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Additions to the popular evaporation rate determination laboratory of alcohol-based hand-rub sanitizers are described here. Significantly, inquiry-driven protocols for formulating hand-rub sanitizers, falling-ball viscometry, and sanitizer efficacy are included. Incorporation of familiar substances (“molecular relevance”) enables students to assimilate knowledge and connect to and recognize interrelated disciplines. To stimulate engagement and project ownership, students were given the opportunity to alter sanitizer formulations by varying the amount and molecular weight of the gelling agent. Viscosities were determined using a falling-ball viscometer to integrate physics and fluid dynamics concepts into these experiments. Lastly, to stress microbiological applications and determine the efficacy of their hand sanitizers, students tested the formulated hand sanitizers for qualitative efficacy using agar plating of skin secretions and comparing colonies formed from sanitized versus nonsanitized hands. Chemi...

Myxococcus xanthus can vary its phenotype or 'phase' to produce colonies that contain predominantly yellow or tan cells that differ greatly in their abilities to swarm, survive and develop. Yellow variants are proficient at swarming (++) and tend to lyse in liquid during stationary phase. In contrast, tan variants are deficient in swarming (+) and persist beyond stationary phase. The phenotypes and transcriptomes of yellow and tan variants were compared with mutants affected in phase variation. Thirty-seven genes were upregulated specifically in yellow variants including those for production of the yellow pigment, DKxanthene. A mutant in DKxanthene synthesis produced non-pigmented (tan) colonies but still phase varied for swarming suggesting that pigmentation is not the cause of phase variation. Disruption of a gene encoding a HTH-Xre-like regulator, highly expressed in yellow variants, abolished pigment production and blocked the ability of cells to switch from a swarm ++ to a swarm (+) phenotype, showing that HTH-Xre regulates phase variation. Among the four genes whose expression was increased in tan variants was pkn14, which encodes a serine-threonine kinase that regulates programmed cell death in Myxococcus via the MrpC-MazF toxin-antitoxin complex. High levels of phosphorylated Pkn14 may explain why tan cells enjoy enhanced survival.

The bacterium Myxococcus xanthus has two motility systems: S motility, which is powered by type IV pilus retraction, and A motility, which is powered by unknown mechanism(s). We found that A motility involved transient adhesion complexes that remained at fixed positions relative to the substratum as cells moved forward. Complexes assembled at leading cell poles and dispersed at the rear of the cells. When cells reversed direction, the A-motility clusters relocalized to the new leading poles together with S-motility proteins. The Frz chemosensory system coordinated the two motility systems. The dynamics of protein cluster localization suggest that intracellular motors and force transmission by dynamic focal adhesions can power bacterial motility.

Ixotrophy is a process that enables certain microbes to prey on other cells. The ability of cells to aggregate or adhere is thought to be a significant initial step in ixotrophy. The gliding, multicellular filamentous bacterium Aureispira sp. CCB-QB1 belongs to the family Saprospiraceae and preys on bacteria such as Vibrio sp. in seawater. Adhesion and cell aggregation were coincident with preying and were hypothesized to play an important role in the ixotrophy in this bacterium. To test this hypothesis, experiments to elucidate the mechanisms of aggregation or adhesion in this bacterium were performed. The ability of Aureispira QB1 to adhere and aggregate to prey bacterium, Vibrio sp., required divalent cations, especially calcium ions. In the presence of calcium, Aureispira QB1 cells captured 99 % of Vibrio sp. cells after 60 min of incubation. Toluidine blue O, which binds acidic polysaccharides, bound to Aureispira QB1 and inhibited adhesion of Aureispira QB1. These results suggest that acidic polysaccharides are needed for aggregation or adhesion of Aureispira and that calcium ions play a significant role in these phenomena.

