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PubMed2002

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.

Amino Acid Sequence↗
PubMed2004

Operator design and mechanism for CarA repressor-mediated down-regulation of the photoinducible carB operon in Myxococcus xanthus.

The carB operon encodes all except one of the enzymes involved in light-induced carotenogenesis in Myxococcus xanthus. Expression of its promoter (P(B)) is repressed in the dark by sequence-specific DNA binding of CarA to a palindrome (pI) located between positions -47 and -64 relative to the transcription start site. This promotes subsequent binding of CarA to additional sites that remain to be defined. CarS, produced in the light, interacts physically with CarA, abrogates CarA-DNA binding, and thereby derepresses P(B). In this study, we delineate the operator design that exists for CarA by precisely mapping out the second operator element. For this, we examined how stepwise deletions and site-directed mutagenesis in the region between the palindrome and the transcription start site affect CarA binding around P(B) in vitro and expression of P(B) in vivo. These revealed the second operator element to be an imperfect interrupted palindrome (pII) spanning positions -26 to -40. In vitro assays using purified M. xanthus RNA polymerase showed that CarA abolishes P(B)-RNA polymerase binding and runoff transcription and that both were restored by CarS, thus rationalizing the observations in vivo. CarA binding to pII (after association with pI) effectively occludes RNA polymerase from P(B) and so provides the operative mechanism for the repression of the carB operon by CarA. The bipartite operator design, whereby transcription is blocked by the low affinity CarA-pII binding and is readily restored by CarS, may have evolved to match the needs for a rapid and an effective response to light.

Bacterial Proteins↗
PubMed2004

The N terminus of Myxococcus xanthus CarA repressor is an autonomously folding domain that mediates physical and functional interactions with both operator DNA and antirepressor protein.

Expression of the Myxococcus xanthus carB operon, which encodes the majority of the enzymes involved in light-induced carotenogenesis, is down-regulated in the dark by the CarA repressor binding to its bipartite operator. CarS, produced on illumination, relieves repression of carB by physically interacting with CarA to dis-mantle CarA-DNA complexes. Here, we demonstrate that the N- and C-terminal portions of CarA are organized as distinct structural and functional domains. Specifically, we show that the 78 N-terminal residues of CarA, CarA(Nter), form a monomeric, highly helical, autonomously folding unit with significant structural stability. Significantly, CarA(Nter) houses both the operator and CarS binding specificity determinants of CarA. CarA(Nter) binds operator with a lower affinity than whole CarA, and the CarA(Nter)-CarS complex has a 1:1 stoichiometry. In vitro, sufficiently high concentrations of CarA(Nter) block M. xanthus RNA polymerase-promoter binding, and this is relieved by CarS. In vivo, substitution of the gene carA by that for CarA(Nter) results in constitutive expression of carB just as in a carA-deleted background. However, re-engineering the latter strain to overexpress CarA(Nter) restores repression of carB. Thus, the 78-residue N-terminal portion of CarA is an autonomously folded, dual function domain that orchestrates specific DNA-protein and protein-protein interactions and, when overexpressed, can be functionally competent in vivo.

Amino Acid Sequence↗
PubMed2005

Identification of a gene involved in polysaccharide export as a transcription target of FruA, an essential factor for Myxococcus xanthus development.

