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PubMed2006

Mutational analysis of the Myxococcus xanthus Omega4406 promoter region reveals an upstream negative regulatory element that mediates C-signal dependence.

C signaling plays a key role in coordinating cell movement and differentiation during the multicellular developmental process of Myxococcus xanthus. C signaling regulates expression of genes induced after about 6 h into development, when cells are forming mounds. One gene whose expression depends absolutely on C signaling was identified by insertion of a transposable element at site Omega4406 which generated a transcriptional fusion between lacZ and an upstream promoter. We have investigated regulation of the Omega4406 promoter. A 5' deletion revealed a negative regulatory element located between bp -533 and -100 relative to the transcriptional start site. In the absence of this element, the promoter was still developmentally regulated but about fourfold more active. Also, the truncated promoter region retained normal dependence on two developmental regulators, FruA and DevS, but lost its dependence on the C-signaling protein CsgA. We infer that C signaling partially overcomes the negative effect of the upstream element on activity of the Omega4406 promoter. Deletion of downstream DNA between bp 50 and 140 caused a threefold loss in expression, suggesting that a positive regulatory element lies in this region. Additional positive and negative regulatory elements are present in the region from bp -69 to -49, based on the effects of multiple-base-pair mutations. Within this region, a 5-bp element and a C-box-like sequence resemble sequences found in other developmentally regulated M. xanthus promoter regions, but the effects of single-base-pair changes in these sequences suggest that each functions uniquely. We conclude that regulation of the Omega4406 promoter involves multiple positive and negative regulatory elements located upstream and downstream of the region typically bound by RNA polymerase.

Bacterial Proteins↗
PubMed2006

Contribution of the cyclic nucleotide phosphodiesterases PdeA and PdeB to adaptation of Myxococcus xanthus cells to osmotic or high-temperature stress.

A tBLASTn search of the Myxococcus xanthus genome database at The Institute for Genomic Research (TIGR) identified three genes (pdeA, pdeB, and pdeC) that encode proteins homologous to 3',5'-cyclic nucleotide phosphodiesterase. pdeA, pdeB, and pdeC mutants, constructed by replacing a part of the gene with the kanamycin or tetracycline resistance gene, showed normal growth, development, and germination under nonstress conditions. However, the spores of mutants, especially the pdeA and pdeB mutants, placed under osmotic stress germinated earlier than the wild-type spores. The phenotype was the opposite of that of the receptor-type adenylyl cyclase (cyaA or cyaB) mutant. Also, pdeA and pdeB mutants were found to have impaired growth under the condition of high-temperature stress. Intracellular cyclic AMP (cAMP) levels of pdeA or pdeB mutant cells under these stressful conditions were about 1.3-fold to 2.0-fold higher than those of wild-type cells. These results suggest that PdeA and PdeB may be involved in osmotic adaptation during spore germination and temperature adaptation during vegetative growth through the regulation of cAMP levels.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed2006

Nla18, a key regulatory protein required for normal growth and development of Myxococcus xanthus.

NtrC-like activators regulate the transcription of a wide variety of adaptive genes in bacteria. Previously, we demonstrated that a mutation in the ntrC-like activator gene nla18 causes defects in fruiting body development in Myxococcus xanthus. In this report, we describe the effect that nla18 inactivation has on gene expression patterns during development and vegetative growth. Gene expression in nla18 mutant cells is altered in the early stages of fruiting body development. Furthermore, nla18 mutant cells are defective for two of the earliest events in development, production of the intracellular starvation signal ppGpp and production of A-signal. Taken together, these results indicate that the developmental program in nla18 mutant cells goes awry very early. Inactivation of nla18 also causes a dramatic decrease in the vegetative growth rate of M. xanthus cells. DNA microarray analysis revealed that the vegetative expression patterns of more than 700 genes are altered in nla18 mutant cells. Genes coding for putative membrane and membrane-associated proteins are among the largest classes of genes whose expression is altered by nla18 inactivation. This result is supported by our findings that the profiles of membrane proteins isolated from vegetative nla18 mutant and wild-type cells are noticeably different. In addition to genes that code for putative membrane proteins, nla18 inactivation affects the expression of many genes that are likely to be important for protein synthesis and gene regulation. Our data are consistent with a model in which Nla18 controls vegetative growth and development by activating the expression of genes involved in gene regulation, translation, and membrane structure.

Bacterial Proteins↗
PubMed2006

DNA replication during aggregation phase is essential for Myxococcus xanthus development.

