Cytoplasmic Chemoreceptor Research Articles

An aerotaxis receptor influences invasion of Agrobacterium tumefaciens into its host.

Agrobacterium tumefaciens is a soil-borne pathogenic bacterium that causes crown gall disease in many plants. Chemotaxis offers A. tumefaciens the ability to find its host and establish infection. Being an aerobic bacterium, A. tumefaciens possesses one chemotaxis system with multiple potential chemoreceptors. Chemoreceptors play an important role in perceiving and responding to environmental signals. However, the studies of chemoreceptors in A. tumefaciens remain relatively restricted. Here, we characterized a cytoplasmic chemoreceptor of A. tumefaciens C58 that contains an N-terminal globin domain. The chemoreceptor was designated as Atu1027. The deletion of Atu1027 not only eliminated the aerotactic response of A. tumefaciens to atmospheric air but also resulted in a weakened chemotactic response to multiple carbon sources. Subsequent site-directed mutagenesis and phenotypic analysis showed that the conserved residue His100 in Atu1027 is essential for the globin domain's function in both chemotaxis and aerotaxis. Furthermore, deleting Atu1027 impaired the biofilm formation and pathogenicity of A. tumefaciens. Collectively, our findings demonstrated that Atu1027 functions as an aerotaxis receptor that affects agrobacterial chemotaxis and the invasion of A. tumefaciens into its host.

A cytoplasmic chemoreceptor and reactive oxygen species mediate bacterial chemotaxis to copper

Chemotaxis is a widespread strategy used by unicellular and multicellular living organisms to maintain their fitness in stressful environments. We previously showed that bacteria can trigger a negative chemotactic response to a copper (Cu)-rich environment. Cu ion toxicity on bacterial cell physiology has been mainly linked to mismetallation events and reactive oxygen species (ROS) production, although the precise role of Cu-generated ROS remains largely debated. Here, using inductively coupled plasma optical emission spectrometry on cell fractionates, we found that the cytoplasmic Cu ion content mirrors variations of the extracellular Cu ion concentration. ROS-sensitive fluorescent probe and biosensor allowed us to show that the increase of cytoplasmic Cu ion content triggers a dose-dependent oxidative stress, which can be abrogated by superoxide dismutase and catalase overexpression. The inhibition of ROS production in the cytoplasm not only improves bacterial growth but also impedes Cu chemotaxis, indicating that ROS derived from cytoplasmic Cu ions mediate the control of bacterial chemotaxis to Cu. We also identified the Cu chemoreceptor McpR, which binds Cu ions with low affinity, suggesting a labile interaction. In addition, we demonstrate that the cysteine 75 and histidine 99 within the McpR sensor domain are key residues in Cu chemotaxis and Cu coordination. Finally, we discovered that invitro both Cu(I) and Cu(II) ions modulate McpR conformation in a distinct manner. Overall, our study provides mechanistic insights on a redox-based control of Cu chemotaxis, indicating that the cellular redox status can play a key role in bacterial chemotaxis.

The Only Chemoreceptor Encoded by che Operon Affects the Chemotactic Response of Agrobacterium to Various Chemoeffectors.

Chemoreceptor (also called methyl-accepting chemotaxis protein, MCP) is the leading signal protein in the chemotaxis signaling pathway. MCP senses and binds chemoeffectors, specifically, and transmits the sensed signal to downstream proteins of the chemotaxis signaling system. The genome of Agrobacterium fabrum (previously, tumefaciens) C58 predicts that a total of 20 genes can encode MCP, but only the MCP-encoding gene atu0514 is located inside the che operon. Hence, the identification of the exact function of atu0514-encoding chemoreceptor (here, named as MCP514) will be very important for us to understand more deeply the chemotaxis signal transduction mechanism of A. fabrum. The deletion of atu0514 significantly decreased the chemotactic migration of A. fabrum in a swim plate. The test of atu0514-deletion mutant (Δ514) chemotaxis toward single chemicals showed that the deficiency of MCP514 significantly weakened the chemotactic response of A. fabrum to four various chemicals, sucrose, valine, citric acid and acetosyringone (AS), but did not completely abolish the chemotactic response. MCP514 was localized at cell poles although it lacks a transmembrane (TM) region and is predicted to be a cytoplasmic chemoreceptor. The replacement of residue Phe328 showed that the helical structure in the hairpin subdomain of MCP514 is a direct determinant for the cellular localization of MCP514. Single respective replacements of key residues indicated that residues Asn336 and Val353 play a key role in maintaining the chemotactic function of MCP514.