Fruiting body development in Myxococcus xanthus is a multicellular event that is coordinated by exchanging intercellular signals. FruA is a transcription factor essential for fruiting body development and is thought to play a key role in the C-signal pathway. Here we present the first identification of a gene regulated by FruA. The gene was isolated from a genomic library via in vitro selection in a DNA binding assay by using the DNA-binding domain of FruA tagged with His(8) at the C-terminal end (FruA-DBD-H(8)). The gene, named fdgA (FruA-dependent gene A), encodes a protein homologous to the outer-membrane auxiliary family protein involved in the polysaccharide export system. FruA-DBD-H(8) bound the upstream promoter region of the fdgA gene from nucleotide -89 to nucleotide -64 with respect to the transcription initiation site, which was required for the induction of fdgA expression during development. fdgA mRNA induced during development was absent in a fruA deletion strain. The deletion of fdgA resulted in defective fruiting body formation and reduced sporulation efficiency (1% that of the parent strain). Moreover, FruA was required for the developmental expression of sasA, which is also involved in the biosynthesis of the lipopolysaccharide O-antigen and is required for fruiting body development. Furthermore, the expression of both fdgA and sasA was partially dependent on the C-signal. These findings expand our understanding of the signal transduction pathway mediated by FruA during development in M. xanthus.

Amino Acid Sequence↗
PubMed2006

Crystal structure of protoporphyrinogen oxidase from Myxococcus xanthus and its complex with the inhibitor acifluorfen.

Protoporphyrinogen IX oxidase, a monotopic membrane protein, which catalyzes the oxidation of protoporphyrinogen IX to protoporphyrin IX in the heme/chlorophyll biosynthetic pathway, is distributed widely throughout nature. Here we present the structure of protoporphyrinogen IX oxidase from Myxococcus xanthus, an enzyme with similar catalytic properties to human protoporphyrinogen IX oxidase that also binds the common plant herbicide, acifluorfen. In the native structure, the planar porphyrinogen substrate is mimicked by a Tween 20 molecule, tracing three sides of the macrocycle. In contrast, acifluorfen does not mimic the planarity of the substrate but is accommodated by the shape of the binding pocket and held in place by electrostatic and aromatic interactions. A hydrophobic patch surrounded by positively charged residues suggests the position of the membrane anchor, differing from the one proposed for the tobacco mitochondrial protoporphyrinogen oxidase. Interestingly, there is a discrepancy between the dimerization state of the protein in solution and in the crystal. Conserved structural features are discussed in relation to a number of South African variegate porphyria-causing mutations in the human enzyme.

Crystallography, X-Ray↗
PubMed2006

Unidirectional movement of flares of cells of Myxococcus xanthus.

Amongst other modes, Myxococcal cells move in swarms that are flares or columns of cells. It has been argued that this is a strategy allowing a large enough number of them to encounter food bacteria. Then, the combined large amount of extracellular lytic enzymes from the mass of cells can provide adequate nutrient resources from the food bacteria for all the myxococci of the swarm. However, how they move as a coherent column has not been adequately explained. Here based on the idea that a rare cell can experience a special mutation such that it moves only unidirectionally, a proposal to account for this aspect of Myxococcus cell movement is suggested. Although wild type individual organisms of this species engage in forward and back movements, a mutant cell that moves unidirectionally can bias the movement of associated wild type cells and lead to the formation of a column of cells, headed by such a unique mutated cell. The non-mutated cells follow along it is suggested because of the S-motility (or social motility) system. This may link them to this single unidirectionally moving mutant cell to give a coherent movement to the column. This proposed type of mutation back mutates to wild type and the column no longer functions as such and only wild-type cells are present.

Mutation↗
PubMed2000

A large family of eukaryotic-like protein Ser/Thr kinases of Myxococcus xanthus, a developmental bacterium.

Myxococcus xanthus is a gram-negative bacterium that forms multicellular fruiting bodies upon starvation. Here, we demonstrate that it contains at least 13 eukaryotic-like protein Ser/Thr kinases (Pkn1 to Pkn13) individually having unique features. All contain the kinase domain of approximately 280 residues near the N-terminal end, which share highly conserved features in eukaryotic Ser/Thr kinases. The kinase domain is followed by a putative regulatory domain consisting of 185 to 692 residues. These regulatory domains share no significant sequence similarities. The C-terminal regions of 11 kinases contain at least 1 transmembrane domain, suggesting that they function as transmembrane sensor kinases. From the recent genomic analysis, protein Ser/Thr kinases were found in various pathogenic bacteria and coexist with protein His kinases. Phylogenetic analysis of these Ser/Thr kinases reveals that all bacterial Ser/Thr kinases were evolved from a common ancestral kinase together with eukaryotic Tyr and Ser/Thr kinases. Coexistence of both Ser/Thr and His kinases in some organisms may be significant in terms of functional differences between the two kinases. We argue that both kinases are essential for some bacteria to adapt optimally to severe environmental changes.

