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PubMed2005

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.

Chemotaxis↗
PubMed2006

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.

Bacterial Proteins↗
PubMed2006

Polar assembly of the type IV pilus secretin in Myxococcus xanthus.

The type IV pilus filament of Myxococcus xanthus penetrates the outer membrane through a gated channel--the PilQ secretin. Assembly of the channel and formation of PilQ multimeric complexes that resist disassembly in heated detergent is correlated with the release of a 50 kDa fragment of PilQ. Tgl lipoprotein is required for PilQ assembly in M. xanthus, because PilQ monomers but no heat and detergent-resistant complexes are present in a strain from which tgl has been deleted. PilQ protein is often found in single patches at both poles of the cell. Tgl, however, is found in a patch at only one pole that most likely identifies the piliated cell pole. Tgl protein that has been transferred from another cell by contact stimulation leads to secretin assembly in the recipient. Pilus proteins PilQ, PilG, PilM, PilN, PilO and PilP are also required for the donation of Tgl by contact stimulation to a stimulation recipient. We suggest that these proteins are parts of a polar superstructure that holds PilQ monomers in a cluster and ready for Tgl to bring about secretin assembly.

Bacterial Proteins↗
PubMed2006

Four signalling domains in the hybrid histidine protein kinase RodK of Myxococcus xanthus are required for activity.

In prokaryotes, the principal signal transduction systems operating at the level of protein phosphorylation are the two-component systems. A number of hybrid histidine protein kinases in these systems contain several receiver domains, however, the function of these receiver domains is unknown. The RodK kinase in Myxococcus xanthus has an unconventional domain composition with a putative N-terminal sensor domain followed by a histidine kinase domain and three receiver domains. RodK is essential for the spatial coupling of the two morphogenetic events underlying fruiting body formation in M. xanthus, aggregation of cells into nascent fruiting bodies and the subsequent sporulation of these cells. RodK kinase activity is indispensable for RodK activity. By systematically substituting the conserved, phosphorylatable aspartate residues in the three receiver domains, genetic evidence is provided that each receiver domain is important for RodK function and that each receiver domain has a distinct function, which depends on phosphorylation. Biochemical analyses provided indirect evidence for phosphotransfer from the RodK kinase domain to the third receiver domain. This is the first example of a hybrid histidine protein kinase in which four signalling domains have been shown to be required for full activity.

Histidine Kinase↗
PubMed2006

A protein Ser/Thr kinase cascade negatively regulates the DNA-binding activity of MrpC, a smaller form of which may be necessary for the Myxococcus xanthus development.

The developmental process of Myxococcus xanthus is achieved by the expression of a specific set of genes under the influence of developmental signals. MrpC is a member of the CRP family of transcription regulators, essential for fruA expression during development. The Pkn8-Pkn14 protein kinase cascade negatively regulates mrpC expression (H. Nariya and S. Inouye, 2005. Mol Microbiol 58: 367-379). Elevated levels of mrpC in pkn8 and pkn14 deletion strains (Deltapkn8 and Deltapkn14) induce untimely FruA production during vegetative growth resulting in significantly faster fruiting body development. mrpC expression is presumably activated by MrpA and MrpB which belong to a two-component His-Asp phosphorelay system and is proposed to require MrpC on the basis of the genetic analysis. In the present study, we demonstrate that MrpC binds to at least eight sites in the upstream region of its promoter. Based on analysis of MrpC binding sites in the mrpC and fruA promoter regions, there are two types of MrpC-specific binding sequences. Importantly, MrpC-binding activity was greatly reduced upon its phosphorylation by Pkn14. MrpC2, a transcription activator for fruA expression, lacks the N-terminal 25 residues of MrpC and exhibited four- and eightfold greater binding activity to the mrpC and fruA promoter regions respectively. Pkn14 was not able to phosphorylate MrpC2 and phosphorylates MrpC at Thr residue(s), thus Thr-21 and/or Thr-22 is (are) the likely site(s) of MrpC phosphorylation. MrpC2 was not detected in a lonD mutant in which fruA expression is low. Thus, the LonD protease essential for development may play an important role for the activation of MrpC-binding activity through its proteolytic processing to MrpC2, required for developmental progression. MrpC2, only detectable during development in DZF1, was present at high levels during vegetative growth in Deltapkn8 and Deltapkn14, thus MrpC phosphorylation may inhibit its proteolytic processing. Based on these results, we propose a mechanism by which two transcription factors essential to development, MrpC and FruA, are regulated during the M. xanthus life cycle.