Previous studies have demonstrated that fruiting body-derived Myxococcus xanthus myxospores contain two fully replicated copies of its genome, implying developmental control of chromosome replication and septation. In this study, we employ DNA replication inhibitors to determine if chromosome replication is essential to development and the exact time frame in which chromosome replication occurs within the developmental cycle. Our results show that DNA replication during the aggregation phase is essential for developmental progression, implying the existence of a checkpoint that monitors chromosome integrity at the end of the aggregation phase.

Amino Acid Sequence↗
PubMed2006

Role of sigmaD in regulating genes and signals during Myxococcus xanthus development.

Starvation-induced development of Myxococcus xanthus is an excellent model for biofilm formation because it involves cell-cell signaling to coordinate formation of multicellular mounds, gene expression, and cellular differentiation into spores. The role of sigma(D), an alternative sigma factor important for viability in stationary phase and for stress responses, was investigated during development by measuring signal production, gene expression, and sporulation of a sigD null mutant alone and upon codevelopment with wild-type cells or signaling mutants. The sigD mutant responded to starvation by inducing (p)ppGpp synthesis normally but was impaired for production of A-signal, an early cell density signal, and for production of the morphogenetic C-signal. Induction of early developmental genes was greatly reduced, and expression of those that depend on A-signal was not restored by codevelopment with wild-type cells, indicating that sigma(D) is needed for cellular responses to A-signal. Despite these early developmental defects, the sigD mutant responded to C-signal supplied by codeveloping wild-type cells by inducing a subset of late developmental genes. sigma(D) RNA polymerase is dispensable for transcription of this subset, but a distinct regulatory class, which includes genes essential for sporulation, requires sigma(D) RNA polymerase or a gene under its control, cell autonomously. The level of sigD transcript in a relA mutant during growth is much lower than in wild-type cells, suggesting that (p)ppGpp positively regulates sigD transcription in growing cells. The sigD transcript level drops in wild-type cells after 20 min of starvation and remains low after 40 min but rises in a relA mutant after 40 min, suggesting that (p)ppGpp negatively regulates sigD transcription early in development. We conclude that sigma(D) synthesized during growth occupies a position near the top of a regulatory hierarchy governing M. xanthus development, analogous to sigma factors that control biofilm formation of other bacteria.

Bacterial Proteins↗
PubMed1999

Gliding motility in bacteria: insights from studies of Myxococcus xanthus.

Gliding motility is observed in a large variety of phylogenetically unrelated bacteria. Gliding provides a means for microbes to travel in environments with a low water content, such as might be found in biofilms, microbial mats, and soil. Gliding is defined as the movement of a cell on a surface in the direction of the long axis of the cell. Because this definition is operational and not mechanistic, the underlying molecular motor(s) may be quite different in diverse microbes. In fact, studies on the gliding bacterium Myxococcus xanthus suggest that two independent gliding machineries, encoded by two multigene systems, operate in this microorganism. One machinery, which allows individual cells to glide on a surface, independent of whether the cells are moving alone or in groups, requires the function of the genes of the A-motility system. More than 37 A-motility genes are known to be required for this form of movement. Depending on an additional phenotype, these genes are divided into two subclasses, the agl and cgl genes. Videomicroscopic studies on gliding movement, as well as ultrastructural observations of two myxobacteria, suggest that the A-system motor may consist of multiple single motor elements that are arrayed along the entire cell body. Each motor element is proposed to be localized to the periplasmic space and to be anchored to the peptidoglycan layer. The force to glide which may be generated here is coupled to adhesion sites that move freely in the outer membrane. These adhesion sites provide a specific contact with the substratum. Based on single-cell observations, similar models have been proposed to operate in the unrelated gliding bacteria Flavobacterium johnsoniae (formerly Cytophaga johnsonae), Cytophaga strain U67, and Flexibacter polymorphus (a filamentous glider). Although this model has not been verified experimentally, M. xanthus seems to be the ideal organism with which to test it, given the genetic tools available. The second gliding motor in M. xanthus controls cell movement in groups (S-motility system). It is dependent on functional type IV pili and is operative only when cells are in close proximity to each other. Type IV pili are known to be involved in another mode of bacterial surface translocation, called twitching motility. S-motility may well represent a variation of twitching motility in M. xanthus. However, twitching differs from gliding since it involves cell movements that are jerky and abrupt and that lack the organization and smoothness observed in gliding. Components of this motor are encoded by genes of the S-system, which appear to be homologs of genes involved in the biosynthesis, assembly, and function of type IV pili in Pseudomonas aeruginosa and Neisseria gonorrhoeae. How type IV pili generate force in S-motility is currently unknown, but it is to be expected that ongoing physiological, genetic, and biochemical studies in M. xanthus, in conjunction with studies on twitching in P. aeruginosa and N. gonorrhoeae, will provide important insights into this microbial motor. The two motility systems of M. xanthus are affected to different degrees by the MglA protein, which shows similarity to a small GTPase. Bacterial chemotaxis-like sensory transduction systems control gliding motility in M. xanthus. The frz genes appear to regulate gliding movement of individual cells and movement by the S-motility system, suggesting that the two motors found in this bacterium can be regulated to result in coordinated multicellular movements. In contrast, the dif genes affect only S-system-dependent swarming.