Amino Acid Sequence↗
PubMed2001

Characterization of the mac-1 gene encoding a putative ABC transporter from Myxococcus xanthus.

The mac-1 gene of Myxococcus xanthus TA, an antibiotic TA producer, encoded a protein with strong sequence similarity to the antibiotic ATP-binding cassette (ABC) transporter for macrolide antibiotics. The mac-1 gene encoding protein (Mac-1) had two ATP-binding domains containing Walker A and B motifs, and no hydrophobic transmembrane regions. Insertional inactivation of mac-1 caused enhanced sensitivity to oleandomycin, a macrolide antibiotic, while the mac-1 mutant showed normal export of antibiotic TA into the extracellular fluid. The mac-1 mutant could form mounds, but was unable to form fruiting bodies or sporulate under nutrient starvation. A primary role for Mac-1 in M. xanthus may be as a transporter which exports or imports a molecule required for the sporulation process.

ATP-Binding Cassette Transporters↗
PubMed2003

Competitive fates of bacterial social parasites: persistence and self-induced extinction of Myxococcus xanthus cheaters.

Cooperative biological systems are susceptible to disruption by cheating. Using the social bacterium Myxococcus xanthus, we have tested the short-term competitive fates of mixed cheater and wild-type strains over multiple cycles of cooperative development. Cheater/wild-type mixes underwent several cycles of starvation-induced multicellular development followed by spore germination and vegetative population growth. The population sizes of cheater and wild-type strains in each pairwise mixture were measured at the end of each developmental phase and each growth phase. Cheater genotypes showed several distinct competitive fates, including cheater persistence at high frequencies with little effect on total population dynamics, cheater persistence after major disruption of total population dynamics, self-extinction of cheaters with wild-type survival, and total population extinction. Our results empirically demonstrate that social exploitation can destabilize a cooperative biological system and increase the risk of local extinction events.

Biological Evolution↗
PubMed1996

Two multifunctional peptide synthetases and an O-methyltransferase are involved in the biosynthesis of the DNA-binding antibiotic and antitumour agent saframycin Mx1 from Myxococcus xanthus.

Saframycin Mx1 is a DNA-binding antibiotic and antitumour agent produced by Myxococcus xanthus. It is a heterocyclic quinone, thought to be synthesized via the linear peptide intermediate AlaGlyTyrTyr. Analysis of 14.1 kb DNA sequence involved in saframycin production revealed genes for two large multifunctional peptide synthetases of 1770 and 2605 amino acids, respectively, and a putative O-methyltransferase of 220 amino acids. The three ORFs read in the same direction and are separated by short non-translated gaps of 44 and 49 bp. The peptide synthetases contain two amino-acid-activating domains each. The first domain lacks two of the most conserved 'core' sequences, and the last domain is followed by a putative reductase functionality, not previously seen in peptide synthetases. Complementation tests showed that antibiotic-non-producing mutant strains lacking one of the peptide synthetases secrete a substrate, presumably a modified amino acid precursor, that can be used by O-methyltransferase-deficient mutant strains to synthesize saframycin Mx1.

Amino Acid Sequence↗
PubMed1997

A mutation that affects fibril protein, development, cohesion and gene expression in Myxococcus xanthus.