Mechanism of Signalling and Adaptation through the Rhodobacter sphaeroides Cytoplasmic Chemoreceptor Cluster.

Rhodobacter sphaeroides has two chemotaxis clusters, an Escherichia coli-like cluster with membrane-spanning chemoreceptors and a less-understood cytoplasmic cluster. The cytoplasmic CheA is split into CheA4, a kinase, and CheA3, a His-domain phosphorylated by CheA4 and a phosphatase domain, which together phosphorylate and dephosphorylate motor-stopping CheY6. In bacterial two-hybrid analysis, one major cytoplasmic chemoreceptor, TlpT, interacted with CheA4, while the other, TlpC, interacted with CheA3. Both clusters have associated adaptation proteins. Deleting their methyltransferases and methylesterases singly and together removed chemotaxis, but with opposite effects. The cytoplasmic cluster signal overrode the membrane cluster signal. Methylation and demethylation of specific chemoreceptor glutamates controls adaptation. Tandem mass spectroscopy and bioinformatics identified four putative sites on TlpT, three glutamates and a glutamine. Mutating each glutamate to alanine resulted in smooth swimming and loss of chemotaxis, unlike similar mutations in E. coli chemoreceptors. Cells with two mutated glutamates were more stoppy than wild-type and responded and adapted to attractant addition, not removal. Mutating all four sites amplified the effect. Cells were non-motile, began smooth swimming on attractant addition, and rapidly adapted back to non-motile before attractant removal. We propose that TlpT responds and adapts to the cell’s metabolic state, generating the steady-state concentration of motor-stopping CheY6~P. Membrane-cluster signalling produces a pulse of CheY3/CheY4~P that displaces CheY6~P and allows flagellar rotation and smooth swimming before both clusters adapt.

Chemotaxis Allows Bacteria To Overcome Host-Generated Reactive Oxygen Species That Constrain Gland Colonization.

The epithelial layer of the gastrointestinal tract contains invaginations, called glands or crypts, which are colonized by symbiotic and pathogenic microorganisms and may function as designated niches for certain species. Factors that control gland colonization are poorly understood, but bacterial chemotaxis aids occupation of these sites. We report here that a Helicobacter pylori cytoplasmic chemoreceptor, TlpD, is required for gland colonization in the stomach. tlpD mutants demonstrate gland colonization defects characterized by a reduction in the percentage of glands colonized but not in the number of bacteria per gland. Consistent with TlpD's reported role in reactive oxygen species (ROS) avoidance, tlpD mutants showed hallmarks of exposure to high ROS. To assess the role of host-generated ROS in TlpD-dependent gland colonization, we utilized mice that lack either the ability to generate epithelial hydrogen peroxide or immune cell superoxide. tlpD gland colonization defects were rescued to wild-type H. pylori levels in both of these mutants. These results suggest that multiple types of innate immune-generated ROS production limit gland colonization and that bacteria have evolved specific mechanisms to sense and direct their motility in response to this signal and thus spread throughout tissue.

Essential Role of the Cytoplasmic Chemoreceptor TlpT in the De Novo Formation of Chemosensory Complexes in Rhodobacter sphaeroides.

