GidA is an FAD-binding protein involved in development of Myxococcus xanthus.

A gene encoding a homologue of the Escherichia coli GidA protein (glucose-inhibited division protein A) lies immediately upstream of aglU, a gene encoding a WD-repeat protein required for motility and development in Myxococcus xanthus. The GidA protein of M. xanthus shares about 48% identity overall with the small (approximately equal to 450 amino acid) form of GidA from eubacteria and about 24% identity overall with the large (approximately equal to 620 amino acid) form of GidA from eubacteria and eukaryotes. Each of these proteins has a conserved dinucleotide-binding motif at the N-terminus. To determine if GidA binds dinucleotide, the M. xanthus gene was expressed with a His6 tag in E. coli cells. Purified rGidA is a yellow protein that absorbs maximally at 374 and 450 nm, consistent with FAD or FMN. Thin-layer chromatography (TLC) showed that rGidA contains an FAD cofactor. Fractionation and immunocytochemical localization show that full length GidA protein is present in the cytoplasm and transported to the periplasm of vegetative-grown M. xanthus cells. In cells that have been starved for nutrients, GidA is found in the cytoplasm. Although GidA lacks an obvious signal sequence, it contains a twin arginine transport (Tat) motif, which is conserved among proteins that bind cofactors in the cytoplasm and are transported to the periplasm as folded proteins. To determine if GidA, like AglU, is involved in motility and development, the gidA gene was disrupted. The gidA- mutant has wild-type gliding motility and initially is able to form fruiting bodies like the wild type when starved for nutrients. However, after several generations, a stable derivative arises, gidA*, which is indistinguishable from the gidA- parent on vegetative medium, but is no longer able to form fruiting bodies. The gidA* mutant releases a heat-stable, protease-resistant, small molecular weight molecule that acts in trans to inhibit aggregation and gene expression of wild-type cells during development.

H(2)O(2)-forming NADH oxidase with diaphorase (cytochrome) activity from Archaeoglobus fulgidus.

An enzyme exhibiting NADH oxidase (diaphorase) activity was isolated from the hyperthermophilic sulfate-reducing anaerobe Archaeoglobus fulgidus. N-terminal sequence of the protein indicates that it is coded for by open reading frame AF0395 in the A. fulgidus genome. The gene AF0395 was cloned and its product was purified from Escherichia coli. Like the native NADH oxidase (NoxA2), the recombinant NoxA2 (rNoxA2) has an apparent molecular mass of 47 kDa, requires flavin adenine dinucleotide for activity, has NADH-specific activity, and is thermostable. Hydrogen peroxide is the product of bivalent oxygen reduction by rNoxA2 with NADH. The rNoxA2 is an oxidase with diaphorase activity in the presence of electron acceptors such as tetrazolium and cytochrome c. During purification NoxA2 remains associated with the enzyme responsible for D-lactate oxidation, the D-lactate dehydrogenase (Dld), and the genes encoding NoxA2 and Dld are in the same transcription unit. Together these results suggest that NADH oxidase may be involved in electron transfer reactions resulting in sulfate respiration.

AglU, a protein required for gliding motility and spore maturation of Myxococcus xanthus, is related to WD-repeat proteins.

The aglU gene of Myxococcus xanthus encodes a protein similar to Het-E1 (vegetative incompatibility) from Podospora anserina, acylaminoacyl-peptidase from Bacillus subtilis, and TolB from Escherichia coli. These proteins all have evenly spaced SPDG repeats that are characteristic of a larger motif called the WD-repeat. The WD-repeat is predicted to form a beta-propeller structure that mediates the assembly of heteromeric protein complexes. AglU has a consensus lipoprotein attachment motif that includes a type II signal sequence followed by a cysteine residue. This suggests that AglU is matured, then attached to the outer membrane via fatty acid acylation at this Cys. Cells carrying a mutation in aglU are blocked in adventurous gliding and can swarm only if cells are in contact with one another. When starved of nutrients, the aglU mutant aggregates and forms multicellular fruiting bodies like the wild-type strain, but is unable to produce heat-resistant spores. This suggests that adventurous gliding motility, per se, is not required for development, but that AglU is essential for a terminal step of spore differentiation.

The Archaeoglobus fulgidus D-lactate dehydrogenase is a Zn(2+) flavoprotein.

