Site-specific receptor methylation of FrzCD in Myxococcus xanthus is controlled by a tetra-trico peptide repeat (TPR) containing regulatory domain of the FrzF methyltransferase

Myxococcus xanthus is a gliding bacterium with a complex life cycle that includes swarming, predation and fruiting body formation. Directed movements in M. xanthus are regulated by the Frz chemosensory system, which controls cell reversals. The Frz pathway requires the activity of FrzCD, a cytoplasmic methyl-accepting chemotaxis protein, and FrzF, a methyltransferase (CheR) containing an additional domain with three tetra trico-peptide repeats (TPRs). To investigate the role of the TPRs in FrzCD methylation, we used full-length FrzF and FrzF lacking its TPRs (FrzF^CheR) to methylate FrzCD in vitro . FrzF methylated FrzCD on a single residue, E182, while FrzF^CheR methylated FrzCD on three residues, E168, E175 and E182, indicating that the TPRs regulate site-specific methylation. E168 and E182 were predicted consensus methylation sites, but E175 is methylated on an HE pair. To determine the roles of these sites in vivo , we substituted each methylatable glutamate with either an aspartate or an alanine residue and determined the impact of the point mutants on single cell reversals, swarming and fruiting body formation. Single, double and triple methylation site mutants revealed that each site played a unique role in M. xanthus behaviour and that the pattern of receptor methylation determined receptor activity. This work also shows that methylation can both activate and inactivate the receptor.     

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.     

The Dif chemosensory pathway is directly involved in phosphatidylethanolamine sensory transduction in Myxococcus xanthus

Myxococcus xanthus cells glide on solid surfaces and are chemotactically stimulated by certain phosphatidylethanolamine species. The dif gene cluster consists of six genes, difABCDEG, five of which encode proteins homologous to known chemotaxis proteins. DifA and DifE are required for the biosynthesis of fibrils, an extracellular matrix comprised of polysaccharide and protein. Chemotactic stimulation by 1,2-O-Bis[11-(Z)-hexadecenoyl]-sn-glycero-3-phosphatidylethanolamine (16:1 PE) and dilauroyl PE (12:0 PE) requires fibrils. Although previous work has shown that difA and difE mutants are not stimulated by 12:0 PE, these results do not distinguish between a dependence on fibrils or a direct role in chemosensory transduction. Here we provide evidence that the Dif chemosensory pathway directly mediates PE sensory transduction. First, stimulation by and adaptation to 16:1 PE requires all of the dif genes, including difBDG, which are not essential for fibril biogenesis. Second, a specific residue within the first putative methylation domain of DifA is required for stimulation by 16:1 PE but not fibril biogenesis. Transmembrane signalling through a chimeric NarX-DifA chemoreceptor is required for fibril formation but not for stimulation by or adaptation to 16:1 PE. Third, difD and difE are required for stimulation by dioleoyl PE (18:1 PE) although the response does not require fibrils. Taken together these results argue that the Dif pathway mediates both matrix formation and lipid chemotaxis.     

Phospholipid directed motility of surface-motile bacteria

Myxococcus xanthus is a surface-motile bacterium that has adapted at least one chemosensory system to allow directed movement towards the slowly diffusible lipid phosphatidylethanolamine (PE). The Dif chemosensory pathway is remarkable because it has at least three inputs coupled to outputs that control extracellular matrix (ECM) production and lipid chemotaxis. The methyl-accepting chemotaxis protein, DifA, has two different sensor inputs that have been localized by mutagenesis. The Dif chemosensory pathway employs a novel protein that slows adaptation. Lipid chemotaxis may play important roles in the M. xanthus life cycle where prey-specific and development-specific attractants have been identified. Lipid chemotaxis may also be an important mechanism for locating nutrients by lung pathogens such as Pseudomonas aeruginosa.     

Methylation of FrzCD, a methyl-accepting taxis protein of Myxococcus xanthus, is correlated with factors affecting cell behavior.