Bacterial chemosensory proteins form large hexagonal arrays. Several key features of chemotactic signaling depend on these large arrays, namely, cooperativity between receptors, sensitivity, integration of different signals, and adaptation. The best-studied arrays are the membrane-associated arrays found in most bacteria. Rhodobacter sphaeroides has two spatially distinct chemosensory arrays, one is transmembrane and the other is cytoplasmic. These two arrays work together to control a single flagellum. Deletion of one of the soluble chemoreceptors, TlpT, results in the loss of the formation of the cytoplasmic array. Here, we show the expression of TlpT in a tlpT deletion background results in the reformation of the cytoplasmic array. The number of arrays formed is dependent on the cell length, indicating spatial limitations on the number of arrays in a cell and stochastic assembly. Deletion of PpfA, a protein required for the positioning and segregation of the cytoplasmic array, results in slower array formation upon TlpT expression and fewer arrays, suggesting it accelerates cluster assembly.IMPORTANCE Bacterial chemosensory arrays are usually membrane associated and consist of thousands of copies of receptors, adaptor proteins, kinases, and adaptation enzymes packed into large hexagonal structures. Rhodobacter sphaeroides also has cytoplasmic arrays, which divide and segregate using a chromosome-associated ATPase, PpfA. The expression of the soluble chemoreceptor TlpT is shown to drive the formation of the arrays, accelerated by PpfA. The positioning of these de novo arrays suggests their position is the result of stochastic assembly rather than active positioning.

Chemotaxis cluster 1 proteins form cytoplasmic arrays in Vibrio cholerae and are stabilized by a double signaling domain receptor DosM

Nearly all motile bacterial cells use a highly sensitive and adaptable sensory system to detect changes in nutrient concentrations in the environment and guide their movements toward attractants and away from repellents. The best-studied bacterial chemoreceptor arrays are membrane-bound. Many motile bacteria contain one or more additional, sometimes purely cytoplasmic, chemoreceptor systems. Vibrio cholerae contains three chemotaxis clusters (I, II, and III). Here, using electron cryotomography, we explore V. cholerae's cytoplasmic chemoreceptor array and establish that it is formed by proteins from cluster I. We further identify a chemoreceptor with an unusual domain architecture, DosM, which is essential for formation of the cytoplasmic arrays. DosM contains two signaling domains and spans the two-layered cytoplasmic arrays. Finally, we present evidence suggesting that this type of receptor is important for the structural stability of the cytoplasmic array.

The Helicobacter pylori CZB Cytoplasmic Chemoreceptor TlpD Forms an Autonomous Polar Chemotaxis Signaling Complex That Mediates a Tactic Response to Oxidative Stress.

Cytoplasmic chemoreceptors are widespread among prokaryotes but are far less understood than transmembrane chemoreceptors, despite being implicated in many processes. One such cytoplasmic chemoreceptor is Helicobacter pylori TlpD, which is required for stomach colonization and drives a chemotaxis response to cellular energy levels. Neither the signals sensed by TlpD nor its molecular mechanisms of action are known. We report here that TlpD functions independently of the other chemoreceptors. When TlpD is the sole chemoreceptor, it is able to localize to the pole and recruits CheW, CheA, and at least two CheV proteins to this location. It loses the normal membrane association that appears to be driven by interactions with other chemoreceptors and with CheW, CheV1, and CheA. These results suggest that TlpD can form an autonomous signaling unit. We further determined that TlpD mediates a repellent chemotaxis response to conditions that promote oxidative stress, including being in the presence of iron, hydrogen peroxide, paraquat, and metronidazole. Last, we found that all tested H. pylori strains express TlpD, whereas other chemoreceptors were present to various degrees. Our data suggest a model in which TlpD coordinates a signaling complex that responds to oxidative stress and may allow H. pylori to avoid areas of the stomach with high concentrations of reactive oxygen species. Helicobacter pylori senses its environment with proteins called chemoreceptors. Chemoreceptors integrate this sensory information to affect flagellum-based motility in a process called chemotaxis. Chemotaxis is employed during infection and presumably aids H. pylori in encountering and colonizing preferred niches. A cytoplasmic chemoreceptor named TlpD is particularly important in this process, and we report here that this chemoreceptor is able to operate independently of other chemoreceptors to organize a chemotaxis signaling complex and mediate a repellent response to oxidative stress conditions. H. pylori encounters and must cope with oxidative stress during infection due to oxygen and reactive oxygen species produced by host cells. TlpD's repellent response may allow the bacteria to escape niches experiencing inflammation and elevated reactive oxygen species (ROS) production.