Genes, Bacterial↗
PubMed1992

Control of cell density and pattern by intercellular signaling in Myxococcus development.

Myxococcus xanthus cells feed, move, and develop cooperatively. Genetic, biochemical, and cell mosaic studies demonstrate that cells coordinate their multicellular behavior by transmission of intercellular signals. Starvation for amino acids at sufficiently high density on a solid surface initiates a series of events culminating in the formation of a multicellular structure called a fruiting body filled with dormant, environmentally resistant spores. This review discusses how myxobacteria use extracellular signals to sequentially check the density and arrangement of cells at different stages during development. For at least one early and one late developmental signal, cell density determines the efficiency of intercellular signaling. In turn, proper signaling insures that the appropriate cell density exists, thus controlling the progress of multicellular development in M. xanthus.

Colony Count, Microbial↗
PubMed1999

Intercellular signaling during fruiting-body development of Myxococcus xanthus.

The myxobacterium Myxococcus xanthus has a life cycle that is dominated by social behavior. During vegetative growth, cells prey on other bacteria in large groups that have been likened to wolf packs. When faced with starvation, cells form a macroscopic fruiting body containing thousands of spores. The social systems that guide fruiting body development have been examined through the isolation of conditional developmental mutants that can be stimulated to develop in the presence of wild-type cells. Extracellular complementation is due to the transfer of soluble and cell contact-dependent intercellular signals. This review describes the current state of knowledge concerning cell-cell signaling during development.

Gene Expression Regulation, Bacterial↗
PubMed2003

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.

Bacterial Proteins↗
PubMed2003

Analysis of fruE, a novel developmental gene of Myxococcus xanthus.

Myxococcus xanthus is a gram-negative soil bacterium that undergoes multicellular development upon nutrient starvation. In the present study, a TnV insertion developmental mutation, Omega773, of M. xanthus was analyzed. The TnV Omega773 insertion was found to be located within a novel developmental gene, fruE. The FruE protein is composed of 140 amino acid residues and bears an N-terminal signal peptide. The amino acid sequence of FruE shared no significant similarity with any other known protein in the databases. The fruE mutant displayed a development-delayed phenotype. The formation of tightly aggregated mounds in the fruE mutant was slower than that in the wild-type strain. The initiation of spore production in the fruE mutant was delayed by 12 h in comparison to the wild-type strain, and the process of spore formation was more asynchronous than that of the wild-type strain. The transcription initiation sites of the fruE gene were located 81 bp (P1) and 57 bp (P2) upstream of the fruE initiation codon. Although both promoters were active during vegetative growth and development, the P1 promoter was more active during development and the P2 promoter was more active during vegetative growth. The expression of the fruE gene increased to a peak at 6 h poststarvation and then decreased. The decrease in fruE expression was not observed in the D and E signal mutants.

Amino Acid Sequence↗
PubMed2005

Factors that modulate the Pkn4 kinase cascade in Myxococcus xanthus.

Myxococcus xanthus, a gram-negative developmental bacterium, contains a large number of protein Ser/Thr kinases (PSTKs). Among these PSTKs, Pkn4 is shown to be 6-phosphofructokinase (PFK) kinase. PFK associates with the regulatory domain of Pkn4 (Pkn4RD) and is activated 2.7-fold upon phosphorylation at Thr-226 by Pkn4. The activation of PFK is required to consume glycogen accumulated during early development and is essential for efficient sporulation. Three new factors, MkapA, MkapB and MkapC have been identified that associate with Pkn4 by the yeast two-hybrid screen and each contains well-known protein-protein interaction domains. MkapB interacts with Pkn4 in a phosphorylation-dependent manner and remains associated with Pkn4 after its phosphorylation. Binding of MkapB to Pkn4 prevents the interaction of Pkn4 with PFK and consequently PFK phosphorylation and activation. A pfk-pkn4 deletion mutant accumulates glycogen at a rate two folds higher than the parent strain, DZF1, at the stationary phase and early development stage, it is unable to consume glycogen during development and produces only 3.4% of the DZF1 spore yield. In contrast, an mkapB deletion mutant exhibits a 24 h delay in fruiting body formation, accumulates less glycogen in the stationary phase and gives rise to 6.4% of the DZF1 spore yield. In addition to Pkn4, MkapA associates with other membrane-associated PSTKs, Pkn1, Pkn2, Pkn8 and Pkn9, while MkapB associates with Pkn8 and Pkn9, and MkapC with Pkn8. These results indicate that there are complex PSTK networks in M. xanthus sharing common modulating factors.