Amino Acid Sequence↗
PubMed2006

Two Ser/Thr protein kinases essential for efficient aggregation and spore morphogenesis in Myxococcus xanthus.

Myxococcus xanthus has a complex life cycle that involves vegetative growth and development. Previously, we described the espAB locus that is involved in timing events during the initial stages of fruiting body formation. Deletion of espA caused early aggregation and sporulation, whereas deletion of espB caused delayed aggregation and sporulation resulting in reduced spore yields. In this study, we describe two genes, pktA5 and pktB8, that flank the espAB locus and encode Ser/Thr protein kinase (STPK) homologues. Cells deficient in pktA5 or pktB8 formed translucent mounds and produced low spore yields, similar in many respects to espB mutants. Double mutant analysis revealed that espA was epistatic to pktA5 and pktB8 with respect to aggregation and fruiting body morphology, but that pktA5 and pktB8 were epistatic to espA with respect to sporulation efficiency. Expression profiles of pktA5-lacZ and pktB8-lacZ fusions and Western blot analysis showed that the STPKs are expressed under vegetative and developmental conditions. In vitro kinase assays demonstrated that the RD kinase, PktA5, autophosphorylated on threonine residue(s) and phosphorylated the artificial substrate, myelin basic protein. In contrast, autophosphorylation of the non-RD kinase, PktB8, was not observed in vitro; however, the phenotype of a pktB8 kinase-dead point mutant resembled the pktB8 deletion mutant, indicating that this residue was important for function and that it likely functions as a kinase in vivo. Immunoprecipitation of Tap-tagged PktA5 and PktB8 revealed an interaction with EspA during development in M. xanthus. These results, taken together, suggest that PktA5 and PktB8 are STPKs that function during development by interacting with EspA and EspB to regulate M. xanthus development.

Amino Acid Sequence↗
PubMed2006

Type IV pili function upstream of the Dif chemotaxis pathway in Myxococcus xanthus EPS regulation.

The developmental bacterium Myxococcus xanthus utilizes gliding motility to aggregate during the formation of multicellular fruiting bodies. The social (S) component of M. xanthus gliding motility requires at least two extracellular surface structures, type IV pili (Tfp) and the fibril polysaccharide or exopolysaccharide (EPS). Retraction of Tfp is proposed to power S motility and EPS from neighbouring cells is suggested to provide an anchor and trigger for Tfp retraction. The production of EPS in M. xanthus is regulated in part by the Dif chemosensory pathway; however, the input signal for the Dif pathway in EPS regulation remains to be uncovered. Using a genetic approach combined with quantitative and qualitative analysis, we demonstrate here that Tfp function upstream of the Dif proteins in regulating EPS production. The requirement of Tfp for the production of EPS was verified using various classes of Tfp mutants. Construction and examination of double and triple mutants indicated that mutations in dif are epistatic to those in pil. Furthermore, extracellular complementation between various Tfp and dif mutants suggests that Tfp, instead of being signals, may constitute the sensor or part of the sensor responsible for mediating signal input into the Dif pathway. We propose that S motility involves a regulatory loop in which EPS triggers Tfp retraction and Tfp provide proximity signals to the Dif pathway to modulate EPS production.

Bacterial Proteins↗
PubMed2006

Recruitment of a novel zinc-bound transcriptional factor by a bacterial HMGA-type protein is required for regulating multiple processes in Myxococcus xanthus.