Hypothetical Protein BB0569 Is Essential for Chemotaxis of the Lyme Disease Spirochete Borrelia burgdorferi.

The Lyme disease spirochete Borrelia burgdorferi has five putative methyl-accepting chemotaxis proteins (MCPs). In this report, we provide evidence that a hypothetical protein, BB0569, is essential for the chemotaxis of B. burgdorferi. While BB0569 lacks significant homology to the canonical MCPs, it contains a conserved domain (spanning residues 110 to 170) that is often evident in membrane-bound MCPs such as Tar and Tsr of Escherichia coli. Unlike Tar and Tsr, BB0569 lacks transmembrane regions and recognizable HAMP and methylation domains and is similar to TlpC, a cytoplasmic chemoreceptor of Rhodobacter sphaeroides. An isogenic mutant of BB0569 constantly runs in one direction and fails to respond to attractants, indicating that BB0569 is essential for chemotaxis. Immunofluorescence, green fluorescent protein (GFP) fusion, and cryo-electron tomography analyses demonstrate that BB0569 localizes at the cell poles and is required for chemoreceptor clustering at the cell poles. Protein cross-linking studies reveal that BB0569 forms large protein complexes with MCP3, indicative of its interactions with other MCPs. Interestingly, analysis of B. burgdorferi mcp mutants shows that inactivation of either mcp2 or mcp3 reduces the level of BB0569 substantially and that such a reduction is caused by protein turnover. Collectively, these results demonstrate that the domain composition and function of BB0569 are similar in some respects to those of TlpC but that these proteins are different in their cellular locations, further highlighting that the chemotaxis of B. burgdorferi is unique and different from the Escherichia coli and Salmonella enterica paradigm. Spirochete chemotaxis differs substantially from the Escherichia coli and Salmonella enterica paradigm, and the basis for controlling the rotation of the bundles of periplasmic flagella at each end of the cell is unknown. In recent years, Borrelia burgdorferi, the causative agent of Lyme disease, has been used as a model organism to understand spirochete chemotaxis and its role in infectious processes of the disease. In this report, BB0569, a hypothetical protein of B. burgdorferi, has been investigated by using an approach of genetic, biochemistry, and cryo-electron tomography analyses. The results indicate that BB0569 has a distinct role in chemotaxis that may be unique to spirochetes and represents a novel paradigm.

Structural conservation of chemotaxis machinery across Archaea and Bacteria.

Chemotaxis allows cells to sense and respond to their environment. In Bacteria, stimuli are detected by arrays of chemoreceptors that relay the signal to a two-component regulatory system. These arrays take the form of highly stereotyped super-lattices comprising hexagonally packed trimers-of-receptor-dimers networked by rings of histidine kinase and coupling proteins. This structure is conserved across chemotactic Bacteria, and between membrane-bound and cytoplasmic arrays, and gives rise to the highly cooperative, dynamic nature of the signalling system. The chemotaxis system, absent in eukaryotes, is also found in Archaea, where its structural details remain uncharacterized. Here we provide evidence that the chemotaxis machinery was not present in the last archaeal common ancestor, but rather was introduced in one of the waves of lateral gene transfer that occurred after the branching of Eukaryota but before the diversification of Euryarchaeota. Unlike in Bacteria, the chemotaxis system then evolved largely vertically in Archaea, with very few subsequent successful lateral gene transfer events. By electron cryotomography, we find that the structure of both membrane-bound and cytoplasmic chemoreceptor arrays is conserved between Bacteria and Archaea, suggesting the fundamental importance of this signalling architecture across diverse prokaryotic lifestyles.