Archaeoglobus fulgidus, a hyperthermophilic, archaeal sulfate reducer, is one of the few organisms that can utilize D-lactate as a sole source for both carbon and electrons. The A. fulgidus open reading frame, AF0394, which is predicted to encode a D-(-)-lactate dehydrogenase (Dld), was cloned, and its product was expressed in Escherichia coli as a fusion with the maltose binding protein (MBP). The 90-kDa MBP-Dld fusion protein was more efficiently expressed in E. coli when coexpressed with the E. coli dnaY gene, encoding the arginyl tRNA for the codons AGA and AGG. When cleaved from the fusion protein by treatment with factor Xa, the recombinant Dld (rDld) has an apparent molecular mass of 50 kDa, similar to that of the native A. fulgidus Dld enzyme. Both the purified MBP-Dld fusion protein and its rDld cleavage fragment have lactate dehydrogenase activities specific for D-lactate, are stable at 80 degrees C, and retain activity after exposure to oxygen. The flavin cofactor FAD, which binds rDld apoprotein with a 1:1 stoichiometry, is essential for activity.

The aadA gene of plasmid R100 confers resistance to spectinomycin and streptomycin in Myxococcus xanthus.

Plasmids with the aadA gene from plasmid R100, which confers resistance to the aminoglycosides spectinomycin and streptomycin in Escherchia coli, can be introduced into wild-type Myxococcus xanthus, strain DK1622, by electroporation. Recombinant M. xanthus strains with integrated plasmids carrying the aadA gene acquire resistance to high levels of these antibiotics. Selection for aadA in M. xanthus can be carried out independently of, or simultaneously with, selection for resistance to kanamycin. The kinds and frequencies of recombination events observed between integrative plasmids with aadA and the M. xanthus chromosome are similar to those observed after the transformation of yeast. Cleavage of integrative plasmid DNA at a site adjacent to a region of homology between the plasmid and the M. xanthus genome favors the targeted disruption of M. xanthus genes by allele replacement.

A chaperone in the HSP70 family controls production of extracellular fibrils in Myxococcus xanthus.

Three independent Tn5-lac insertions in the S1 locus of Myxococcus xanthus inactivate the sglK gene, which is nonessential for growth but required for social motility and multicellular development. The sequence of sglK reveals that it encodes a homologue of the chaperone HSP70 (DnaK). The sglK gene is cotranscribed with the upstream grpS gene, which encodes a GrpE homologue. Unlike sglK, grpS is not required for social motility or development. Wild-type M. xanthus is encased in extracellular polysaccharide filaments associated with the multimeric fibrillin protein. Mutations in sglK inhibit cell cohesion, the binding of Congo red, and the synthesis or secretion of fibrillin, indicating that sglK mutants do not make fibrils. The fibR gene, located immediately upstream of the grpS-sglK operon, encodes a product which is predicted to have a sequence similar to those of the repressors of alginate biosynthesis in Pseudomonas aeruginosa and Pseudomonas putida. Inactivation of fibR leads to the overproduction of fibrillin, suggesting that M. xanthus fibril production and Pseudomonas alginate production are regulated in analogous ways. M. xanthus and Pseudomonas exopolysaccharides may play similar roles in a mechanism of social motility conserved in these gram-negative bacteria.

Acetyl-CoA decarbonylase/synthase complex from Archaeoglobus fulgidus.

The acetyl-CoA decarbonylase/synthase (ACDS) multienzyme complex catalyzes the reversible cleavage and synthesis of acetyl-CoA in methanogens. This report of the enzyme complex in Archaeoglobus fulgidus demonstrates the existence of a functional ACDS complex in an organism that is not a methanogen. The A. fulgidus enzyme complex contained five subunits of 89, 72, 50, 49.5, and 18.5 kDa, and it catalyzed the overall synthesis of acetyl-CoA according to the following reaction: CO2 + 2 Fdred(Fe2+) + 2 H+ + CH3 - H4SPt + CoA <==> acetyl-CoA + H4SPt + 2 Fdox(Fe3+) + H2O where Fd is ferredoxin, and CH3-H4SPt and H4SPt denote N5-methyl-tetrahydrosarcinapterin and tetrahydrosarcinapterin, respectively.

Genetic determinants of immunity and integration of temperate Myxococcus xanthus phage Mx8.