The aglZ gene of Myxococcus xanthus was identified from a yeast two-hybrid assay in which MglA was used as bait. MglA is a 22-kDa cytoplasmic GTPase required for both adventurous and social gliding motility and sporulation. Genetic studies showed that aglZ is part of the A motility system, because disruption or deletion of aglZ abolished movement of isolated cells and aglZ sglK double mutants were nonmotile. The aglZ gene encodes a 153-kDa protein that interacts with purified MglA in vitro. The N terminus of AglZ shows similarity to the receiver domain of two-component response regulator proteins, while the C terminus contains heptad repeats characteristic of coiled-coil proteins, such as myosin. Consistent with this motif, expression of AglZ in Escherichia coli resulted in production of striated lattice structures. Similar to the myosin heavy chain, the purified C-terminal coiled-coil domain of AglZ forms filament structures in vitro.

Myxococcus xanthus glides over solid surfaces without the use of flagella, dependent upon two large sets of adventurous (A) and social (S) genes, using two different mechanisms of gliding motility. Myxococcus xanthus A-S- double mutants form non-motile colonies lacking migratory cells at their edges. We have isolated 115 independent mutants of M. xanthus with insertions of transposon magellan-4 in potential A genes by screening for insertions that reduce the motility of a mutant S- parental strain. These insertions are found not only in the three loci known to be required for A motility, mglBA, cglB, and aglU, but also in 30 new genes. Six of these new genes encode different homologues of the TolR, TolB, and TolQ transport proteins, suggesting that adventurous motility is dependent on biopolymer transport. Other insertions which affect both A and S motility suggest that both systems share common energy and cell wall determinants. Because the spectrum of magellan-4 insertions in M. xanthus is extraordinarily broad, transposon mutagenesis with this eukaryotic genetic element permits the rapid genetic analysis of large sets of genes that contribute to a complex microbial behaviors such as A motility.

Summary The mglA gene encodes a 22 kDa GTPase that is critical for single-cell (A) gliding, type IV pili-mediated (S) gliding and development of Myxococcus xanthus. To identify components that interact with MglA to control these processes, second-site mutations that restore movement to non-motile mglA mutants were sought. An allele-specific extragenic suppressor of mglA8, named mas815 (mglA8 suppressor 15), was obtained. mas815 does not bypass the requirement for MglA, yet it restores type IV pili-mediated motility and starvation-induced development. Single-cell (A) motility is not restored. The suppressing mutation maps to the 3′ end of a gene, masK, in an operon immediately upstream of the mglBA operon. masK encodes a protein of the STY kinase family. When the masK gene was used as bait against a library carrying M. xanthus DNA in the yeast two-hybrid system, eight positive, independent clones containing fusions of mglA to GAL4 were obtained, thus confirming the interaction between MglA and MasK. MasK, expressed in Escherichia coli, was shown to phosphorylate at a tyrosine residue(s). The gain-of-function in the masK815 mutant was correlated with increased production of extracellular fibrils, which are required for adhesion, cell–cell contact and sensing phosphatidylethanolamine chemoattractants. These data suggest that the interaction between MasK and MglA is an essential part of a signal transduction pathway controlling motility and development.

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 β-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.

Members of the genus Archaeoglobus are hyperthermophilic sulfate reducers with an optimal growth temperature of 83 °C. Archaeoglobus fulgidus can utilize simple compounds including D-lactate, L-lactate and pyruvate as the sole substrate for carbon and electrons for dissimilatory sulfate reduction. Previously we showed that this organism makes a D-lactate dehydrogenase (Dld) that requires FAD and Zn2+ for activity. To determine the cellular location and topology of Dld and to identify proteins that interact with Dld, an antibody directed against Dld was prepared. Immunocytochemical studies using gold particle-coated secondary antibodies show that more than 85% of Dld is associated with the membrane. A truncated form of Dld was detected in immunoblots of whole cells treated with protease, showing that Dld is an integral membrane protein and that a significant portion of Dld, including part of the FAD-binding pocket, is outside the membrane facing the S-layer. The gene encoding Dld is part of an operon that includes noxA2, which encodes one of several NADH oxidases in A. fulgidus. Previous studies have shown that NoxA2 remains bound to Dld during purification. Thin sections of A. fulgidus probed simultaneously with antibodies against Dld and NoxA2 show that both proteins co-localized to the same sites in the membrane. Although these data show a tight interaction between NoxA2 and Dld, the role of NoxA2 in electron transport reactions is unknown. Rather, NoxA2 may protect proteins involved in electron transfer by reducing O2 to H2O2 or H2O.