Myxococcus xanthus, a nonflagellated gliding bacterium, exhibits multicellular behavior during vegetative growth and fruiting body formation. The frizzy (frz) genes are required to control directed motility for these interactions. The frz genes encode proteins that are homologous to all of the major enteric chemotaxis proteins, with the exception of CheZ. In this study, we characterized FrzCD, a protein which is homologous to the methyl-accepting chemotaxis proteins from the enteric bacteria. FrzCD, unlike the other methyl-accepting chemotaxis proteins, was found to be localized primarily in the cytoplasmic fraction of cells. FrzCD migrates as a ladder of bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, reflecting heterogeneity due to methylation or demethylation and to deamidation. FrzCD was shown to be methylated in vivo when cells were exposed to yeast extract or Casitone and demethylated when starved in buffer. We used the methylation state of FrzCD as revealed by Western blot (immunoblot) analyses to search for stimuli that are recognized by the frz signal transduction system. Common amino acids, nucleotides, vitamins, and sugars were not recognized, but certain lipids and alcohols were recognized. For example, the saturated fatty acids capric acid and lauric acid stimulated FrzCD methylation, whereas a variety of other saturated fatty acids did not. Lauryl alcohol and lipoic acid also stimulated methylation, as did phospholipids containing lauric acid. In contrast, several short-chain alcohols, such as isoamyl alcohol, and some other solvents caused demethylation. The relatively high concentrations of the chemicals required for a response may indicate that these chemicals are not the relevant signals recognized by M. xanthus in nature. Isoamyl alcohol and isopropanol also had profound effects on the behavior of wild-type cells, causing them to reverse continuously. Cells of frzB, frzF, and frzG mutants also reversed continuously in the presence of isoamyl alcohol, whereas cells of frzA, frzCD, or frzE mutants did not. On the basis of the data presented, we propose a model for the frz signal transduction pathway in M. xanthus.     

Sensory adaptation during negative chemotaxis in Myxococcus xanthus.

Myxococcus xanthus exhibits many tactic movements that require the frz signal transduction system, such as colony swarming and cellular aggregation during fruiting body formation. Previously we demonstrated that the Frz proteins control the chemotactic movements of M. xanthus (W. Shi, T. Köhler, and D. R. Zusman, Mol. Microbiol. 9:601-611, 1993). However it was unclear from that study how chemotaxis might be achieved at the cellular level. In this study, we showed that M. xanthus cells not only modulate the reversal frequency of cell movement in response to repellent stimuli but also exhibit sensory adaptation in response to the continuous presence of nonsaturating repellent stimuli. The sensory adaptation behavior requires FrzF (a putative methyltransferase) and is correlated with the methylation-demethylation of FrzCD, a methyl-accepting chemotaxis protein. These results indicate that negative chemotaxis in M. xanthus is achieved by chemokinesis plus sensory adaptation in a manner analogous to that of the free-swimming enteric bacteria.     

Isolation and phenotypic characterization of Myxococcus xanthus mutants which are defective in sensing negative stimuli.

Myxococcus xanthus is a gram-negative gliding bacterium that exhibits a complex life cycle. Exposure of M. xanthus to chemicals like dimethyl sulfoxide (DMSO) at nondeleterious concentrations or the depletion of nutrients caused several negative responses by the cells. DMSO (> 0.1 M) or nutrient depletion triggered a repellent response: cell swarming was inhibited and FrzCD (a methyl-accepting chemotaxis protein) was demethylated; higher concentrations of DMSO (> 0.3 M) or prolonged starvation induced an additional response which involved cellular morphogenesis: DMSO caused cells to convert from rod-shaped vegetative cells to spherical, environmentally resistant "DMSO spores," and starvation induced myxospore formation in the fruiting bodies. In order to investigate the nature of these responses, we isolated a number of mutants defective in negative chemotaxis and/or sporulation. Characterization of these mutants indicated that negative chemotaxis plays an important role in colony swarming and in developmental aggregation. In addition, the results revealed some of the major interrelationships between the signal transduction pathways which respond to negative stimuli: (i) DMSO exposure and starvation were initially sensed by different systems, the neg system for DMSO and the stv system for starvation; (ii) the repellent response signals triggered by DMSO or starvation were then relayed by the frz signal transduction system; mutants defective in these responses showed altered FrzCD methylation patterns; and (iii) the morphogenesis signals in response to DMSO or starvation utilize a group of genes involved in sporulation (spo).     