An 8.1-kb fragment of the temperate Myxococcus xanthus phage Mx8 genome, when cloned into a plasmid vector, permits site-specific integration of the plasmid and confers superinfection immunity. Sequence analysis of a 9.5-kb region of Mx8 DNA containing this fragment reveals 19 densely packed open reading frames, four of which have predicted products with known or suspected activities. The Mx8 imm gene, required for superinfection immunity, has a sequence similar to that of Arabidopsis thaliana G-box-binding factor 1. Mx8 makes a DNA adenine methylase, Mox, and integrase, Int, related to other methylases and integrases. The int gene has two alternate translation initiation codons within the extensively overlapping uoi (upstream of int) gene. Comparison of the predicted product of the uoi gene with Salmonella phage P22 and Streptomyces plasmid Xis proteins shows that temperate phage excisionases may use variations of a helix-turn-helix motif to recognize specific DNA sequences.

Complementation of sporulation and motility defects in a prokaryote by a eukaryotic GTPase.

The complex prokaryote, Myxococcus xanthus, undergoes a program of multicellular development when starved for nutrients, culminating in sporulation. M. xanthus makes MglA, a 22-kDa, soluble protein that is required for both multicellular development and gliding motility. MglA is similar in sequence to the Saccharomyces cerevisiae SAR1 protein, a member of the Ras/Rab/Rho superfamily of small eukaryotic GTPases. The SAR1 gene, when integrated into the M. xanthus genome, complements the sporulation defect of a DeltamglA strain. A forward, second-site mutation on the M. xanthus chromosome, rpm, in combination with SAR1, restores fruiting body morphogenesis and gliding motility to a DeltamglA strain. The result that the rpm mutation suppresses the substitution of SAR1 for mglA suggests that Sar1p interacts with other M. xanthus proteins to control the motility-dependent aggregation of cells during development.

Stress-Induced Production of Biofilm in the Hyperthermophile Archaeoglobus fulgidus.

Archaeoglobus fulgidus, an anaerobic marine hyperthermophile, forms a biofilm in response to environmental stresses. The biofilm is a heterogeneous, morphologically variable structure containing protein, polysaccharide, and metals. Production of the biofilm can be induced by nonphysiological extremes of pH and temperature, by high concentrations of metals, and by addition of antibiotics, xenobiotics, or oxygen. Cells within the biofilm show an increased tolerance to otherwise toxic environmental conditions. Metals sequestered within the biofilm stimulate growth of A. fulgidus cells in metal-depleted medium. These data suggest that cells may produce biofilm as a mechanism for concentrating cells and attaching to surfaces, as a protective barrier, and as a reserve nutrient. Because similar biofilms are formed by Archaeoglobus profundus, Methanococcus jannaschii, and Methanobacterium thermoautotrophicum, biofilm formation might be a common stress response mechanism among the archaea.

Temperate Myxococcus xanthus phage Mx8 encodes a DNA adenine methylase, Mox.

Temperate bacteriophage Mx8 of Myxococcus xanthus encapsidates terminally repetitious DNA, packaged as circular permutations of its 49-kbp genome. During both lytic and lysogenic development, Mx8 expresses a nonessential DNA methylase, Mox, which modifies adenine residues in occurrences of XhoI and PstI recognition sites, CTCGAG and CTGCAG, respectively, on both phage DNA and the host chromosome. The mox gene is necessary for methylase activity in vivo, because an amber mutation in the mox gene abolishes activity. The mox gene is the only phage gene required for methylase activity in vivo, because ectopic expression of mox as part of the M. xanthus mglBA operon results in partial methylation of the host chromosome. The predicted amino acid sequence of Mox is related most closely to that of the methylase involved in the cell cycle control of Caulobacter crescentus. We speculate that Mox acts to protect Mx8 phage DNA against restriction upon infection of a subset of natural M. xanthus hosts. One natural isolate of M. xanthus, the lysogenic source of related phage Mx81, produces a restriction endonuclease with the cleavage specificity of endonuclease BstBI.

Genetics of gliding motility and development in Myxococcus xanthus.

