CheB(1), suggesting that signaling through CheA(1) cannot initiate feedback receptor adaptation via CheB(1)-P.     

Identification, sequence, and expression of Treponema denticola mcpA, a putative chemoreceptor gene

The nucleotide sequence of a methyl-accepting chemotaxis protein gene, mcpA, from Treponema denticola has been determined. The mcpA gene encodes a 729-amino acid protein whose deduced amino acid sequence has significant homology with several bacterial MCPs. T. denticola McpA contains two N-terminal transmembrane regions and two C-terminal putative methylation sequences that are separated by a highly conserved signaling domain. The organization of these structural features is characteristic of MCPs. The observed molecular mass of the in vitro synthesized McpA (76.0 kDa) correlates with the predicted molecular mass of the protein (80.1 kDa).     

The roles of the multiple CheW and CheA homologues in chemotaxis and in chemoreceptor localization in Rhodobacter sphaeroides

Rhodobacter sphaeroides has multiple homologues of most of the Escherichia coli chemotaxis genes, organized in two major operons and other, unlinked, loci. These include cheA1 and cheW1 (che Op1) and cheA2, cheW2 and cheW3 (che Op2). We have deleted each of these cheA and cheW homologues in-frame and examined the chemosensory behaviour of these strains on swarm plates and in tethered cell assays. In addition, we have examined the effect of these deletions on the polar localization of the chemoreceptor McpG. In E. coli, deletion of either cheA or cheW results in a non-chemotactic phenotype, and these strains also show no receptor clustering. Here, we demonstrate that CheW2 and CheA2 are required for the normal localization of McpG and for normal chemotactic responses under both aerobic and photoheterotrophic conditions. Under aerobic conditions, deletion of cheW3 has no significant effect on McpG localization and only has an effect on chemotaxis to shallow gradients in swarm plates. Under photoheterotrophic conditions, however, CheW3 is required for McpG localization and also for chemotaxis both on swarm plates and in the tethered cell assay. These phenotypes are not a direct result of delocalization of McpG, as this chemoreceptor does not mediate chemotaxis to any of the compounds tested and can therefore be considered a marker for general methyl-accepting chemotaxis protein (MCP) clustering. Thus, there is a correlation between the normal localization of McpG (and presumably other chemoreceptors) and chemotaxis. We propose a model in which the multiple different MCPs in R. sphaeroides are contained within a polar chemoreceptor cluster. Deletion of cheW2 and cheA2 under both aerobic and photoheterotrophic conditions, and cheW3 under photoheterotrophic conditions, disrupts the cluster and hence reduces chemotaxis to any compound sensed by these MCPs.     

Dual recognition of the bacterial chemoreceptor by chemotaxis-specific domains of the CheR methyltransferase

Adaptation to persisting stimulation is required for highly sensitive detection of temporal changes of stimuli, and often involves covalent modification of receptors. Therefore, it is of vital importance to understand how a receptor and its cognate modifying enzyme(s) modulate each other through specific protein-protein interactions. In the chemotaxis of Escherichia coli, adaptation requires methylation of chemoreceptors (e.g. Tar) catalyzed by the CheR methyltransferase. CheR binds to the C-terminal NWETF sequence of a chemoreceptor that is distinct from the methylation sites. However, little is known about how CheR recognizes its methylation sites or how it is distributed in a cell. In this study, we used comparative genomics to demonstrate that the CheR chemotaxis methyltransferase contains three structurally and functionally distinct modules: (i) the catalytic domain common to a methyltransferase superfamily; (ii) the N-terminal domain; and (iii) the beta-subdomain of the catalytic domain, both of which are found exclusively in chemotaxis methyltransferases. The only evolutionary conserved motif specific to CheR is the positively charged face of helix alpha2 in the N-terminal domain. The disulfide cross-linking analysis suggested that this face interacts with the methylation helix of Tar. We also demonstrated that CheR localizes to receptor clusters at cell poles via interaction of the beta-subdomain with the NWETF sequence. Thus, the two chemotaxis-specific modules of CheR interact with distinct regions of the chemoreceptor for targeting to the receptor cluster and for recognition of the substrate sites, respectively.     

Analysis of the Frz signal transduction system of Myxococcus xanthus shows the importance of the conserved C-terminal region of the cytoplasmic chemoreceptor FrzCD in sensing signals

The Frz chemosensory system controls directed motility in Myxococcus xanthus by regulating cellular reversal frequency. M. xanthus requires the Frz system for vegetative swarming on rich media and for cellular aggregation during fruiting body formation on starvation media. The Frz signal transduction pathway is formed by proteins that share homology with chemotaxis proteins from enteric bacteria, which are encoded in the frzA-F putative operon and the divergently transcribed frzZ gene. FrzCD, the Frz system chemoreceptor, contains a conserved C-terminal module present in methyl-accepting chemotaxis proteins (MCPs); but, in contrast to most MCPs, FrzCD is localized in the cytoplasm and the N-terminal region of FrzCD does not contain transmembrane or sensing domains, or even a linker region. Previous work on the Frz system was limited by the unavailability of deletion strains. To understand better how the Frz system functions, we generated a series of in-frame deletions in each of the frz genes as well as regions encoding the N-terminal portion of FrzCD. Analysis of mutants containing these deletions showed that FrzCD (MCP), FrzA (CheW) and FrzE (CheA-CheY) control vegetative swarming, responses to repellents and directed movement during development, thus constituting the core components of the Frz pathway. FrzB (CheW), FrzF (CheR), FrzG (CheB) and FrzZ (CheY-CheY) are required for some but not all responses. Furthermore, deletion of approximately 25 amino acids from either end of the conserved C-terminal region of FrzCD results in a constitutive signalling state of FrzCD, which induces hyper-reversals with no net cell movement. Surprisingly, deletion of the N-terminal region of FrzCD shows only minor defects in swarming. Thus, signal input to the Frz system must be sensed by the conserved C-terminal module of FrzCD and not the usual N-terminal region. These results indicate an alternative mechanism for signal sensing with this cytoplasmic MCP.     

