Zhaomin Yang

Chemosensory pathways, motility and development in Myxococcus xanthus

The complex life cycle of Myxococcus xanthus includes predation, swarming, fruiting-body formation and sporulation. The genome of M. xanthus is large and comprises an estimated 7,400 open reading frames, of which approximately 605 code for regulatory genes. These include eight clusters of chemotaxis-like genes that define eight chemosensory pathways, most of which have dedicated functions. Although many of these chemosensory pathways have a role in controlling motility, at least two of these pathways control gene expression during development.

A new set of chemotaxis homologues is essential for Myxococcus xanthus social motility

Myxococcus xanthus cells aggregate and develop into multicellular fruiting bodies in response to starvation. A new M. xanthus locus, designated dif for defective in fruiting, was identified by the characterization of a mutant defective in fruiting body formation. Molecular cloning, DNA sequencing and sequence analysis indicate that the dif locus encodes a new set of chemotaxis homologues of the bacterial chemotaxis proteins MCPs (methyl‐accepting chemotaxis proteins), CheW, CheY and CheA. The dif genes are distinct genetically and functionally from the previously identified M. xanthus frz chemotaxis genes, suggesting that multiple chemotaxis‐like systems are required for the developmental process of M. xanthus fruiting body formation. Genetic analysis and phenotypical characterization indicate that the M. xanthus dif locus is required for social (S) motility. This is the first report of a M. xanthus chemotaxis‐like signal transduction pathway that could regulate or co‐ordinate the movement of M. xanthus cells to bring about S motility.

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.

Exopolysaccharide biosynthesis genes required for social motility in Myxococcus xanthus.

Social (S)‐motility in Myxococcus xanthus is a flagellum‐independent gliding motility system that allows bacteria to move in groups on solid surfaces. S‐motility has been shown to require type IV pili (TFP), exopolysaccharide (EPS; a component of fibrils) and lipopolysaccharide (LPS). Previously, information concerning EPS biogenesis in M. xanthus was lacking. In this study, we screened 5000 randomly mutagenized colonies for defects in S‐motility and EPS and identified two genetic regions essential for EPS biogenesis: the EPS synthesis (eps) region and the EPS‐associated (eas) region. Mutants with insertions in the eps and eas regions were defective in S‐motility and fruiting body formation. These mutants failed to bind the dye calcofluor white, indicating that they lacked EPS; however, they retained normal TFP and LPS. Analysis of the eps locus showed several open reading frames (ORFs) that encode homologues to glycosyltransferases, glucanases and EPS transporters as well as regulatory proteins; the eas locus contains two ORFs: one exhibits homology to hypothetical proteins with a conserved domain of unknown function and the other displays no apparent homology to other proteins in the database. Further genetic mutagenesis analysis indicates that the whole eps region is involved in the biosynthesis of fibrils and fibril EPS. The operon at the proximal end of the eps region was analysed by generating in‐frame deletion mutations. These mutants showed varying degrees of defects in the bacteriums ability to produce EPS or perform EPS‐related functions, confirming the involvement of these genes in M. xanthus EPS biogenesis.

Myxococcus xanthus Chemotaxis Homologs DifD and DifG Negatively Regulate Fibril Polysaccharide Production

The extracellular matrix fibrils of Myxococcus xanthus are essential for the social lifestyle of this unusual bacterium. These fibrils form networks linking or encasing cells and are tightly correlated with cellular cohesion, development, and social (S) gliding motility. Previous studies identified a set of bacterial chemotaxis homologs encoded by the dif locus. It was determined that difA, difC, and difE, encoding respective homologs of a methyl-accepting chemotaxis protein, CheW, and CheA, are required for fibril production and therefore S motility and development. Here we report the studies of three additional genes residing at the dif locus, difB, difD, and difG. difD and difG encode homologs of chemotaxis proteins CheY and CheC, respectively. difB encodes a positively charged protein with limited homology at its N terminus to conserved bacterial proteins with unknown functions. Unlike the previously characterized dif genes, none of these three newly studied dif genes are essential for fibril production, S motility, or development. The difB mutant showed no obvious defects in any of the processes examined. In contrast, the difD and the difG mutants were observed to overproduce fibril polysaccharides in comparison with production by the wild type. The observation that DifD and DifG negatively regulate fibril polysaccharide production strengthens our hypothesis that the M. xanthus dif genes define a chemotaxis-like signal transduction pathway which regulates fibril biogenesis. To our knowledge, this is the first report of functional studies of a CheC homolog in proteobacteria. In addition, during this study, we slightly modified previously developed assays to easily quantify fibril polysaccharide production in M. xanthus.

Gliding Motility Revisited: How Do the Myxobacteria Move without Flagella?

SUMMARY In bacteria, motility is important for a wide variety of biological functions such as virulence, fruiting body formation, and biofilm formation. While most bacteria move by using specialized appendages, usually external or periplasmic flagella, some bacteria use other mechanisms for their movements that are less well characterized. These mechanisms do not always exhibit obvious motility structures. Myxococcus xanthus is a motile bacterium that does not produce flagella but glides slowly over solid surfaces. How M. xanthus moves has remained a puzzle that has challenged microbiologists for over 50 years. Fortunately, recent advances in the analysis of motility mutants, bioinformatics, and protein localization have revealed likely mechanisms for the two M. xanthus motility systems. These results are summarized in this review.

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.

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.

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.

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.