Differential effects of chemoreceptor methylation-domain mutations on swarming and development in the social bacterium Myxococcus xanthus

The soil bacterium Myxococcus xanthus is a model organism for the study of multicellular behaviour and development in bacteria. M. xanthus cells move on solid surfaces by gliding motility, periodically reversing their direction of movement. Motility is co-ordinated to allow cells to effectively feed on macromolecules or prey bacteria when nutrients are plentiful and to form developmental fruiting bodies when nutrients are limiting. The Frz signal transduction pathway regulates cellular movements by modulating cell reversal frequency. Input to the Frz pathway is controlled by the cytoplasmic receptor, FrzCD, a methyl-accepting chemotaxis protein (MCP). FrzCD lacks the transmembrane and periplasmic domains common to MCPs but contains a unique N-terminal domain, the predicted ligand-binding domain. As deletion of the N-terminal domain of FrzCD only results in minor defects in motility, we investigated the possibility that the methylation of the conserved C-terminal domain of FrzCD plays a central role in regulating the pathway. For this study, each of the potential methylation sites of FrzCD were systematically modified by site-directed mutagenesis, substituting glutamine/glutamate pairs for alanines. Four of the seven mutations produced dramatic phenotypes; two of the mutations had a stimulatory effect on the pathway, as evidenced by cells hyper-reversing, whereas another two had an inhibitory effect, causing these cells to rarely reverse. These four mutants displayed defects in vegetative swarming and developmental aggregation. These results suggests a model in which the methylation domain can both activate and inhibit the Frz pathway depending on which residues are methylated. The diversity of phenotypes suggests that specific modifications of FrzCD act to differentially regulate motility and developmental aggregation in M. xanthus.     

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.     

FrzCD, a methyl-accepting taxis protein from Myxococcus xanthus, shows modulated methylation during fruiting body formation.

The frizzy (frz) genes of Myxococcus xanthus are required to control directed motility during vegetative growth and fruiting body formation. FrzCD, a protein homologous to the methyl-accepting chemotaxis proteins from enteric bacteria, is modified by methylation in response to environmental conditions. Transfer of cells from rich medium to fruiting medium initially caused rapid demethylation of FrzCD. Subsequently, the amount of FrzCD increased, but most remained unmethylated. At about the time of mound formation (9 h), most of the FrzCD was converted to methylated forms. Dispersal of developing cells (10 h) in buffer led to the demethylation of FrzCD, whereas concentration of these cells caused methylation of FrzCD. Some mutants which were unable to form fruiting bodies still modified their FrzCD during incubation under conditions of starvation on a surface.     

Developmental sensory transduction in Myxococcus xanthus involves methylation and demethylation of FrzCD.

Myxococcus xanthus is a bacterium that moves by gliding motility and exhibits multicellular development (fruiting body formation). The frizzy (frz) mutants aggregate aberrantly and therefore fail to form fruiting bodies. Individual frz cells cannot control the frequency at which they reverse direction while gliding. Previously, FrzCD was shown to exhibit significant sequence similarity to the enteric methyl-accepting chemotaxis proteins. In this report, we show that FrzCD is modified by methylation and that frzF encodes the methyltransferase. We also identify a new gene, frzG, whose predicted product is homologous to that of the cheB (methylesterase) gene from Escherichia coli. Thus, although M. xanthus is unflagellated, it appears to have a sensory transduction system which is similar in many of its components to those found in flagellated bacteria.     

Lipid chemotaxis and signal transduction in Myxococcus xanthus

The lipid phosphatidylethanolamine (PE) is the first chemoattractant to be described for a surface-motile bacterium. In Myxococcus xanthus, the specific activity of PE is determined by its fatty acid components. Two active species have been identified: dilauroyl PE and dioleoyl PE. Excitation to dilauroyl PE requires fibril appendages and the presence of two cytoplasmic chemotaxis systems, of which one (Dif) appears to mediate excitation and the other (Frz) appears to mediate adaptation. A possible mechanism for fibril-mediated signal transduction is discussed, along with the potential roles for PE chemotaxis in the context of the M. xanthus life cycle.     