Successful development in multicellular eukaryotes requires cell-cell communication and the coordinated spatial and temporal movements of cells. The complex array of networks required to bring eukaryotic development to fruition can be modeled by the development of the simpler prokaryote Myxococcus xanthus. As part of its life cycle, M. xanthus forms multicellular fruiting bodies containing differentiated cells. Analysis of the genes essential for M. xanthus development is possible because strains with mutations that block development can be maintained in the vegetative state. Development in M. xanthus is induced by starvation, and early events in development suggest that signaling stages have evolved to monitor the metabolic state of the developing cell. In the absence of these signals, which include amino acids, alpha-keto acids, and other intermediary metabolites, the ability of cells to differentiate into myxospores is impaired. Mutations that block genes controlling gliding motility disrupt the morphogenesis of fruiting bodies and sporogenesis in surprising ways. In this review, we present data that encourage future genetic and biochemical studies of the relationships between motility, cell-cell signaling, and development in M. xanthus.

Genetic suppression and phenotypic masking of a Myxococcus xanthus frzF- defect.

An insertion of transposon Tn5-lac, omega 4519, generates a lacZ fusion with a Myxococcus xanthus promoter expressed during both vegetative growth and development. Sequence analysis of the junction of omega 4519 with M. xanthus DNA shows that the insertion is in frzF, a homologue of cheR from Salmonella typhimurium. When frzF- (or frzCD-) cells are starved for nutrients at modest densities, they aggregate to form a radial pattern and produce fewer than 1% of the wild-type complement of spores. At higher densities, frzF::omega 4519 cells form 'frizzy' aggregates and produce 80-90% of the wild-type complement of spores. In contrast, when cells with both a frzF- (or frzCD-) and an sglA1 mutation are allowed to develop at either low or high cell densities, they produce frizzy aggregates containing a near wild-type complement of heat-resistant spores. In addition to suppressing the density dependence of fruiting-body morphogenesis, the sglA1 mutation also suppresses the sporulation defect caused by two different frzF- mutations and a frzCD- mutation. In contrast, a mutation in a different S motility gene, sglG1, does not suppress the frz- mutations. Thus, the suppression of frz- mutations by sgl- mutations is allele-specific, and depends on the sgl allele, but not the frz allele. Because the phenotypes of frz- mutations have been determined in a (suppressing) sglA1 genetic background, the frz genes may play more central roles in development than initially recognized.

Genes required for both gliding motility and development in Myxococcus xanthus.

Myxococcus xanthus cells can glide both as individual cells, dependent on Adventurous motility (A motility), and as groups of cells, dependent upon Social motility (S motility). Tn5-lac mutagenesis was used to generate 16 new A- and nine new S- mutations. In contrast with previous results, we find that subsets of A- mutants are defective in fruiting body morphogenesis and/or myxospore differentiation. All S- mutants are defective in fruiting body morphogenesis, consistent with previous results. Whereas some S- mutants produce a wild-type complement of spores, others are defective in the differentiation of myxospores. Therefore, a subset of the A genes and all of the S genes are critical for fruiting body morphogenesis. Subsets of both A and S genes are essential for sporulation. Three S::Tn5-lac insertions result in surprising phenotypes. Colonies of two S- mutants glide on 'swim' (0.35% agar) plates to form fractal patterns. These S- mutants are the first examples of a bacterium in which mutations result in fractal patterns of colonial spreading. An otherwise wild-type strain with one S- insertion resembles the frz- sglA1- mutants upon development, suggesting that this S- gene defines a new chemotaxis component in M. xanthus.

New clusters of genes required for gliding motility in Myxococcus xanthus.

Gliding is the directed movement of cells across surfaces which occurs in the absence of external organelles such as flagella. Gliding of the complex prokaryote, Myxococcus xanthus, results from the action of two independent sets of genes known as the A (adventurous motility) and S (social motility) genes. Strains with mutations in both systems (A-S-) do not spread on agar surfaces because both individual and group movement is abolished. To generate regulated, transcriptional fusions with operons including A and S genes, we introduced TN5-lac into A- and S- strains to obtain non-motile A-S::Tn5-lac and A::Tn5-lac S- double mutants. These insertions identify five separate clusters of A genes and three separate clusters of S genes on the M. xanthus genome. Some Tn5-lac insertions map near two of the five previously identified motility gene clusters, but at least five new clusters were identified in this search. Single mutations at only one locus, mglA, block motility; the mglA locus is epistatic to A and S motility genes. A- and S- Tn5-lac insertions were transduced into mgl+ and delta mgl strains. The levels of beta-galactosidase activity produced from each A- or S- Tn5-lac insertion are similar in otherwise isogenic mgl+ and delta mgl strains, showing that MglA does not regulate the transcription of many A and S genes.