Bacillus subtilis CheD is a chemoreceptor modification enzyme required for chemotaxis

The chemotaxis machinery of Bacillus subtilis is similar to that of the well characterized system of Escherichia coli. However, B. subtilis contains several chemotaxis genes not found in the E. coli genome, such as cheC and cheD, indicating that the B. subtilis chemotactic system is more complex. In B. subtilis, CheD is required for chemotaxis; the cheD mutant displays a tumbly phenotype, has abnormally methylated chemoreceptors, and responds poorly to most chemical stimuli. Homologs of B. subtilis CheD have been found in chemotaxis-like operons of a large number of bacteria and archaea, suggesting that CheD plays an important role in chemotactic sensory transduction for many organisms. However, the molecular function of CheD has remained unknown. In this study, we show that CheD catalyzes amide hydrolysis of specific glutaminyl side chains of the B. subtilis chemoreceptor McpA. In addition, we present evidence that CheD deamidates other B. subtilis chemoreceptors including McpB and McpC. Previously, deamidation of B. subtilis receptors was thought to be catalyzed by the CheB methylesterase, as is the case for E. coli receptors. Because cheD mutant cells do not respond to most chemoattractants, we conclude that deamidation by CheD is required for B. subtilis chemoreceptors to effectively transduce signals to the CheA kinase.     

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.     

A CheW homologue is required for Myxococcus xanthus fruiting body development,social gliding motility,and fibril biogenesis

In bacteria with multiple sets of chemotaxis genes, the deletion of homologous genes or even different genes in the same operon can result in disparate phenotypes. Myxococcus xanthus is a bacterium with multiple sets of chemotaxis genes and/or homologues. It was shown previously that difA and difE, encoding homologues of the methyl-accepting chemoreceptor protein (MCP) and the CheA kinase, respectively, are required for M. xanthus social gliding (S) motility and development. Both difA and difE mutants were also defective in the biogenesis of the cell surface appendages known as extracellular matrix fibrils. In this study, we investigated the roles of the CheW homologue encoded by difC, a gene at the same locus as difA and difE. We showed that difC mutations resulted in defects in M. xanthus developmental aggregation, sporulation, and S motility. We demonstrated that difC is indispensable for wild-type cellular cohesion and fibril biogenesis but not for pilus production. We further illustrated the ectopic complementation of a difC in-frame deletion by a wild-type difC. The identical phenotypes of difA, difC, and difE mutants are consistent and supportive of the hypothesis that the Dif chemotaxis homologues constitute a chemotaxis-like signal transduction pathway that regulates M. xanthus fibril biogenesis and S motility.     

Identification of methylation sites in Thermotoga maritima chemotaxis receptors

Adaptation in bacterial chemotaxis involves reversible methylation of specific glutamate residues within the cytoplasmic domains of methyl-accepting chemotaxis proteins. The specific sites of methylation in Salmonella enterica and Escherichia coli chemoreceptors, identified 2 decades ago, established a consensus sequence for methylation by methyltransferase CheR. Here we report the in vitro methylation of chemoreceptors from Thermotoga maritima, a hyperthermophile that has served as a useful source of chemotaxis proteins for structural analysis. Sites of methylation have been identified by liquid chromatography-mass spectrometry/mass spectrometry. Fifteen sites of methylation were identified within the cytoplasmic domains of four different T. maritima chemoreceptors. The results establish a consensus sequence for chemoreceptor methylation sites in T. maritima that is distinct from the previously identified consensus sequence for E. coli and S. enterica. These findings suggest that consensus sequences for posttranslational modifications in one organism may not be directly extrapolated to analogous modifications in other bacteria.     

NahY, a Catabolic Plasmid-Encoded Receptor Required for Chemotaxis of Pseudomonas putida to the Aromatic Hydrocarbon Naphthalene

Pseudomonas putida G7 exhibits chemotaxis to naphthalene, but the molecular basis for this was not known. A new gene, nahY, was found to be cotranscribed with meta cleavage pathway genes on the NAH7 catabolic plasmid for naphthalene degradation. The nahY gene encodes a 538-amino-acid protein with a membrane topology and a C-terminal region that resemble those of chemotaxis transducer proteins. A P. putida G7 nahY mutant grew on naphthalene but was not chemotactic to this aromatic hydrocarbon. The protein NahY thus appears to function as a chemoreceptor for naphthalene or a related compound. The presence of nahY on a catabolic plasmid implies that chemotaxis may facilitate biodegradation.