Myxococcus xanthus is one of the diverse bacteria that display gliding motility, involving two mechanisms, called adventurous (A) and social (S) gliding motility. The genetic and phenotypic analysis of mutants defective in gliding motility has shown that gliding motility is more complex than flagellum-dependent motility. The first molecular insights about the mechanisms of gliding motility were gleaned from the results of genetic studies performed by Hodgkin and Kaiser, who used chemical mutagens to generate mutations that affected gliding. The phenotypes of gliding mutants can be measured in terms of the differences in their velocities of gliding. The more detailed molecular genetic analysis of the two mechanisms of gliding motility in M. xanthus has been facilitated by the use of transposon mutagenesis. M. xanthus cells that have a functional A gliding system are able to move as isolated cells on a solid surface. The majority of mutations that abolish S motility affect the production of type IV pili (TFP), the exopolysaccharide (EPS) component of fibrils, or the lipopolysaccharide (LPS) moiety of O-antigen. The function of EPS in social motility has been elucidated by phenotypic analysis of mutants of M. xanthus lacking EPS, a secreted polymer comprised primarily of N-acetylglucosamine (GlcNAc) and glucosamine (GlcN). M. xanthus has a complex life cycle. In the presence of adequate nutrients, the cells undergo vegetative growth and divide, but when the cells are starved of nutrients, they aggregate and form fruiting bodies containing myxospores.

Myxococcus xanthus undergoes phase variation during growth to produce predominantly two colony phenotypes. The majority are yellow colonies containing swarm-proficient cells and a minority are tan colonies containing swarm-deficient cells. Comparison of the transcriptomes of a yellow variant, a tan variant, and three tan mutants led to the identification of differentially-regulated genes that define key segments of the phase variation pathway. For example, expression of genes for the yellow pigment DKxanthene and the antibiotic myxovirescin was increased significantly in yellow variants. In contrast, expression of the siderophore myxochelin, hemin binding proteins, and iron transport proteins was increased specifically in tan strains. Thus, a consequence of phase variation is that yellow cells shift from producing antibiotic and pigment to producing components involved in acquisition of iron, which may increase fitness during periods of iron limitation. Multiple protein kinases and HTH-Xre DNA-binding proteins identified in this study may be involved in the regulatory hierarchy that governs phase variation.

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.

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.

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.

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 Δ mglA 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 Δ mglA 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.

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 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.

Gliding motility is critical for normal development of spore-filled fruiting bodies in the soil bacterium Myxococcus xanthus. Mutations in mgl block motility and development but one mgl allele can be suppressed by a mutation in masK, the last gene in an operon adjacent to the mgl operon. Deletion of the entire 5.5 kb masABK operon crippled gliding and fruiting body development and decreased sporulation. Expression of pilAGHI, which encodes type IV pili (TFP) components essential for social (S) gliding, several cryptic pil genes, and a LuxR family protein were reduced significantly in the Δmas mutant while expression of the myxalamide operon was increased significantly. Localization and two-hybrid analysis suggest that the three Mas proteins form a membrane complex. MasA-PhoA fusions confirmed that MasA is an integral cytoplasmic membrane protein with a ≈100 amino acid periplasmic domain. Results from yeast two-hybrid assays showed that MasA interacts with the lipoprotein MasB and MasK, a protein kinase and that MasB and MasK interact with one another. Additionally, yeast two-hybrid analysis revealed a physical interaction between two gene products of the mas operon, MasA and MasB, and PilA. Deletion of mas may be accompanied by compensatory mutations since complementation of the Δmas social gliding and developmental defects required addition of both pilA and masABK.

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.