Structure of bacterial cytoplasmic chemoreceptor arrays and implications for chemotactic signaling

Most motile bacteria sense and respond to their environment through a transmembrane chemoreceptor array whose structure and function have been well-studied, but many species also contain an additional cluster of chemoreceptors in their cytoplasm. Although the cytoplasmic cluster is essential for normal chemotaxis in some organisms, its structure and function remain unknown. Here we use electron cryotomography to image the cytoplasmic chemoreceptor cluster in Rhodobacter sphaeroides and Vibrio cholerae. We show that just like transmembrane arrays, cytoplasmic clusters contain trimers-of-receptor-dimers organized in 12-nm hexagonal arrays. In contrast to transmembrane arrays, however, cytoplasmic clusters comprise two CheA/CheW baseplates sandwiching two opposed receptor arrays. We further show that cytoplasmic fragments of normally transmembrane E. coli chemoreceptors form similar sandwiched structures in the presence of molecular crowding agents. Together these results suggest that the 12-nm hexagonal architecture is fundamentally important and that sandwiching and crowding can replace the stabilizing effect of the membrane. DOI: http://dx.doi.org/10.7554/eLife.02151.001.

New Insights into Bacterial Chemoreceptor Array Structure and Assembly from Electron Cryotomography

Bacterial chemoreceptors cluster in highly ordered, cooperative, extended arrays with a conserved architecture, but the principles that govern array assembly remain unclear. Here we show images of cellular arrays as well as selected chemoreceptor complexes reconstituted in vitro that reveal new principles of array structure and assembly. First, in every case, receptors clustered in a trimers-of-dimers configuration, suggesting this is a highly favored fundamental building block. Second, these trimers-of-receptor dimers exhibited great versatility in the kinds of contacts they formed with each other and with other components of the signaling pathway, although only one architectural type occurred in native arrays. Third, the membrane, while it likely accelerates the formation of arrays, was neither necessary nor sufficient for lattice formation. Molecular crowding substituted for the stabilizing effect of the membrane and allowed cytoplasmic receptor fragments to form sandwiched lattices that strongly resemble the cytoplasmic chemoreceptor arrays found in some bacterial species. Finally, the effective determinant of array structure seemed to be CheA and CheW, which formed a “superlattice” of alternating CheA-filled and CheA-empty rings that linked receptor trimers-of-dimer units into their native hexagonal lattice. While concomitant overexpression of receptors, CheA, and CheW yielded arrays with native spacing, the CheA occupancy was lower and less ordered, suggesting that temporal and spatial coordination of gene expression driven by a single transcription factor may be vital for full order, or that array overgrowth may trigger a disassembly process. The results described here provide new insights into the assembly intermediates and assembly mechanism of this massive macromolecular complex.

ParA-like protein uses nonspecific chromosomal DNA binding to partition protein complexes

Recent data have shown that plasmid partitioning Par-like systems are used by some bacterial cells to control localization of protein complexes. Here we demonstrate that one of these homologs, PpfA, uses nonspecific chromosome binding to separate cytoplasmic clusters of chemotaxis proteins upon division. Using fluorescent microscopy and point mutations, we show dynamic chromosome binding and Walker-type ATPase activity are essential for cluster segregation. The N-terminal domain of a cytoplasmic chemoreceptor encoded next to ppfA is also required for segregation, probably functioning as a ParB analog to control PpfA ATPase activity. An orphan ParA involved in segregating protein clusters therefore uses a similar mechanism to plasmid-segregating ParA/B systems and requires a partner protein for function. Given the large number of genomes that encode orphan ParAs, this may be a common mechanism regulating segregation of proteins and protein complexes.

Localization of a bacterial cytoplasmic receptor is dynamic and changes with cell-cell contacts

Directional motility in the gliding bacterium Myxococcus xanthus requires controlled cell reversals mediated by the Frz chemosensory system. FrzCD, a cytoplasmic chemoreceptor, does not form membrane-bound polar clusters typical for most bacteria, but rather cytoplasmic clusters that appear helically arranged and span the cell length. The distribution of FrzCD in living cells was found to be dynamic: FrzCD was localized in clusters that continuously changed their size, number, and position. The number of FrzCD clusters was correlated with cellular reversal frequency: fewer clusters were observed in hypo-reversing mutants and additional clusters were observed in hyper-reversing mutants. When moving cells made side-to-side contacts, FrzCD clusters in adjacent cells showed transient alignments. These events were frequently followed by one of the interacting cells reversing. These observations suggest that FrzCD detects signals from a cell contact-sensitive signaling system and then re-localizes as it directs reversals to distributed motility engines.