DifA, a methyl-accepting chemoreceptor protein-like sensory protein, uses a novel signaling mechanism to regulate exopolysaccharide production in Myxococcus xanthus

DifA is a methyl-accepting chemotaxis protein (MCP)-like sensory transducer that regulates exopolysaccharide (EPS) production in Myxococcus xanthus. Here mutational analysis and molecular biology were used to probe the signaling mechanisms of DifA in EPS regulation. We first identified the start codon of DifA experimentally; this identification extended the N terminus of DifA for 45 amino acids (aa) from the previous bioinformatics prediction. This extension helped to address the outstanding question of how DifA receives input signals from type 4 pili without a prominent periplasmic domain. The results suggest that DifA uses its N-terminus extension to sense an upstream signal in EPS regulation. We suggest that the perception of the input signal by DifA is mediated by protein-protein interactions with upstream components. Subsequent signal transmission likely involves transmembrane signaling instead of direct intramolecular interactions between the input and the output modules in the cytoplasm. The basic functional unit of DifA for signal transduction is likely dimeric as mutational alteration of the predicted dimeric interface of DifA significantly affected EPS production. Deletions of 14-aa segments in the C terminus suggest that the newly defined flexible bundle subdomain in MCPs is likely critical for DifA function because shortening of this bundle can lead to constitutively active mutations.     

Nitrate-dependent activation of the Dif signaling pathway of Myxococcus xanthus mediated by a NarX-DifA interspecies chimera

Myxococcus xanthus fibril exopolysaccharide (EPS), essential for the social gliding motility and development of this bacterium, is regulated by the Dif chemotaxis-like pathway. DifA, an MCP homolog, is proposed to mediate signal input to the Dif pathway. However, DifA lacks a prominent periplasmic domain, which in classical chemoreceptors is responsible for signal perception and for initiating transmembrane signaling. To investigate the signaling properties of DifA, we constructed a NarX-DifA (NafA) chimera from the sensory module of Escherichia coli NarX and the signaling module of M. xanthus DifA. We report here the first functional chimeric signal transducer constructed using genes from organisms in two different phylogenetic subdivisions. When expressed in M. xanthus, NafA restored fruiting body formation, EPS production, and S-motility to difA mutants in the presence of nitrate. Studies with various double mutants indicate that NafA requires the downstream Dif proteins to function. We propose that signal inputs to the Dif pathway and transmembrane signaling by DifA are essential for the regulation of EPS production in M. xanthus. Despite the apparent structural differences, DifA appears to share similar transmembrane signaling mechanisms with enteric sensor kinases and chemoreceptors.     

Demonstration of interactions among <Emphasis Type="Italic">Myxococcus xanthus</Emphasis> Dif chemotaxis-like proteins by the yeast two-hybrid system

The Myxococcus xanthus dif locus encodes several bacterial chemotaxis homologues that are crucial for fibril exopolysaccharide (EPS) production, social gliding motility, and fruiting body development. In primary sequence, DifA is homologous to methyl-accepting chemotaxis protein, DifC to CheW, DifD to CheY, DifE to CheA, and DifG to CheC. In this study, the interactions among the Dif chemotaxis-like proteins were investigated using the yeast two-hybrid (Y2H) system. DifC was found to interact with both DifA and DifE. Using a modified Y2H or a three-hybrid system, it was demonstrated that DifC is capable of mediating the formation of DifA, DifC, and DifE ternary protein complexes. The conserved domains of DifE, based on sequence analysis, likely reflect functional conservations of CheA-type kinases, because its P2 domain interacts with DifD, P5 with DifC, and the P3 domain appears to dimerize. Similarly, C-terminal regions of DifA appear to dimerize as well. In addition, DifG was found to interact with DifD, which is consistent with the hypothesis that DifG is a phosphatase of DifD-phosphate. These findings support the models in which Dif proteins constitute a unique chemotaxis-like signal transduction pathway with central functions in regulating EPS production in M. xanthus.     

Sensory transduction in the gliding bacterium Myxococcus xanthus

Sensory transduction in the gliding bacterium Myxococcus xanthus is mediated by the frz genes. These genes are homologous to the chemotaxis genes of enteric bacteria and control the rate of cell reversal during gliding. Sensory transduction is hypothesized to involve the recognition of substances present in the medium at the cell surface and the subsequent stimulation of a cytoplasmic methyl-accepting protein, FrzCD. Phosphorylation of FrzE is also involved in the sensory transduction pathway. Despite the similarities between the chemotaxis proteins of enteric bacteria and M. xanthus Frz proteins, fundamental differences exist between these different bacteria in terms of the ability of cells to recognize and respond to substances in their environment. The mechanism of directional switching and the nature of the gliding motor remain obscure. It is hoped that the study of the interaction of the Frz proteins will allow greater understanding of these problems.     

