Patricia L. Hartzell

Identification of genes required for adventurous gliding motility in Myxococcus xanthus with the transposable element mariner

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.

AglZ Is a Filament-Forming Coiled-Coil Protein Required for Adventurous Gliding Motility of Myxococcus xanthus

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.

MglA, a small GTPase, interacts with a tyrosine kinase to control type IV pili‐mediated motility and development of Myxococcus xanthus

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.

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 prokaryoteMyxococcus xanthus. As part of its life cycle,M. xanthus forms multicellular fruiting bodies containing differentiated cells. Analysis of the genes essential forM. xanthus development is possible because strains with mutations that block development can be maintained in the vegetative state. Development inM. 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, α-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 inM. xanthus.

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 (≈ 450 amino acid) form of GidA from eubacteria and about 24% identity overall with the large (≈ 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.

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

Myxococcus xanthus has two different mechanisms of motility, adventurous (A) motility, which permits individual cells to glide over solid surfaces, and social (S) motility, which permits groups of cells to glide. To identify the genes involved in S-gliding motility, we mutagenized a 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.

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 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.

H2O2-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.

Acetyl-CoA decarbonylase/synthase complex from Archaeoglobus fulgidus

Abstract 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:w CO2 + 2 Fdred(Fe2+) + 2 H+ + CH3– H4SPt + CoA ⇌ acetyl-CoA + H4SPt + 2 Fdox(Fe3+) + H2Owhere Fd is ferredoxin, and CH3–H4SPt and H4SPt denote N5-methyl-tetrahydrosarcinapterin and tetrahydrosarcinapterin, respectively.

Biofilm formation in hyperthermophilic Archaea.

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 .