Enhanceosome assembly in eukaryotes often requires high mobility group A (HMGA) proteins. In prokaryotes, the only known transcriptional regulator with HMGA-like physical, structural and DNA-binding properties is Myxococcus xanthus CarD. Here, we report that every CarD-regulated process analysed also requires the product of gene carG, located immediately downstream of and transcriptionally coupled to carD. CarG has the zinc-binding H/C-rich metallopeptidase motif found in archaemetzincins, but with Q replacing a catalytically essential E. CarG, a monomer, binds two zinc atoms, shows no apparent metallopeptidase activity, and its stability in vivo absolutely requires the cysteines. This indicates a strictly structural role for zinc-binding. In vivo CarG localizes to the nucleoid but only if CarD is also present. In vitro CarG shows no DNA-binding but physically interacts with CarD via its N-terminal and not HMGA domain. CarD and CarG thus work as a single, physically linked, transcriptional regulatory unit, and if one exists in a bacterium so does the other. Like zinc-associated eukaryotic transcriptional adaptors in enhanceosome assembly, CarG regulates by interacting not with DNA but with another transcriptional factor.

Amino Acid Sequence↗
PubMed2006

FibA and PilA act cooperatively during fruiting body formation of Myxococcus xanthus.

The extracellular matrix (ECM) of Myxococcus xanthus is essential for social (S-) motility and fruiting body formation. An ECM-bound protein, FibA, is homologous to M4 zinc metalloproteases and is important for stimulation by a phosphatidylethanolamine (PE) chemoattractant and for formation of discrete aggregation foci. In this work, we demonstrate that a correlation exists between a reduced ability to respond to PE and the observed defects in fruiting body morphogenesis. Furthermore, the fibA aggregation defect is accentuated by the absence of either PilA, the structural subunit of type IV pili, or DifD, a chemosensory response regulator. The inability to form fruiting bodies is not due to a loss of S-motility, but rather the loss of PilA and pili as pilT fibA mutants form fruiting bodies. The FibA active site residue E342 is important for fruiting body morphogenesis in the absence of PilA. Mutants exhibiting defects in fruiting body morphogenesis also produce fewer viable spores. It is proposed that FibA and PilA act as extracellular sensors for developmental signals.

Amino Acid Sequence↗
PubMed1994

Protein purification, gene cloning and sequencing of an acidic endoprotease from Myxococcus xanthus DK101.

An acidic endoprotease (MAEP) secreted during vegetative growth by Myxococcus xanthus DK101 was purified to homogeneity by a series of chromatographic procedures. The endoprotease cleaved the Phe-Met bond of kappa-casein under acidic conditions (pH 5.9). Its apparent molecular mass and its isoelectric point have been estimated to be 12 kDa and 4.5, respectively. From the N-terminal amino acid sequence, a set of two primers for polymerase chain reaction have been designed. Amplification of the corresponding DNA fragment (84 bp) generated a probe, then used to screen an expression DNA library of M. xanthus and to isolate a recombinant plasmid which contained a 2127-bp insert. The nucleotide sequence included an open reading frame (ORF) of 585 nucleotides, encoding 195 amino acids, that exhibited a high degree of similarity with the N-terminal amino acid sequence of the purified MAEP. The polypeptide sequence inferred from this ORF revealed that the mature enzyme should contain 131 amino acids arising from a 195-amino-acid precursor protein.

Amino Acid Sequence↗
PubMed1994

Purification and characterization of an alkaline elastase from Myxococcus xanthus.