Background The mglA gene from the bacterium Myxococcus xanthus encodes a 22kDa protein related to the Ras superfamily of monomeric GTPases. MglA is required for the normal function of A-motility (a dventurous), S-motility (s ocial), fruiting body morphogenesis, and sporulation. MglA and its homologs differ from all eukaryotic and other prokaryotic GTPases because they have a threonine (Thr78) in place of the highly conserved aspartate residue of the consensus PM3 (p hosphate-m agnesium binding) region. To identify residues critical for MglA function or potential protein interactions, and explore the function of Thr78, the phenotypes of 18 mglA mutants were characterized. Results Nine mutants, with mutations predicted to alter residues that bind the guanine base or coordinate magnesium, did not produce detectable MglA. As expected, these mutants were mot- dev- because MglA is essential for these processes. Of the remaining nine mutants, seven showed a wild-type distribution pattern for MglA but fell into two categories with regard to function. Five of the seven mutants exhibited mild phenotypes, but two mutants, T78D and P80A, abolished motility and development. The localization pattern of MglA was abolished in two mutants that were mot- spo- and dev-. These two mutants were predicted to alter surface residues at Asp52 and Thr54, which suggests that these residues are critical for proper localization and may define a protein interaction site. Improving the consensus match with Ras at Thr78 abolished function of MglA. Only the conservative serine substitution was tolerated at this position. Merodiploid constructs revealed that a subset of alleles, including mglA D52A, were dominant and also illustrated that changing the balance of MglA and its co-transcribed partner, MglB, affects A-motility. Conclusion Our results suggest that GTP binding is critical for stability of MglA because MglA does not accumulate in mutants that cannot bind GTP. The threonine in PM3 of MglA proteins represents a novel modification of the highly conserved GTPase consensus at this position. The requirement for a hydroxyl group at this position may indicate that MglA is subject to modification under certain conditions. Proper localization of MglA is critical for both motility and development and likely involves protein interactions mediated by residues Asp52 and Thr54.

MglA, a 22-kDa protein related to monomeric GTPases, is required for the normal operation of the A (Adventurous) and S (Social) motility and for multicellular development of Myxococcus xanthus. To determine how MglA controls A- and S-motility, MglA was assayed biochemically and its cellular location was determined. His-tagged MglA hydrolyzed GTP slowly in vitro at a rate nearly identical to that of Ras showing that MglA has GTPase activity. Immunofluorescence microscopy of fixed cells from liquid showed that MglA was associated with helical track similar to the MreB spiral that spanned the length of the cell. The distribution pattern of MglA depended on the type of surface from which cells were harvested. In cells gliding on 1.5% (w/v) agar, the helical pattern gave way to punctate clusters of MglA-Yfp at the poles and along the long axis (lateral clusters). The lateral clusters emerged near the leading pole as the cell advanced coincident with a decrease in the intensity of the MglA-Yfp cluster at the leading pole. Newly formed lateral clusters remained fixed with regard to the substratum as the cell moved forward, similar to focal adhesion complexes described for AglZ, a protein partner of MglA. Lateral clusters did not form in cells gliding in methylcellulose, a polymer that stimulates S-motility at low cell density; rather MglA-Yfp was diffuse in the cytoplasm and more concentrated at the poles. The results suggest that conditions that favor S-motility prevent the formation of lateral clusters of MglA, which are associated with A-motility functions. © 2010 Wiley-Liss, Inc.

The bacterium Myxococcus xanthus glides over surfaces using two different locomotive mechanisms, called S (social) and A (adventurous) motility that enable cells to move both as groups and as individuals. Neither mechanism involves flagella. The functions of these two motors are coordinated by the activity of a small Ras-like protein, encoded by the mglA gene. The results of previous studies of a second-site suppressor of the mglA-8 missense mutation masK-815 indicate that MglA interacts with a protein tyrosine kinase, MasK, to control social motility. Sequence analysis of the sites of 12 independent insertions of the transposon magellan-4 that result in the loss of motility in an M. xanthus mglA-8 masK-815 double mutant shows that nine of these 12 insertions are in genes known to be required for S gliding motility. This result confirms that the masK-815 suppressor restores S but not A motility. Three of the 12 insertions define three new genes required for S motility and show that the attachment of heptose to the lipopolysaccharide inner core, an ortholog of the CheR methyltransferase, and a large protein with YD repeat motifs, are required for S motility. When these three insertions are backcrossed into an otherwise wild-type genetic background, their recombinants are found to have defects in S, but not, A motility. The spectrum of magellan-4 insertions that lead to the loss of S motility in the mglA-8 masK-815 double mutant background is different than that resulting from a previous mutant hunt starting with a different (A mutant) genetic background, suggesting that the number of genes required for S motility in M. xanthus is quite large.