Bacterial Proteins↗
PubMed2004

The high-mobility group A-type protein CarD of the bacterium Myxococcus xanthus as a transcription factor for several distinct vegetative genes.

CarD is the only reported prokaryotic protein showing structural and functional features typical of eukaryotic high-mobility group A transcription factors. In prokaryotes, proteins similar to CarD appear to be confined primarily to myxobacteria. In Myxococcus xanthus, CarD has been previously shown to act as a positive element in two different regulatory networks: one for light-induced synthesis of carotenoids and the other for starvation-induced fruiting body formation. We have now tested the effect of a loss-of-function mutation in the carD gene (carD1) on the expression of a random collection of lacZ-tagged genes, which are normally expressed in the dark during vegetative growth in rich medium. Our results indicate that CarD plays a significant role in the transcriptional regulation of various indicated genes. The carD1 mutation downregulates some genes and upregulates others. Also reported here is the isolation of several mutations that suppress the strong effect of carD1 on the expression of a particular vegetative gene. One of them (sud-2) also suppresses the effect of carD1 on other vegetative genes and on fruiting-body formation. Thus, CarD and the sud-2 gene product appear to participate in a single mechanism, which underlies various apparently diverse regulatory phenomena ascribed to CarD.

Amino Acid Sequence↗
PubMed2005

Transposon insertions of magellan-4 that impair social gliding motility in Myxococcus xanthus.

Myxococcus xanthus has two different mechanisms of motility, adventurous (A) motility, which permits individual cells to glide over solid surfaces, and social (S) motility, which permits groups of cells to glide. To identify the genes involved in S-gliding motility, we mutagenized a delta aglU (A-) strain with the defective transposon, magellan-4, and screened for S- mutants that form nonmotile colonies. Sequence analysis of the sites of the magellan-4 insertions in these mutants and the alignment of these sites with the M. xanthus genome sequence show that two-thirds of these insertions lie within 27 of the 37 nonessential genes known to be required for social motility, including those necessary for the biogenesis of type IV pili, exopolysaccharide, and lipopolysaccharide. The remaining insertions also identify 31 new, nonessential genes predicted to encode both structural and regulatory determinants of S motility. These include three tetratricopeptide repeat proteins, several regulators of transcription that may control the expression of genes involved in pilus extension and retraction, and additional enzymes involved in polysaccharide metabolism. Three insertions that abolish S motility lie within genes predicted to encode glycolytic enzymes, suggesting that the signal for pilus retraction may be a simple product of exopolysaccharide catabolism.

Bacterial Proteins↗
PubMed1993

Rhizopodin, a new compound from Myxococcus stipitatus (myxobacteria) causes formation of rhizopodia-like structures in animal cell cultures. Production, isolation, physico-chemical and biological properties.

A new cytostatic compound, rhizopodin, was isolated from the culture broth of the myxobacterium, Myxococcus stipitatus. The compound inhibited growth of various animal cell cultures without killing the cells. The ID50, measured by an MTT assay, was 12 approximately 30 ng/ml, depending on the cell line. Especially cells growing fibroblast-like showed typical morphological changes. They became larger and within hour formed long branching and reticular runners. These morphological changes were irreversible. Rhizopodin suppresses bleb formation in K-562 cells, and therefore could act by interacting with protein phosphorylation.

Animals↗
PubMed2000

The stringent response in Myxococcus xanthus is regulated by SocE and the CsgA C-signaling protein.