An extracellular elastase, termed Myxococcus xanthus alkaline protease 1 (MAP1), has been purified from M. xanthus DK1622 culture supernatants by a combination of ion-exchange and affinity chromatographies. It consists of a single peptide chain of 39 kDa. The elastolytic activity was totally suppressed by 10 mM 1,10-phenanthroline and the enzyme may then be classified as a metalloprotease. Its pH optimum was estimated to be 8.2 with both elastin-orcein and succinyl-Ala3 p-nitroanilide as substrates. Despite its low pI (5.2), MAP1 was adsorbed on elastin at 80%, a result which privileges hydrophobic interactions between MAP1 and elastin rather than salt bridges, as for known basic elastases. About 80% of the original amidasic and elastolytic activities were conserved after a 30-min prior incubation of the enzyme at 40 degrees C; however, 70% of the amidasic activity is measured, instead of 15% for the elastolytic activity, after 30 min at 50 degrees C. Thermal denaturation at this temperature may prevent adsorption of the enzyme on elastin without any important change of the elastase structure. MAP1 readily hydrolyzes the Gly23-Phe24 bond in the oxidized insulin B chain; the peptide bonds Ala14-Leu15, Leu15-Tyr16, Phe24-Phe25, Phe25-Tyr26 are also cleaved, suggesting a primary specificity of the enzyme for hydrophobic or aromatic residues at the first amino acid towards the C-terminus from the cleavage site (P'1 position) [Schechter, I. & Berger, A. (1967) Biochem. Biophys. Res. Commun. 27, 157-162]. This hypothesis is consistent with the fact that Ala2-Phe-Ala and Ala3-Phe-Ala are hydrolyzed even though tri-alanine to hexa-alanine oligomers are not. The evidence of an elastase with the same molecular mass and pI as MAP1 is given during fruiting body development in submerged culture of M. xanthus. The fact that aromatic amino acids have been found to be the most representative of A-signal [Kuspa, A., Plamann, L. & Kaiser, D. (1992) J. Bacteriol. 174, 3319-3326] is consistent with the hypothesis that, regarding its specificity, MAP1 is likely to play a role in development of myxobacteria.

Amino Acid Sequence↗
PubMed1995

A cluster of structural and regulatory genes for light-induced carotenogenesis in Myxococcus xanthus.

In the bacterium Myxococcus xanthus, several genes for carotenoid synthesis lie together at the carA-carB chromosomal locus and are co-ordinately activated by blue light. A 12-kb DNA stretch from wild-type M. xanthus has been sequenced that includes the entire carA-carB gene cluster. According to sequence analysis, the cluster contains 11 different genes. Intergenic distances are very short or nil (implying translational coupling), giving further support to previous evidence indicating that most (or all) of the genes in the cluster form a single operon. At the promoter region, a potential -35 site for the binding of sigma factors is found. However, the -10 region shows little similarity with analogous sites in other bacterial promoters. Five (possibly six) genes in the carA-carB operon code for enzymes acting on early or late steps of the pathway for carotenoid synthesis. Other genes in the operon show no overall similarity with previously known genes. However, peptide stretches in the predicted products of two genes exhibit strong similarity with the DNA binding domain of the MerR family of transcriptional regulators. At least one of the predicted DNA-binding domains is altered in a mutant strain affected in light-regulation of the car genes.

Alkyl and Aryl Transferases↗
PubMed1994

Increases in the intracellular concentration of glycerol during development in Myxococcus xanthus S. Courtney Frasch.

The role of glycerol as a natural morphogen of myxospore formation in Myxococcus xanthus was examined. Glycerol was extracted from cells undergoing development and analyzed by gas chromatography. Glycerol is present in cells, and the intracellular level undergoes a series of transient increases during development. The data suggest a role for glycerol in myxosporulation and fruiting body morphogenesis supporting the notion that this chemical induction of sporulation may represent a physiological pathway in development.

Chromatography, Gas↗
PubMed1996

Behavioral analysis of single cells of Myxococcus xanthus in response to prey cells of Escherichia coli.