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 ΔaglU (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.

The mglA gene encodes a 22 kDa GTPase that is critical for single-cell (A) gliding, type IV pili-mediated (S) gliding and development of Myxococcus xanthus. To identify components that interact with MglA to control these processes, second-site mutations that restore movement to non-motile mglA mutants were sought. An allele-specific extragenic suppressor of mglA8, named mas815 (mglA8 suppressor 15), was obtained. mas815 does not bypass the requirement for MglA, yet it restores type IV pili-mediated motility and starvation-induced development. Single-cell (A) motility is not restored. The suppressing mutation maps to the 3' end of a gene, masK, in an operon immediately upstream of the mglBA operon. masK encodes a protein of the STY kinase family. When the masK gene was used as bait against a library carrying M. xanthus DNA in the yeast two-hybrid system, eight positive, independent clones containing fusions of mglA to GAL4 were obtained, thus confirming the interaction between MglA and MasK. MasK, expressed in Escherichia coli, was shown to phosphorylate at a tyrosine residue(s). The gain-of-function in the masK815 mutant was correlated with increased production of extracellular fibrils, which are required for adhesion, cell-cell contact and sensing phosphatidylethanolamine chemoattractants. These data suggest that the interaction between MasK and MglA is an essential part of a signal transduction pathway controlling motility and development.

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°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.

Publisher Summary Biofilm is produced by other members of the domain Archaea, such as Haloferax, Thermococcus, Methanosarcina, and Methanobacterium , and by eubacterial sulfate reducers such as Desulfovibrio . Archaea contain hydrophobic polyisoprenoid ether-linked lipids instead of typical alkane ester-linked lipids found in eubacteria and eukaryotes. A variety of modifications can be found among the Archaea, particularly in the hyperthermophiles, whose modified lipids likely enhance thermal stability. Lipids in biofilm may contain additional modifications that increase resistance to heat and chemicals. Biofilm from hyperthermophiles, particularly hyperthermophiles in the domain Archaea, is less well characterized. Although biofilm of A. fulgidus contains carbohydrate, the genes involved in the synthesis of polysaccharides in eubacteria have not been identified by sequence analysis in A. fulgidus .

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. The soil bacterium Myxococcus xanthus is a model prokaryotic system for the study of intercellular communication. Cellcell interactions play central roles in both gliding motility and an elaborate multicellular developmental cycle initiated by starvation. Development leads to the morphogenesis of fruiting bodies containing differentiated, heat-resistant spores. Although the change from rod-shaped vegetative cells to spherical spores is rapid, genetic methods have been used successfully to arrange the early steps of this process into an ordered series of molecular events. Many mutations that block M. xanthus development do not affect vegetative growth. Consequently, methods for the analysis of mutations that block development have relied on genetic manipulations with vegetative cells.

Three independent Tn 5-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.

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.

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.

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.

Summary Myxococous 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.

Single mutations in the mglA gene in Myxococcus xanthus render cells incapable of gliding. The mglA strains are unique in that all other nonmotile strains of M. xanthus isolated are the result of at least two independent mutations in separate motility system genes. Translational fusions of trpE, or of lacZ, to mglA were constructed, and the resulting fusion polypeptides were used to generate antibodies. Antibodies specific to MglA protein were purified. Antibody-tagged MglA was found localized to the cytoplasm of M. xanthus cells both by fractionation of cell extracts and by electron microscopy of thin sections of whole cells. Four of the five mglA missense mutants tested failed to produce detectable levels of the MglA antigen in whole cell extracts. Nonmotile double mutants (A-S-), which have one mutation in a gene of system A and one mutation in a gene of system S, have the same phenotype as null mglA mutants but produce wild-type levels of MglA protein. MglA protein is conserved in all strains of myxobacteria tested. The amino acid sequence of MglA protein includes three sequence motifs characteristic of GDP/GTP-binding proteins. On the basis of its genetic properties, intracellular location, and amino acid sequence, it is argued that MglA protein is a regulator in the sequence of functions leading to cell movement.

