Body Formation In Myxococcus Xanthus Research Articles

Two intercellular signals required for fruiting body formation in Myxococcus xanthus act sequentially but non‐hierarchically

Starvation-induced fruiting body formation in Myxococcus xanthus depends on intercellular signalling. A-signal functions after 2 h of starvation and its synthesis depends on the asg genes. C-signal functions after 6 h of starvation and is generated by proteolytic cleavage of a precursor by the protease PopC. Previous gene expression studies suggested that the A- and C-signal lie on a hierarchical pathway. Here we explored the causal relationship between the A- and C-signal. The asgA and asgB mutants have reduced popC expression, PopC accumulation and C-signal accumulation. popC expression was shown not to depend on A-signal but on the AsgA and AsgB proteins. Restored popC expression in the two mutants rescued PopC and C-signal accumulation as well as C-signalling and the developmental defects of the two mutants without restoring A-signalling. Based on these results we suggest that A- and C-signal do not lie on a hierarchical, dependent pathway. Instead the A- and C-signal act sequentially and without a causal relationship suggesting that they are linked by a shared timing mechanism, which ensures the early and late onset of A-signalling and C-signalling, respectively, during starvation. This pathway topology represents a novel architecture for bacterial intercellular signalling systems involving more than one signal.

Regulated Secretion of a Protease Activates Intercellular Signaling during Fruiting Body Formation in M. xanthus

In response to starvation Myxococcus xanthus initiates a developmental program that culminates in fruiting body formation. There are two morphogenetic events in this program, aggregation and sporulation, which are temporally and spatially coordinated by the contact-dependent intercellular C-signal protein (p17). p17 is generated by proteolytic cleavage of the p25 precursor protein, which accumulates in the outer membrane of vegetative and starving cells. However, p17 generation is restricted to starving cells. Here we identify the subtilisin-like protease PopC that is directly responsible for cleavage of p25. PopC accumulates in the cytoplasm of vegetative cells but is selectively secreted during starvation coinciding with the generation of p17. Consequently, p25 and PopC only encounter each other in starving cells. Thus, restriction of p25 cleavage to starving cells occurs by a compartmentalization mechanism that depends on selective secretion of PopC during starvation. Our results provide evidence for regulated proteolysis via regulated secretion.

Proteins Associated with theMyxococcus xanthusExtracellular Matrix

Fruiting body formation of Myxococcus xanthus, like biofilm formation of many other organisms, involves the production of an extracellular matrix (ECM). While the polysaccharide component has been studied, the protein component has been largely unexplored. Proteins associated with the ECM were solubilized from purified ECM by boiling with sodium dodecyl sulfate and were identified by liquid chromatography-tandem mass spectrometry of tryptic fragments. The ECM is enriched in proteins of novel function; putative functions were assigned for only 5 of the 21 proteins. Thirteen putative ECM proteins had lipoprotein secretion signals. The genes for many ECM proteins were disrupted in the wild-type (WT), fibA, and pilA backgrounds. Disruption of the MXAN4860 gene had no effect in the WT or fibA background but in the pilA background resulted in a 24-h delay in aggregation and sporulation compared to its parent. The results of this study show that the M. xanthus ECM proteome is diverse and novel.

Novel Iso-branched Ether Lipids as Specific Markers of Developmental Sporulation in the Myxobacterium Myxococcus xanthus

Iso-fatty acids (FAs) are the dominant FA family in all myxobacteria analyzed. Furthermore, it was postulated that iso-FAs or compounds derived thereof are involved in fruiting body formation in Myxococcus xanthus, since mutants with a reduced level of iso-FA due to a reduced level of the precursor isovaleryl-CoA, are delayed in aggregation and produce only few myxospores. To elucidate the function of iso-FAs and their corresponding lipids we have analyzed the developmental phenotype of mutants having different levels of iso-FAs resulting in a clear correlation between the amount of iso-FAs and the delay of aggregation and reduction in spore yield. Addition of either isovalerate or 13-methyltetradecanoic acid resulted in restoration of the wild-type FA profile and normal development. Detailed analysis of the fatty acid (FA) profile during fruiting body formation in Myxococcus xanthus wild-type revealed the specific accumulation of 13-methyltetradecanal and 1-O-13-methyltetradecylglycerol which were produced specifically in the myxospores and which are derived from 1-O-(13-methyl-1-Z-tetradecenyl)-2-O-(13-methyltetradecanoyl)-glycero-3-phosphatidylethanolamine (VEPE) and 1,2-di-(13-methyltetradecanoyl)-3-(13-methyltetradecyl)glycerol (TG-1), respectively. The structures of these unusual ether lipids have been determined by spectrometric methods and synthesis (for TG-1). Analysis of several mutants blocked at different stages of development indicated that the biosynthesis of TG-1 is developmentally regulated and that VEPE might be an intermediate in the TG-1 biosynthesis. Finally, addition of TG-1 to mutants blocked in the biosynthesis of isovaleryl-CoA could restore aggregation and sporulation emphasizing the important role of iso-branched lipids for myxobacterial development.