Chemotaxis mediated by NarX–FrzCD chimeras and nonadapting repellent responses in Myxococcus xanthus

Myxococcus xanthus requires gliding motility for swarming and fruiting body formation. It uses the Frz chemosensory pathway to regulate cell reversals. FrzCD is a cytoplasmic chemoreceptor required for sensing effectors for this pathway. NarX is a transmembrane sensor for nitrate from Escherichia coli. In this study, two NarX-FrzCD chimeras were constructed to investigate M. xanthus chemotaxis: NazD(F) contains the N-terminal sensory module of NarX fused to the C-terminal signalling domain of FrzCD; NazD(R) is similar except that it contains a G51R mutation in the NarX domain known to reverse the signalling output of a NarX-Tar chimera to nitrate. We report that while nitrate had no effect on the wild type, it decreased the reversal frequency of M. xanthus expressing NazD(F) and increased that of M. xanthus expressing NazD(R). These results show that directional motility in M. xanthus can be regulated independently of cellular metabolism and physiology. Surprisingly, the NazD(R) strain failed to adapt to nitrate in temporal assays as did the wild type to known repellents. The lack of temporal adaptation to negative stimuli appears to be a general feature in M. xanthus chemotaxis. Thus, the appearance of biased movements by M. xanthus in repellent gradients is likely due to the inhibition of net translocation by repellents.

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.

TlpC, a novel chemotaxis protein in Rhodobacter sphaeroides, localizes to a discrete region in the cytoplasm.

TlpC is encoded in the second chemotaxis operon of Rhodobacter sphaeroides. This protein shows some homology to membrane-spanning chemoreceptors of many bacterial species but, unlike these, is essential for R. sphaeroides chemotaxis to all compounds tested. Genomic replacement of tlpC with a C-terminal gfp fusion demonstrated that TlpC localized to a discrete cluster within the cytoplasm. Immunogold electron microscopy also showed that TlpC localized to a cytoplasmic electron-dense region. Correct TlpC-GFP localization depended on the downstream signalling proteins, CheW3, CheW4 and CheA2, and was tightly linked to cell division. Newly divided cells contained a single cluster but, as the cell cycle progressed, a second cluster appeared close to the initial cluster. As elongation continued, these clusters moved apart so that, on septation, each daughter cell contained a single TlpC cluster. The data presented suggest that TlpC is either a cytoplasmic chemoreceptor responding to or integrating global signals of metabolic state or a novel and essential component of the chemotaxis signalling pathway. These data also suggest that clustering is essential for signalling and that a mechanism may exist for targeting and localizing proteins within the bacterial cytoplasm.

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.

The third chemotaxis locus of Rhodobacter sphaeroides is essential for chemotaxis.

The purple photosynthetic bacterium Rhodobacter sphaeroides has three loci encoding multiple homologues of the bacterial chemosensory proteins: 13 putative chemoreceptors, four CheW, four CheA, six CheY, two CheB and three CheR. Previously, studies have shown that, although deletion of cheOp1 led to only minor changes in behaviour, deletion of cheOp2 led to a loss of taxis. The third locus encodes two CheA, one CheR, one CheB, one CheW, one CheY, a putative cytoplasmic chemoreceptor (TlpT) and a protein showing homology to the chromosomal partitioning factor Soj (designated Slp). Here, we show that every protein encoded by this locus is essential for normal chemotaxis. Phototaxis is also dependent upon all the components of this locus, except CheB2 and Slp. The two putative CheA proteins encoded in this locus are unusual. CheA3 has only the P1 domain and the P5 regulatory domain linked by a large internal domain, whereas CheA4 lacks the P1 and P2 domains required for phosphorylation and response regulator binding. These data indicate that the minimal set of proteins required for normal chemotaxis in R. sphaeroides is all the proteins encoded by cheOp2 and the third chemotaxis locus, and that the multiple chemosensory protein homologues found in R. sphaeroides are not redundant.