A simplified methylcoenzyme M methylreductase assay with artificial electron donors and different preparations of component C from Methanobacterium thermoautotrophicum delta H.

Different preparations of the methylreductase were tested in a simplified methylcoenzyme M methylreductase assay with artificial electron donors under a nitrogen atmosphere. ATP and Mg2+ stimulated the reaction. Tris(2,2'-bipyridine)ruthenium (II), chromous chloride, chromous acetate, titanium III citrate, 2,8-diaminoacridine, formamidinesulfinic acid, cob(I)alamin (B12s), and dithiothreitol were tested as electron donors; the most effective donor was titanium III citrate. Methylreductase (component C) was prepared by 80% ammonium sulfate precipitation, 70% ammonium sulfate precipitation, phenyl-Sepharose chromatography, Mono Q column chromatography, DEAE-cellulose column chromatography, or tetrahydromethanopterin affinity column chromatography. Methylreductase preparations which were able to catalyze methanogenesis in the simplified reaction mixture contained contaminating proteins. Homogeneous component C obtained from a tetrahydromethanopterin affinity column was not active in the simplified assay but was active in a methylreductase assay that contained additional protein components.

Incorporation of coenzyme M into component C of methylcoenzyme M methylreductase during in vitro methanogenesis.

Reduction of the methyl group of [methyl-3H,thio-35S]2-methylthioethanesulfonic acid to methane by a reconstituted enzyme system resulted in a slow incorporation of [thio-35S]2-mercaptoethanesulfonic acid (HS-CoM) into component C of the methylreductase system. Only 35S label was associated with component C. The ratio of incorporated HS-CoM to component C was 1.96 to 1. The ratio of HS-CoM to factor F430, the nickel-containing cofactor of component C, was 1.18 to 1. Extraction of factor F430 from the protein resulted in the release of 62 +/- 8% of the 35S label, but the label was not covalently bound to F430. The incorporation of label into component C was coupled to methyl group reduction; no label was found associated with component C from a reconstituted reaction containing unlabeled 2-methylthioethanesulfonic acid and [thio-35S]HS-CoM.

Requirement of the nickel tetrapyrrole F430 for in vitro methanogenesis: reconstitution of methylreductase component C from its dissociated subunits.

The subunits and the nickel tetrapyrrole factor F430 of the methylreductase component C from Methanobacterium thermoautotrophicum have been separated from one another and reassociated to form an intact, active enzyme. The individual subunits were extracted from polyacrylamide gels after NaDodSO4/polyacrylamide gel electrophoresis and reassociated with F430 under a H2 atmosphere at 55 degrees C. When these components were reassociated in the presence of the substrate 2-(methylthio)ethanesulfonic acid, 72% of the original activity was recovered. When F430 was omitted, no activity was detected in the reconstituted methylreductase reaction. Individual subunits were inactive, when incubated in the presence of factor F430, 2-(methylthio)ethanesulfonic acid, magnesium acetate, and ATP. Evidence suggests that F430 binds to the large Mr 68,000 subunit of component C.

Immunocytochemical localization of component C of the methylreductase system in Methanococcus voltae and Methanobacterium thermoautotrophicum.

Antibodies were raised against homogeneous preparations of component C of the methylreductase system from Methanococcus voltae and Methanobacterium thermoautotrophicum. Cells of these organisms were fixed with paraformaldehyde and/or glutaraldehyde, sectioned, and labeled with antibodies and colloidal gold-labeled protein A. In M. voltae the gold particles were predominantly located in the vicinity of the cytoplasmic membrane. In rare cases a similar result was obtained also with M. thermoautotrophicum. However, in all but a few of the ultrathin sections of this bacterium, the label was randomly distributed in the cell interior. If one assumes a reliable fixation of all cell components, these results would suggest that the two distantly related methanogens studied have distinctive patterns for the localization of component C. The results with M. voltae are in agreement with recent findings that the methylreductase system is involved in the generation of a proton-motive force at the membrane.