The mgl operon contains two open reading frames (ORFs) which are transcribed together. A collection of nonmotile mutants helped to define the downstream ORF as the mglA gene. Single mutations at the mglA locus completely abolish motility. A series of deletion mutations was constructed to determine the role of the upstream ORF (now called mglB). A strain carrying a deletion in mglB and with an intact mglA produces small colonies. The cells are motile, but their rate of swarm spreading is reduced. Measurements of cell movement showed that mglB mutant cells advanced, on average, less than 0.1 cell length in 5 min. The mglB+ cells advanced an average of 1.3 cell lengths in the same time. Extracts of delta mglB cells contain 15 to 20% as much of the 22-kDa MglA protein as do mglB+ cells, as measured in Western immunoblots and enzyme-linked immunosorbent assays. However, the amount of mgl transcript is the same in the delta mglB mutants as in the mglB+ strain. Heterozygous partial diploids mglB/mglA with the wild-type alleles in trans have normal motility, demonstrating that the largest of the mglB deletions is not polar on mglA. Like other motility defects, a delta mglB mutation alters fruiting body development and sporulation. The mglB mutants delayed aggregation, produced small immature fruiting bodies, and sporulated at 45 to 50% wild-type levels. All aspects of the mglB mutant phenotype are explained by the reduced levels of mglA protein and the assumption that it limits the amount of gliding.

Summary Component C of the CH 3 -S-CoM methylreductase system has been isolated and purified from four different methanogens. The native protein from Methanobacteriwn thermoautotrophicum has an M r of 300,000 and is composed of 6 subunits with an α 2 (Mr 68,000), β 2 (45,000), γ 2 (38,500) stoichiometry [ Ellefson, W. and Wolfe, R. S. , J. Biol. Chem. 256 , 4259–4262 (1981)]. We have purified component C from three other methanogens, Methanococcus voltae strain PS, Methanococcus jannaschii and Methanosarcina barkeri s strain 227. The native component C from Mc. voltae has an M r of 297,000 with three subunits of Mr 66,000, 44,000 and 34,500. The native protein from Mc. jannaschii has an M r of 300,000 and is also composed of three subunits of M r 65,000, 43,500 and 36,000. Component C from Ms. barkeri has an M r of 305,000 daltons and has three subunits of M r 73,000, 41,500 and 33,000. All of these enzymes contain the chromophore factor F 430 with a spectrum similar to that from Mb. thermoautotrophicum . Antibody raised to component C from Mb. thermoautotrophicum cross reacted slightly with component C from Mc. voltae but did not cross react with the other isolates tested. Antibody to component C from Mc. voltae did not cross react with component C from Mb. thermoautotrophicum or Ms. barkeri but did cross react slightly with component C from Mc. jannaschii . Component C from each of these sources was able to reconstitute the CH 3 -S-CoM methylreductase assay in homologous as well as heterologous reactions at different temperatures.