Coupling of multicellular morphogenesis and cellular differentiation by an unusual hybrid histidine protein kinase in Myxococcus xanthus

We describe an unusual hybrid histidine protein kinase, which is important for spatially coupling cell aggregation and sporulation during fruiting body formation in Myxococcus xanthus. A rodK mutant makes abnormal fruiting bodies and spores develop outside the fruiting bodies. RodK is a soluble, cytoplasmic protein, which contains an N-terminal sensor domain, a histidine protein kinase domain and three receiver domains. In vitro phosphorylation assays showed that RodK possesses kinase activity. Kinase activity is essential for RodK function in vivo. RodK is present in vegetative cells and remains present until the late aggregation stage, after which the level decreases in a manner that depends on the intercellular A-signal. Genetic evidence suggests that RodK may regulate multiple temporally separated events during fruiting body formation including stimulation of early developmental gene expression, inhibition of A-signal production and inhibition of the intercellular C-signal transduction pathway. We speculate that RodK undergoes a change in activity during development, which is reflected in changes in phosphotransfer to the receiver domains.

Regulation of fruA Expression during Vegetative Growth and Development of Myxococcus xanthus

Expression of the fruA gene, encoding a putative transcription factor essential for fruiting body formation of Myxococcus xanthus, is specifically activated during development. In the present study, we have analyzed the mechanism of the transcriptional regulation of fruA expression. From gel retardation and footprinting assays using various fruA regulatory regions as probes and competitors, a protein designated factor X was found to specifically bind to a sequence (xbs) located downstream of the transcription-initiation site (+78 to +94) of the fruA gene. Factor X activity was present during vegetative growth and decreased during early development. Analysis of promoter activities of various segments of the fruA regulatory region using the lacZ reporter gene in vivo indicated that a DNA segment extending 45-bp upstream from the transcription-initiation site was required for developmentally regulated fruA expression at a low level. In addition, cis-acting regulatory regions located upstream and downstream of the fruA promoter region and including C-box and xbs, were found to be involved in regulation of fruA expression during development. When inserted into the vegA gene, the xbs element inhibited vegA expression during vegetative growth. Together with previously reported results, our studies reveal that fruA expression is regulated by both positive and negative mechanisms during the M. xanthus life cycle.

Role of fruA and csgA Genes in Gene Expression during Development of Myxococcus xanthus: ANALYSIS BY TWO-DIMENSIONAL GEL ELECTROPHORESIS

Two genes, fruA and csgA, encoding a putative transcription factor and C-factor, respectively, are essential for fruiting body formation of Myxococcus xanthus. To investigate the role of fruA and csgA genes in developmental gene expression, developing cells as well as vegetative cells of M. xanthus wild-type, fruA::Tc, and csgA731 strains were pulse-labeled with [(35)S]methionine, and the whole cell proteins were analyzed using two-dimensional immobilized pH gradient/SDS-PAGE. Differences in protein synthesis patterns among more than 700 protein spots were detected during development of the three strains. Fourteen proteins showing distinctly different expression patterns in mutant cells were analyzed in more detail. Five of the 14 proteins were identified as elongation factor Tu (EF-Tu), Dru, DofA, FruA, and protein S by immunoblot analysis and mass spectroscopy. A gene encoding DofA was cloned and sequenced. Although both fruA and csgA genes regulate early development of M. xanthus, they were found to differently regulate expression of several developmental genes. The production of six proteins, including DofA and protein S, was dependent on fruA, whereas the production of two proteins was dependent on csgA, and one protein was dependent on both fruA and csgA. To explain the present findings, a new model was presented in which different levels of FruA phosphorylation may distinctively regulate the expression of two groups of developmental genes.