Extracellular matrix fibrils are involved in the cell-cell interactions of the social prokaryote, Myxococcus xanthus. The fibrils are composed of a carbohydrate backbone and a set of five integral fibrillar proteins (IFPs) ranging from 14 to 66 kDa. As part of an attempt to understand the function(s) of the IFPs, a mutant (ifp-1:20) was generated that lacks IFP-1:20, one of the fibril proteins, as shown by Western blot analysis of both whole cells and isolated fibrils. Unlike those of the parent strain, the fibrils of the mutant were removed easily from the cells by shear forces. Development in ifp-1:20 was aberrant--aggregation and early mound formation were delayed by 6-10 h and mature fruiting bodies never formed. Myxospore production was also greatly reduced. Additionally, fibril-mediated cohesion in ifp-1:20 was changed. Cohesion resulted in chains of cells rather than the characteristic clumps of cells seen for the parent strain. Isolated ifp-1:20 fibrils, unlike wild-type fibrils, could not rescue cohesion of non-cohesive, fibril-negative dsp cells, supporting the notion that the fibrils were functionally altered. The mutation also reduced developmental gene expression by three- to fourfold in omega 4521, a transposon insertion mutant expressed early in development. Expression of a later developmental gene fusion was not affected, suggesting that the fibrils may not be required for later developmental gene expression. These data suggest that intact fibrils may function early in development to facilitate close cell proximity for signal exchange.

Bacterial Adhesion↗
PubMed1998

Molecular analysis of the DNA gyrB gene from Myxococcus xanthus.

DNA gyrase, an essential type II topoisomerase, mediates negative supercoiling of the bacterial chromosome, thereby affecting the processes of DNA replication, transcription, recombination and repair. The gyrB gene from the Gram-negative soil bacterium Myxococcus xanthus was sequenced. The sequence predicts a protein of 815 amino acid residues displaying significant homology to all known GyrB proteins. A 6-His-GyrB fusion protein was overexpressed in Escherichia coli and purified to near homogeneity using affinity chromatography on Ni-nitrilotriacetic acid-agarose and novobiocin-Sepharose columns. The fusion protein bound novobiocin and cross-reacted with anti-E. coli GyrB antibodies, indicating structural and functional similarities to the E. coli DNA GyrB. The gene was mapped to the region of the origin of replication (oriC) of M. xanthus.

Amino Acid Sequence↗
PubMed1999

Genetic and functional analysis of genes required for the post-modification of the polyketide antibiotic TA of Myxococcus xanthus.

The antibiotic TA of Myxococcus xanthus is a complex macrocyclic polyketide, produced through successive condensations of acetate by a type I PKS (polyketide synthase) mechanism. The genes encoding TA biosynthesis are clustered on a 36 kb DNA fragment, which has been cloned and analysed. The chemical structure of TA and the mechanism by which it is synthesized indicate the need for several post-modification steps, which are introduced into the carbon chain of the polyketide to form the final bioactive molecule. These include the addition of several carbon atoms originating from acetate carbonyl, three C-methylations, O-methylation and a specific hydroxylation. This paper reports the analysis of five genes which are involved in the post-modification of TA. Their functional analysis, by specific gene disruption, suggests that they may be essential for the production of the active antibiotic. The characteristics and organization of the genes suggest that they may be involved in the addition of the carbon atoms which arise from acetate.

Amino Acid Sequence↗
PubMed1994

Endogenous ADP-ribosylation during development of the prokaryote Myxococcus xanthus.

We examined endogenous ADP-ribosylation of proteins during the development of the prokaryote Myxococcus xanthus. In vivo and in vitro endogenous ADP-ribosylation of M. xanthus proteins was detected and the profile of modified proteins changed during development. Adenosine and nicotinamide inhibited ADP-ribosylation. Nicotinamide stimulated cells at low density to develop, in a manner similar to that previously observed with adenosine. Higher concentrations of nicotinamide inhibited aggregation. The in vivo effects of nicotinamide on developing M. xanthus cells correlate with its in vitro effects on ADP-ribosylation and the developmental profile of putative ADP-ribosylation substrates. These results suggest that ADP-ribosylation may regulate developmental proteins in M. xanthus.