Myxococcus xanthus fruiting body development is induced by amino acid limitation. The decision to grow or develop is established by the RelA-dependent stringent response and A-signaling. We identified two new members of this regulatory hierarchy, socE and the C-signaling gene csgA. SocE depletion arrests growth and induces sporulation under conditions that normally favor growth as well as curtailing DNA and stable RNA synthesis, inhibiting cell elongation, and inducing accumulations of the stringent nucleotides ppGpp and pppGpp [(p)ppGpp]. This system separates C-signaling, which does not occur under these conditions, from CsgA enzyme activity. Amino acid substitutions in the CsgA coenzyme binding pocket or catalytic site eliminate growth arrest. relA mutation also eliminates growth arrest. Eleven pseudorevertants selected for growth following SocE depletion contained mutations in csgA or relA. These results suggest that CsgA induces the stringent response and while SocE inhibits it. Unlike the csgA mutant, wild-type and socE csgA cells maintained high levels of (p)ppGpp throughout development. We suggest that CsgA maintains growth arrest throughout development to divert carbon from A-signaling and other sources into developmental macromolecular synthesis.

Amino Acids↗
PubMed2000

Identification of a substrate for Pkn2, a protein Ser/Thr kinase from Myxococcus xanthus by a novel method for substrate identification.

Eukaryotic cells contain a large number of protein Ser/ Thr kinases, which play important roles in signal transduction required for cell proliferation, differentiation, and stress response and adaptation. It is also known that some prokaryotes contain a family of protein Ser/Thr kinases. A major challenge in the characterization of these kinases is how to identify their specific substrates. Here we developed such a method using a protein Ser/Thr kinase, Pkn2 from Myxococcus xanthus, a Gram-negative soil bacterium. When Pkn2 is inducibly expressed in E. coli, cells are unable to form colonies on agar plates. This lethal effect of Pkn2 was eliminated in an inactive Pkn2 mutant in which the highly conserved Lys residue was changed to Asn, indicating that phosphorylation of a cellular protein(s) in E. coli resulted in growth arrest. Several clones from an E. coli genomic library were found to suppress the lethal effect when co-expressed with pkn2. Four out of seven multi-copy suppressors were identified to encode HU, (3 for HUalpha and 1 for HUB) a histone-like DNA binding protein. Purified HUalpha was found to be specifically phosphorylated by Pkn2 at Thr-59, and the phosphorylated HUalpha became unable to bind to DNA, suggesting that the phosphorylation of endogenous HU proteins by Pkn2 contributed at least in part to the lethal effect in E. coli. The present method termed the STEK method (Suppressors of Toxic Effects of Kinases) may be widely used for the substrate identification not only for prokaryotic protein Ser/Thr kinases but also for eukaryotic kinases.

Bacterial Proteins↗
PubMed2001

SigB, SigC, and SigE from Myxococcus xanthus homologous to sigma32 are not required for heat shock response but for multicellular differentiation.

Myxococcus xanthus has been known to have multiple sigma factors which are considered to play important roles in regulation of gene expression in development. A new gene encoding a putative sigma factor, sigE, was cloned by using a degenerate oligonucleotide corresponding to the conserved region 2.2 of M. xanthus SigA. In the 2.0-kb nucleotide sequence, an open reading frame consisting of 280 amino acid residues was identified. The amino acid sequence of SigE shows high similarity to heat shock sigma factors in bacteria. However, the sigE gene is not induced by heat shock and deletion of sigE does not affect production of heat shock proteins. SigE is expressed during both vegetative growth and fruiting body development. In the deletion mutant of the sigE gene fruiting body formation is initiated earlier and fewer spores are produced than in the parent strain. Interestingly, the deltasigE mutant shows defects in fruiting body formation at 37 degrees C. In addition to SigE, SigB and SigC show high sequence similarity to heat shock sigma factors. However, even if all three sigma factor genes are disrupted, heat shock proteins are still normally induced. A deltasigBdeltasigCdeltasigE triple deletion strain forms fruiting bodies earlier, but sporulats later than the parent strain. Spores from the triple deletion mutant are aberrant and their viability is less than 0.001% compared with that of the parent strain, suggesting that these sigma factors may have redundant functions in multicellular differentiation of M. xanthus.

Amino Acid Sequence↗
PubMed2002

[Isolation and disruption analysis of a developmental gene of Myxococcus xanthus].

FruB is an associated protein of FruA, a transcription factor essential for the development of Myxococcus xanthus. Degenerate oligonucleotide primers were designed based on the N-terminal amino acid sequence of FruB. Using genomic DNA as template, an approximate 110 bp fragment was generated and further served as a probe to screen a small genomic library of M. xanthus. A 4.5 kb SacI fragment was isolated on the basis of its homology to the probe. Disruption of fruB delayed the morphogenesis of fruiting bodies and depressed spore yield, suggesting that to some extent, FruB might be involved in the development of M. xanthus.

Amino Acid Sequence↗