Nonmotile mutants of Myxococcus xanthus (Myxobacterales) failed to execute the morphogenetic movements required to shape a fruiting body. In addition, nonmotile mutants produced very few spores when plated for fruiting body development at cell densities appropriate for wild-type cells. At higher initial cell densities, the proportion of nonmotile cells that sporulate increased, indicating that one important function of motility in fruiting body development is to increase the local cell density. However, even at 10 times normal cell density, nonmotile cells sporulated at only 1% the wild-type level. This sporulation deficiency of nonmotile mutants accompanies an altered pattern of gene expression, monitored by using transcriptional fusions of lacZ to genes expressed at specific times during fruiting body development. Motility was not required for normal expression of five lac fusions that are expressed within the first 6 hr of fruiting-body development. However, the levels of expression from five lac fusions to later-expressed genes were reduced or abolished in nonmotile strains. beta-Galactosidase expression in these late Tn5 lac insertions was increased, and fruiting body development occurred in certain nonmotile strains that can be stimulated to move when mixed with a donor strain. This shows that motility itself is required because the stimulated cells are nonmotile genotypically. The nonmotile mutations had the same effect on developmental beta-galactosidase expression from these 10 lac fusions as an insertion mutation in the csg (formerly spoC) gene. csg mutants have a cell-cell interaction defect that blocks fruiting body development at approximately 6 hr. The similarity in the pattern of developmental expression of motility mutants and csg mutants suggests that motility is required for this csg-mediated cell-cell interaction.

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.

Mutants of Myxococcus xanthus that had lost the ability to glide were examined to elucidate the mechanism of gliding motility. Nonmotile mutants resulting from a single mutational step were all defective at the same locus, mgl, which implied an important role for the mgl product(s) in gliding. Deletion experiments, transposon insertion mutagenesis, and genetic rescue of mgl mutants mapped the locus to a 1.6-kilobase segment of Myxococcus DNA. Two species of RNA that hybridized with mgl DNA were found both during vegetative growth and during the starvation-induced development of fruiting bodies, which also requires cell movement. The two RNA species, of 1.5 and 1.3 kilobases, had the same 5' to 3' orientation and overlapped extensively. The DNA sequences of mgl+ and of seven mgl mutants were determined. Each mutant differed from mgl+ by a single-base-pair change in the sequence. Two adjacent open reading frames were found in the sequence hybridizing to both species of mgl RNA. Six of the single-base-pair changes, each of which would result in a single-amino-acid change, and an insertion-produced mgl mutation were located in the downstream open reading frame. This open reading frame (of 195 amino acids) is therefore an mgl gene, called mglA. The function of the upstream open reading frame is not known with certainty, although it does contain one of the mgl mutant sites and could be a second mgl gene.

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.

To identify the electron acceptor of the methylenetetrahydromethanopterin dehydrogenase of Methanobacterium thermoautotrophicum , we have purified the enzyme to homogeneity. The purified enzyme is absolutely dependent on coenzyme F420 (a 7,8-didemethyl-8-hydroxy-5-deazariboflavin derivative) for activity. Several alternative electron acceptors are ineffectual in the reaction. Changes in the absorption spectra of reaction mixtures indicate that 1.1 mol of coenzyme F420 is reduced per mol of substrate oxidized. The reaction is reversible and the equilibrium favors oxidation of methylenetetra-hydromethanopterin.

Nickel metalloenzymes have been found in all methane-producing bacteria examined. Two hydrogenases from Methanobacterium thermoautotrophicum contain nickel which has been shown to be coordinated by S-containing ligands in one form. A compound called F/sub 430/ has been isolated from M. thermoautotrophicum, strain ..delta..H, and has been identified as the Ni-containing prosthetic group in protein component C of the S-methyl coenzyme-M reductase system. F/sub 430/ has been established to have a Ni(II)-tetrapyrrole structure, constituting the only known biological example of nickel in a tetrahydro reduced corphine. The detailed mechanistic role of F/sub 430/ in methyl group reduction has not been identified. We report here a Ni x-ray absorption spectroscopic (XAS) study of several forms of F/sub 430/ and component C isolated from M. thermoautotrophicum, strain ..delta..H. These preliminary results indicate that free F/sub 430/ is structurally distinct from the F/sub 430/-containing protein. Also, depending upon the conditions used, extraction of F/sub 430/ from the protein yields at least two structurally distinct forms, one similar to that in the intact protein and one similar to free F/sub 430/. Analysis of the extended x-ray absorption fine structure (EXAFS) data and comparison with a number of structurally characterized Ni(II) complexes provide evidence that themore » flexibility of the tetrapyrrole is responsible for the different structural forms.« less