Myxococcus xanthus cells move over surfaces by gliding motility. The frz signal transduction system is used to control the reversal frequency, and thus the overall direction of movement of M. xanthus cells. We analyzed the behavior of wild-type and frz mutant cells in response to prey bacteria (Escherichia coli). Wild-type cells of M. xanthus did not respond to microcolonies of E. coli until they made physical contact. Cells which penetrated a colony remained in the colony until all of the prey cells were digested. Cells of frz mutants also penetrated E. coli microcolonies and digested some of the E. coli cells, but they invariably abandoned the microcolony leaving their food source behind. These observations illustrate the importance of the frz system of signal transduction for the feeding behavior of M. xanthus cells.

Bacterial Adhesion↗
PubMed1996

A Myxococcus xanthus cell density-sensing system required for multicellular development.

Progression through early Myxococcus xanthus multicellular fruiting body development requires the generation of and response to extracellular A signal. Extracellular A signal is a specific set of amino acids at an extracellular concentration greater than 10 muM. It functions as a cell density signal during starvation that allows the cells to sense that a minimal cell density has been reached and development can proceed. The generation of extracellular A signal requires the products of three asg genes. They have recently been identified as AsgA, a fused two-component histidine protein kinase and response regulator; AsgB, a putative DNA-binding protein; and AsgC, the M, xanthus major sigma factor. Other elements of the A signaling pathway map to the sasB locus and appear to be A signal transducers. These elements are regulators of the earliest A signal-dependent gene, whose promoter is a member of the sigma-54 family. Continued study of the A signaling pathway is expected to identify additional components of this network required for the complex behavioural response of fruiting body formation.

Cell Count↗
PubMed1999

A NusG-like transcription anti-terminator is involved in the biosynthesis of the polyketide antibiotic TA of Myxococcus xanthus.

The antibiotic TA of Myxococcus xanthus is synthesized through a type I polyketide synthase mechanism. Previous studies have indicated that several genes essential for TA production are clustered within a 40-kb region and are transcriptionally co-regulated. In this study, we report the genetic analysis of the first gene in the TA gene cluster, identified as a NusG-like transcription anti-terminator. Functional analysis of this NusG-like anti-terminator gene by specific gene disruption confirms that it is essential for TA production but not for normal growth and development.

Amino Acid Sequence↗
PubMed2000

Phenotypic analyses of frz and dif double mutants of Myxococcus xanthus.

Myxococcus xanthus is a Gram-negative gliding bacterium that aggregates and develops into multicellular fruiting bodies in response to starvation. Two chemosensory systems (frz and dif), both of which are homologous to known chemotaxis proteins, were previously identified through characterization of various developmental mutants. This study aims to examine the interaction between these two systems since both of them are required for fruiting body formation of M. xanthus. Through detailed phenotypic analyses of frz and dif double mutants, we found that both frz and dif are involved in cellular reversal and social motility; however, the frz genes are epistatic in controlling cellular reversal, whereas the dif genes are epistatic in controlling social motility. The study suggests that the integration of these two chemotaxis systems may play a central role in controlling the complicated social behaviors of M. xanthus.

Bacterial Proteins↗
PubMed2001

An unusual beta-ketoacyl:acyl carrier protein synthase and acyltransferase motifs in TaK, a putative protein required for biosynthesis of the antibiotic TA in Myxococcus xanthus.

The antibiotic TA of Myxococcus xanthus is produced by a type-I polyketide synthase mechanism. Previous studies have indicated that TA genes are clustered within a 36-kb region. The chemical structure of TA indicates the need for several post-modification steps, which are introduced to form the final bioactive molecule. These include three C-methylations, an O-methylation and a specific hydroxylation. In this study, we describe the genetic analysis of taK, encoding a specific polyketide beta-ketoacyl:acyl carrier protein synthase, which contains an unusual beta-ketoacyl synthase and acyltransferase motifs and is likely to be involved in antibiotic TA post-modification. Functional analysis of this beta-ketoacyl:acyl carrier protein synthase by specific gene disruption suggests that it is essential for the production of an active TA molecule.

3-Oxoacyl-(Acyl-Carrier-Protein) Synthase↗