Chemotaxis plays a role in the social behaviour of Myxococcus xanthus

Myxococcus xanthus is a Gram-negative bacterium that glides on a solid surface and displays a wide range of social behaviour including microbial development. The frz genes are homologues to the chemotaxis genes of Escherichia coli and Salmonella typhimurium and have been shown to be involved in microbial development. However, chemotaxis has never been clearly demonstrated in Myxococcus. In this study, we showed that M. xanthus exhibited tactic movements to many chemicals when they were subjected to steep and stable chemical gradients. M. xanthus was observed to spread into areas with abundant nutrients like yeast extract or Casitone and avoid areas with no nutrients or repellents (short-chain alcohols or DMSO. Responses to attractants and repellents were additive. Movement towards attractants or away from repellents required the frz genes and was correlated with methylation or demethylation of FrzCD, a methyl-accepting taxis protein. Furthermore, the frz genes were found to be required for both fruiting body formation during starvation and swarming in nutrient-rich medium. In wild-type strains, cells near the colony edge were observed to swarm towards the surrounding growth medium and to contain highly methylated FrzCD; cells near the colony centre contained mainly demethylated FrzCD and showed directed movement towards the colony edge. FrzCD was also found to be methylated during the aggregation stage of fruiting body formation on agar but largely demethylated in cells shaken in liquid starvation media. An frzf mutant failed to exhibit directed cell movements and no longer showed modification of FrzCD under these conditions. These observations suggest that M. xanthus does show chemotactic movements, that these movements require the frz genes, and that chemotaxis plays a very important role in the social behaviour of this organism.     

Methylation of FrzCD Defines a Discrete Step in the Developmental Program of Myxococcus xanthus

Myxococcus xanthus is a gram-negative soil bacterium which undergoes fruiting body formation during starvation. The frz signal transduction system has been found to play an important role in this process. FrzCD, a methyl-accepting taxis protein homologue, shows modulated methylation during cellular aggregation, which is thought to be part of an adaptation response to an aggregation signal. In this study, we assayed FrzCD methylation in many known and newly isolated mutants defective in fruiting body formation to determine a possible relationship between the methylation response and fruiting morphology. The results of our analysis indicated that the developmental mutants could be divided into two groups based on their ability to show normal FrzCD methylation during development. Many mutants blocked early in development, i.e., nonaggregating or abnormally aggregating mutants, showed poor FrzCD methylation. The well-characterized asg, bsg, csg, and esg mutants were found to be of this type. The defects in FrzCD methylation of these signaling mutants could be partially rescued by extracellular complementation with wild-type cells or addition of chemicals which restore their fruiting body formation. Mutants blocked in late development, i.e., translucent mounds, showed normal FrzCD methylation. Surprisingly, some mutants blocked in early development also exhibited a normal level of FrzCD methylation. The characterized mutants in this group were found to be defective in social motility. This indicates that FrzCD methylation defines a discrete step in the development of M. xanthus and that social motility mutants are not blocked in these early developmental steps.     

Myxococcus xanthus dif genes are required for biogenesis of cell surface fibrils essential for social gliding motility

Myxococcus xanthus social (S) gliding motility has been previously reported by us to require the chemotaxis homologues encoded by the dif genes. In addition, two cell surface structures, type IV pili and extracellular matrix fibrils, are also critical to M. xanthus S motility. We have demonstrated here that M. xanthus dif genes are required for the biogenesis of fibrils but not for that of type IV pili. Furthermore, the developmental defects of dif mutants can be partially rescued by the addition of isolated fibril materials. Along with the chemotaxis genes of various swarming bacteria and the pilGHIJ genes of the twitching bacterium Pseudomonas aeruginosa, the M. xanthus dif genes belong to a unique class of bacterial chemotaxis genes or homologues implicated in the biogenesis of structures required for bacterial surface locomotion. Genetic studies indicate that the dif genes are linked to the M. xanthus dsp region, a locus known to be crucial for M. xanthus fibril biogenesis and S gliding.