Programmed Death in Bacteria

Programmed cell death (PCD) in bacteria plays an important role in developmental processes, such as lysis of the mother cell during sporulation of Bacillus subtilis and lysis of vegetative cells in fruiting body formation of Myxococcus xanthus. The signal transduction pathway leading to autolysis of the mother cell includes the terminal sporulation sigma factor Esigma(K), which induces the synthesis of autolysins CwlC and CwlH. An activator of autolysin in this and other PCD processes is yet to be identified. Autolysis plays a role in genetic exchange in Streptococcus pneumoniae, and the gene for the major autolysin, lytA, is located in the same operon with recA. DNA from lysed cells is picked up by their neighbors and recombined into the chromosome by RecA. LytA requires an unknown activator controlled by a sensory kinase, VncS. Deletion of vncS inhibits autolysis and also decreases killing by unrelated antibiotics. This observation suggests that PCD in bacteria serves to eliminate damaged cells, similar to apoptosis of defective cells in metazoa. The presence of genes affecting survival without changing growth sensitivity to antibiotics (vncS, lytA, hipAB, sulA, and mar) indicates that bacteria are able to control their fate. Elimination of defective cells could limit the spread of a viral infection and donate nutrients to healthy kin cells. An altruistic suicide would be challenged by the appearance of asocial mutants without PCD and by the possibility of maladaptive total suicide in response to a uniformly present lethal factor or nutrient depletion. It is proposed that a low rate of mutation serves to decrease the probability that asocial mutants without PCD will take over the population. It is suggested that PCD is disabled in persistors, rare cells that are resistant to killing, to ensure population survival. It is suggested that lack of nutrients leads to the stringent response that suppresses PCD, producing a state of tolerance to antibiotics, allowing cells to discriminate between nutrient deprivation and unrepairable damage. High levels of persistors are apparently responsible for the extraordinary survival properties of bacterial biofilms, and genes affecting persistence appear to be promising targets for development of drugs aimed at eradicating recalcitrant infections. PCD in unicellular eukaryotes is also considered, including aging in Saccharomyces cerevisiae. Apoptosis-like elimination of defective cells in S. cerevisiae and protozoa suggests that all unicellular life forms evolved altruistic programmed death that serves a variety of useful functions.

The guanosine nucleotide (p)ppGpp initiates development and A-factor production in myxococcus xanthus.

Guanosine 3'-di-5'-(tri)di-phosphate nucleotides [(p)ppGpp], synthesized in response to amino acid limitation, induce early gene expression leading to multicellular fruiting body formation in Myxococcus xanthus. A mutant (DK527) that fails to accumulate (p)ppGpp in response to starvation was found to be blocked in development prior to aggregation. By use of a series of developmentally regulated Tn5lac transcriptional fusion reporters, the time of developmental arrest in DK527 was narrowed to within the few hours of development, the period of starvation recognition. The mutant is also defective in the production of A-factor, an early extracellular cell-density signal. The relA gene from Escherichia coli, which encodes a ribosome-dependent (p)ppGpp synthetase, rescues this mutant. We also demonstrate that inactivation of the M. xanthus relA homolog blocks development and the accumulation of (p)ppGpp. Moreover, the wild-type allele of Myxococcus relA rescues DK527. These observations support a model in which accumulation of (p)ppGpp, in response to starvation, initiates the program of fruiting body development, including the production of A-factor.

Methionine inhibits developmental aggregation of Myxococcus xanthus by blocking the biosynthesis of S-adenosyl methionine

Previous studies showed that high concentrations of methionine (> 1 mM) inhibited aggregation and fruiting body formation in Myxococcus xanthus (E. Rosenberg, D. Filer, D. Zafriti, and S. H. Kindler, J. Bacteriol. 115: 29-34, 1973, and J. M. Campos and D. R. Zusman, Proc. Natl. Acad. Sci. USA 72:518-522, 1975). However, the mechanism for the inhibition was unclear. In this study, we found that high levels of methionine inhibited the biosynthesis of S-adenosylmethionine (SAM) and that reduced intracellular levels of SAM are correlated with defective chemotactic movements and reduced developmental gene expression. In addition, we found that methionine analogs and high concentrations of amino acids which are known to affect SAM synthesis in other bacteria, such as threonine, lysine, and isoleucine, also caused reduced cellular levels of SAM and blocked fruiting body formation in M. xanthus. These results indicate that SAM is required for development of M. xanthus and the inhibitory effect of methionine on development results, at least in part, from its blocking of the biosynthesis of SAM.

Myxococcus xanthus protein C is a major spore surface protein

Fruiting body formation in Myxococcus xanthus involves the aggregation of cells to form mounds and the differentiation of rod-shaped cells into spherical myxospores. The surface of the myxospore is composed of several sodium dodecyl sulfate (SDS)-soluble proteins, the best characterized of which is protein S (Mr, 19,000). We have identified a new major spore surface protein called protein C (Mr, 30,000). Protein C is not present in extracts of vegetative cells but appears in extracts of developing cells by 6 h. Protein C, like protein S, is produced during starvation in liquid medium but is not made during glycerol-induced sporulation. Its synthesis is blocked in certain developmental mutants but not others. When examined by SDS-polyacrylamide gel electrophoresis, two forms of protein C are observed. Protein C is quantitatively released from spores by treatment with 0.1 N NaOH or by boiling in 1% SDS. It is slowly washed from the spore surface in water but is stabilized by the presence of magnesium. Protein C binds to the surface of spores depleted of protein C and protein S. Protein C is a useful new marker for development in M. xanthus because it is developmentally regulated, spore associated, abundant, and easily purified.