Adenosine↗
PubMed1995

Starvation yields a drastic decrease in outer-membrane permeability to a periplasmic foreign protein in Myxococcus xanthus.

A recombinant Myxococcus xanthus strain was constructed that constitutively produces two proteins from Escherichia coli, the cytoplasmic beta-galactosidase and the periplasmic pH 2.5 acid phosphatase (AppA protein). We have previously shown that during vegetative growth, AppA protein is partly accumulated in the periplasm of M. xanthus and partly released into the medium. We demonstrate here that during starvation-induced development, release of periplasmic AppA protein to the medium did not occur over a period of 20 h. This was coincident with, but not caused by, the arrest of the synthesis of the foreign proteins. We have shown that this lack of secretion could be attributed to starvation per se and did not depend on the ability of the cells to undergo development. Our findings suggest that protein secretion which occurs during the first hours of starvation-induced development might therefore take place via a different route from that which occurs in vegetative cells.

Acid Phosphatase↗
PubMed1995

A new Myxococcus xanthus gene cluster for the biosynthesis of the antibiotic saframycin Mx1 encoding a peptide synthetase.

The gene cluster for the biosynthesis of the heterocyclic quinone antibiotic saframycin Mx1 of Myxococcus xanthus DM504/15 was inactivated and tagged by Tn5 insertions. The tagged genes were cloned in Escherichia coli and used to select overlapping cosmid clones spanning 58 kb of the M. xanthus genome. Gene disruption experiments defined a > or = 18 kb contiguous DNA region involved in saframycin biosynthesis. Sequencing of part of this region revealed a large ORF containing two 600-amino-acid domains with similarity to peptide synthetase amino-acid-activating sequences, suggesting that saframycin Mx1 is synthesized by a nonribosomal multienzyme complex, similar to other bioactive peptides.

Amino Acid Sequence↗
PubMed1999

Genetic suppression analysis of an asgA missense mutation in Myxococcus xanthus.

The asgA gene is required for generation of extracellular A signal, which serves as a cell-density signal for fruiting body development in Myxococcus xanthus. The AsgA protein is a histidine protein kinase and consists of a receiver domain that is conserved among response regulators of two-component signal transduction systems, followed by a histidine protein kinase domain that is conserved among sensor proteins of two-component systems. AsgA is thought to function in a signal transduction pathway that leads to expression of genes required for A-signal generation. A genetic suppression analysis of an asgA missense mutation was undertaken in order to identify genes that may provide information regarding the role of AsgA in A-signal generation and fruiting body formation. Twenty-two independent strains containing mutations that suppress asgA473 were isolated by selecting for production of heat-resistant spores under conditions that promote fruiting body development in wild-type cells. Ten of the 22 suppressor strains contained bypass suppressors. All the suppressor strains had direct spore counts at least three to four times greater than the original asgA473 mutant, and three strains had viable counts that exceeded wild-type by more than one order of magnitude. Surprisingly, none of the suppressor strains produced wild-type levels of extracellular A-signal.

Bacterial Proteins↗
PubMed2006

Byssovorax cruenta gen. nov., sp. nov., nom. rev., a cellulose-degrading myxobacterium: rediscovery of 'Myxococcus cruentus' Thaxter 1897.

A rare, cellulose-decomposing myxobacterium is described, and a new genus name, Byssovorax, is proposed for it. The organism is almost certainly identical to the species 'Myxococcus cruentus' Thaxter 1897, and that species epithet is therefore revived for the novel bacterium: the type strain of Byssovorax cruenta gen. nov., sp. nov., nom. rev. is strain By c2(T) (=DSM 14553(T)=CIP 108850(T)). The G+C content of its DNA is 69.9 mol%. The 16S rRNA gene sequence shows that the species belongs to the family Polyangiaceae, suborder 'Sorangineae', of the Myxococcales. An emended description of the organism is given.

Base Composition↗