Site-specific integration and expression of a developmental promoter in Myxococcus xanthus.

A series of intercellular signals are involved in the regulation of gene expression during fruiting body formation of Myxococcus xanthus. Mutations which block cell interactions, such as csgA (formerly known as spoC), also prevent expression of certain developmentally regulated promoters. csgA+ cells containing Tn5 lac omega DK4435, a developmentally regulated promoter fused to lacZ, began synthesizing lacZ mRNA 12 to 18 h into the developmental cycle. beta-Galactosidase specific activity increased about 12 h later. Neither lacZ mRNA nor beta-galactosidase activity was detected in a developing csgA mutant containing omega DK4435. The developmental promoter and its fused lacZ reporter gene were cloned into a pBR322-derived plasmid vector containing a portion of bacteriophage Mx8. These plasmids preferentially integrated into the M. xanthus chromosome by site-specific recombination at the bacteriophage Mx8 attachment site and maintained a copy number of 1 per chromosome. The integrated plasmids were relatively stable, segregating at a frequency of 0.0007% per generation in the absence of selection. The cloned and integrated promoter behaved like the native promoter, expressing beta-galactosidase at the proper time during wild-type development and failing to express the enzyme during development of a csgA mutant. The overall level of beta-galactosidase expression in merodiploid cells containing one native promoter and one promoter fused to lacZ was about half that of cells containing a single promoter fused to lacZ. These results suggest that the timing of developmentally regulated gene expression is largely independent of the location of this gene within the chromosome. Furthermore, they show that site-specific recombination can be a useful tool for establishing assays for promoter or gene function in M. xanthus.     

Rippling is a predatory behavior in Myxococcus xanthus

Cells of Myxococcus xanthus will, at times, organize their movement such that macroscopic traveling waves, termed ripples, are formed as groups of cells glide together on a solid surface. The reason for this behavior has long been a mystery, but we demonstrate here that rippling is a feeding behavior which occurs when M. xanthus cells make direct contact with either prey or large macromolecules. Rippling has been observed during two fundamentally distinct environmental conditions: (i) starvation-induced fruiting body development and (ii) predation of other organisms. Our results indicate that case (i) does not occur in all wild-type strains and is dependent on the intrinsic level of autolysis. Analysis of predatory rippling indicates that rippling behavior is inducible during predation on proteobacteria, gram-positive bacteria, yeast (such as Saccharomyces cerevisiae), and phage. Predatory efficiency decreases under genetic and physiological conditions in which rippling is inhibited. Rippling will also occur in the presence of purified macromolecules such as peptidoglycan, protein, and nucleic acid but does not occur in the presence of the respective monomeric components and also does not occur when the macromolecules are physically separated from M. xanthus cells. We conclude that rippling behavior is a mechanism utilized to efficiently consume nondiffusing growth substrates and that developmental rippling is a result of scavenging lysed cell debris.     

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.     

Spatial cues play a role in the development of Myxococcus xanthus

Intercellular signaling plays an important role in spatially regulated developmental processes. Myxococcus xanthus C signal transmission during fruiting body formation requires motile, densely packed, well aligned cells. tThe fruiting body consists of two domains: an outer domain which has densely packed, well aligned, motile cells: and an inner domain of more loosely packed, non-motile, sporulating cells. The two domains are characterized by different patterns of C-dependent gene expression, which begins in the outer domain where C-signaling is most efficient, and reaches its maximum in the inner domain. These domains may be maintained by a dynamic mechanism which relies on passive transport of the sporulating cells from the outer domain, where sporulation is initiated, to the inner domain by the motile cells in the outer domain.     

Tyrosine phosphorylation in Myxococcus xanthus, a multicellular prokaryote.

Tyrosine phosphorylation is an extremely rare event in prokaryotes, occurring almost exclusively in multicellular eukaryotes. We have identified, for the first time, by the use of antiphosphotyrosine monoclonal antibody and Western blot (immunoblot) analysis, two tyrosine-phosphorylated membrane proteins in the multicellular prokaryote Myxococcus xanthus. The pattern of tyrosine phosphorylation was shown to change during development, indicating a possible role for this regulatory modification during two stages of development, i.e., aggregation and sporulation. Furthermore, the altered pattern of tyrosine phosphorylation observed in a variety of signaling mutants was shown to differ from that observed in the wild type, suggesting further the possible involvement of tyrosine phosphorylation during the development program.     

mlpB, a gene encoding a new lipoprotein in Myxococcus xanthus

AIMS: To search for and study the genes involved in the regulation of phosphate in the soil developmental bacterium Myxococcus xanthus. METHODS AND RESULTS: The mlpB gene encoding a 149 residue polypeptide was identified while screening for genes with products related to phosphate metabolism. The amino terminal 19 residues of MlpB encode a typical prokaryotic signal sequence with a putative lipoprotein cleavage site. CONCLUSIONS: In this study, a new myxobacterial putative lipoprotein is reported. The data suggest that MlpB may be involved in the secretion of phosphate-related proteins. SIGNIFICANCE AND IMPACT OF THE STUDY: Soil bacteria have complex regulatory systems for using inorganic phosphate. This nutrient is limiting in the environment, and has a critical importance for growth and in the initiation of differentiation for developmental bacteria. A number of proteins are involved in all these processes, including membrane lipoproteins, which are being increasingly studied in M. xanthus.     

nsd, a locus that affects the Myxococcus xanthus cellular response to nutrient concentration

Expression of the previously reported Tn5lac Omega4469 insertion in Myxococcus xanthus cells is regulated by the starvation response. Interested in learning more about the starvation response, we cloned and sequenced the region containing the insertion. Our analysis shows that the gene fusion is located in an open reading frame that we have designated nsd (nutrient sensing/utilizing defective) and that its expression is driven by a sigma70-like promoter. Sequence analysis of the nsd gene product provides no information on the potential structure or function of the encoded protein. In a further effort to learn about the role of nsd in the starvation response, we closely examined the phenotype of cells carrying the nsd::Tn5lac Omega4469 mutation. Our analysis showed that these cells initiate development on medium that contains nutrients sufficient to sustain vegetative growth of wild-type cells. Furthermore, in liquid media these same nutrient concentrations elicit a severe impairment of growth of nsd cells. The data suggest that the nsd cells launch a starvation response when there are enough nutrients to prevent one. In support of this hypothesis, we found that, when grown in these nutrient concentrations, nsd cells accumulate guanosine tetraphosphate, the cellular starvation signal. Therefore, we propose that nsd is used by cells to respond to available nutrient levels.