Alkaline, acid, and neutral phosphatase activities are induced during development in Myxococcus xanthus

One of the signals that has been reported to be important in stimulating fruiting body formation of Myxococcus xanthus is starvation for phosphate. We therefore chose to study phosphatase activity during M. xanthus development. Many phosphatases can cleave the substrate p-nitrophenol phosphate. Using this substrate in buffers at various pHs, we obtained a profile of phosphatase activities during development and germination of M. xanthus. These experiments indicated that there are five patterns of phosphatase activity in M. xanthus: two vegetative and three developmental. The two uniquely vegetative activities have pH optima at 7.2 and 8.5. Both require magnesium and both are inhibited by the reducing agent dithiothreitol. The developmental (spores) patterns of activity have pH optima of 5.2, 7.2, and 8.5. All three activities are Mg independent. Only the alkaline phosphatase activity is inhibited by dithiothreitol. The acid phosphatase activity is induced very early in development, within the first 2 to 4 h. Both the neutral and alkaline phosphatase Mg-independent activities are induced much later, about the time that myxospores become evident (24 to 30 h). The three activities are greatly diminished upon germination; however, the kinetics of loss differ for all three. The acid phosphatase activity declines very rapidly, the neutral activity begins to decline only after spores begin to convert to rods, and the alkaline phosphatase activity remains high until the time the cells begin to divide. All three developmental activities were measured in the developmental signalling mutants carrying asg, csg, and dsg. The pattern of expression obtained in the mutants was consistent with that of other developmentally regulated genes which exhibit similar patterns of expression during development. The ease with which phosphatases can be assayed should make the activities described in this report useful biochemical markers of stages of both fruiting body formation and germination.

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.

Evidence for long-lived mRNA during fruiting body formation in myxococcus xanthus.

Half-lives of Myxococcus xanthus mRNA were determined by inhibiting RNA polymerase with rifampin and then measuring the rate of [35S]methionine incorporation into protein. Vegetative cells resuspended in clone fruiting liquid culture showed an average mRNA half-life of approximately 3.5 min. Developmental cells (24 or 48 hr) exhibited biphasic decay curves with apparent mRNA half-lives of approximately 3.5 min and 20-30 min. The more-stable mRNA species accounted for about 30% of all mRNA present in 24-hr cells and as much as 40% of all mRNA of 48-hr cells. Analysis of the 35S-labeled proteins by NaDodSO4/polyacrylamide gel electrophoresis showed that only 5-10 major polypeptides are synthesized after 30 min of rifampin treatment. One of these is protein S, the spore surface coat protein, because immunoprecipitation of the 35S-labeled proteins with antisera specific for protein S showed continued synthesis of protein S in the presence of rifampin. The half-life for its mRNA was calculated to be 15-30 min. RNA from vegetative and developing cells was pulse labeled with 32PO4 followed by rifampin treatment. Analysis of the labeled RNA on 3% NaDodSO4/polyacrylamide gels showed 5-10 long-lived mRNA bands in developing cells. These results show that there are several abundant mRNA species synthesized developmentally that are exceptionally long lived. The fact that the majority of the mRNA species show the shorter half-life suggests that developing cells retain the normal mechanism for mRNA degradation.

Excreted adenosine is a cell density signal for the initiation of fruiting body formation in Myxococcus xanthus

Myxococcus xanthus is a bacterium that forms multicellular fruiting bodies in response to starvation. The initiation of fruiting body formation is cell density dependent, and we suggest that cells measure their cell density by titering the extracellular concentration of excreted adenosine. Our evidence is as follows: (1) At low cell densities fruiting body formation does not occur unless adenosine is added. (2) Norit, a substance that binds purines, inhibits fruiting body formation, and this inhibition is reversed by adenosine. (3) Cells labeled with [ 14C]carbonate excrete [ 14C]adenosine which is bound by the Norit. Furthermore, [ 14C]adenosine is excreted by developing cells at a concentration that will induce fruiting body formation at low cell density. The extracellular adenosine concentration increases with the cell density over a broad range of densities. (4) Hadacidin, an inhibitor of de novo AMP synthesis, inhibits fruiting body formation, and inhibition by hadacidin can be reversed with adenosine. Adenosine also appears to be involved in the aggregation process because the shape and size of the fruiting bodies are sensitive to the external concentration of adenosine.

Inhibition of vegetative growth and fruiting body formation in Myxococcus xanthus

Inhibition of vegetative growth and fruiting body formation in Myxococcus xanthus

Inhibition of vegetative growth and fruiting body formation inMyxococcus xanthus

Inhibition of vegetative growth and fruiting body formation in<i>Myxococcus xanthus</i>

Inhibition of fruiting body formation in Myxococcus xanthus by cytokinins

Inhibition of fruiting body formation in Myxococcus xanthus by cytokinins