Identification and localization of Myxococcus xanthus porins and lipoproteins

Myxococcus xanthus DK1622 contains inner (IM) and outer membranes (OM) separated by a peptidoglycan layer. Integral membrane, β-barrel proteins are found exclusively in the OM where they form pores allowing the passage of nutrients, waste products and signals. One porin, Oar, is required for intercellular communication of the C-signal. An oar mutant produces CsgA but is unable to ripple or stimulate csgA mutants to develop suggesting that it is the channel for C-signaling. Six prediction programs were evaluated for their ability to identify β-barrel proteins. No program was reliable unless the predicted proteins were first parsed using Signal P, Lipo P and TMHMM, after which TMBETA-SVM and TMBETADISC-RBF identified β-barrel proteins most accurately. 228 β-barrel proteins were predicted from among 7331 protein coding regions, representing 3.1% of total genes. Sucrose density gradients were used to separate vegetative cell IM and OM fractions, and LC-MS/MS of OM proteins identified 54 β-barrel proteins. Another class of membrane proteins, the lipoproteins, are anchored in the membrane via a lipid moiety at the N-terminus. 44 OM proteins identified by LC-MS/MS were predicted lipoproteins. Lipoproteins are distributed between the IM, OM and ECM according to an N-terminal sorting sequence that varies among species. Sequence analysis revealed conservation of alanine at the +7 position of mature ECM lipoproteins, lysine at the +2 position of IM lipoproteins, and no noticable conservation within the OM lipoproteins. Site directed mutagenesis and immuno transmission electron microscopy showed that alanine at the +7 position is essential for sorting of the lipoprotein FibA into the ECM. FibA appears at normal levels in the ECM even when a +2 lysine is added to the signal sequence. These results suggest that ECM proteins have a unique method of secretion. It is now possible to target lipoproteins to specific IM, OM and ECM locations by manipulating the amino acid sequence near the +1 cysteine processing site.     

Identification of heat-stable A-factor from Myxococcus xanthus.

The asg mutants of Myxococcus xanthus fail to produce a set of related substances called A-factor. A-factor is released into the medium and is required early in fruiting body development. Lacking A-factor, the asg mutants are defective in aggregation, sporulation, and expression of most genes whose products appear later than 1 h after development is induced by starvation. Previous work has shown that these defects are reversed when A-factor, released by developing wild-type cells, is added to asg mutant cells. Part of the material in conditioned medium with A-factor activity is heat stable and dialyzable. This low-molecular-weight A-factor consists of a mixture of amino acids and peptides. Fifteen single amino acids have A-factor activity, and 11 of these are found in conditioned medium. Mixtures of amino acids have a total activity approximately equal to the sum of the activities of their constituents. Conditioned medium also contains peptides with A-factor activity. Pure peptides have A-factor activity, and their specific activities are equal to or less than the sum of the activities of their constituent amino acids. There is no evidence for a specialized A-factor peptide in conditioned medium, one with a specific activity greater than the sum of its constituent amino acids. About half of the heat-stable A-factor activity in conditioned medium can be accounted for by free amino acids, and the remaining half can be accounted for by peptides. It is argued that heat-stable A-factor induces A-dependent gene expression not by the nutritional action of amino acids but through a chemosensory circuit.     

Identification and characterization of the Myxococcus xanthus bsgA gene product.

The bsgA mutants of Myxococcus xanthus are blocked at a very early stage of the developmental program. They fail to produce fruiting bodies or to sporulate under normal conditions but can be rescued by extracellular complementation in mixtures with wild-type cells. A bsgA-lacZ gene fusion was constructed and expressed in Escherichia coli. The resulting fusion protein, which has beta-galactosidase enzyme activity, was partially purified by affinity chromatography and preparative polyacrylamide gel electrophoresis. The protein was used to immunize mice, which produced a hybridoma secreting monoclonal antibody that was specific for the bsgA gene product. The monoclonal antibody was used in Western blot (immunoblot) experiments to determine the apparent cellular location of the bsgA protein in M. xanthus and to compare the level of this protein at various times in the Myxococcus life cycle.     

Spectroscopic and Microscopic Characterization of Uranium Biomineralization in Myxococcus xanthus

In this work, synchrotron-based X-ray absorption spectroscopy (XAS) and transmission electron microscopy (TEM) studies were carried out to elucidate at molecular scale the interaction mechanisms of Myxococcus xanthus with uranium at different pH values. Extended X-ray absorption fine structure (EXAFS) spectroscopic measurements showed that there are significant differences in the structural parameters of the U complexes formed by this bacterium at pH 2 and 4.5. At very low acidic pH of 2, the cells accumulated U(VI) as organic phosphate-metal complexes. At pH 4.5, however, the cells of this bacterium precipitated U(VI) as meta-autunite-like phase. TEM analyses demonstrated that at pH 2 the uranium accumulates were located mainly at the cell surface, whereas at pH 4.5 a uranium precipitation occurred on the cell wall and within the extracellular polysaccharides (EPS) characteristic of this bacterium. Dead/live staining studies showed that 30% and 50% of the uranium treated cell populations were alive at pH 2 and 4.5, respectively. The precipitation of U(VI) as mineral meta-autunite-like phase is possibly due to the bacterial acidic phosphatase activity. The precipitation of uranium as mineral phases may lead to more stable U(VI) sequestration that may be suitable for remediation purposes. These observations, combined with the very high uranium accumulation capability of the studied bacterial cells indicate that M. xanthus may significantly influence the fate of uranium in soil environments where these bacterial species are mainly found.     

一种粘细菌发育相关基因的分离及敲除分析

FruB是与粘细菌(Myxococcus xanthus)发育特异性转录因子FruA具有亲和力的蛋白因子,协同FruA参与对靶基因的调控。根据FruB氨基端氨基酸序列,设计简并性寡核苷酸引物对染色体DNA进行PCR扩增,以扩增产物为探针自粘细菌小型基因文库筛选出同源的4.5kb SacI阳性片段。fruB基因阻断分析表明,fruB功能缺失延缓子实体发育并降低粘孢子产率,提示fruB与粘细菌发育分化有一定联系。     

Untersuchungen an Myxococcus xanthus

Ohne Zusammenfassung     

Fatty Acids of Myxococcus xanthus

Fatty acids were extracted from saponified vegetative cells and myxospores of Myxococcus xanthus and examined as the methyl esters by gas-liquid chromatography. The acids consisted mainly of C₁₄ to C₁₇ species. Branched acids predominated, and iso-pentadecanoic acid constituted half or more of the mixture. The other leading component (11–28%) was found to be 11-n-hexadecenoic acid. Among the unsaturated acids were two diunsaturated ones, an n-hexadecadienoic acid and an iso-heptadecadienoic acid. No significant differences between the fatty acid compositions of the vegetative cells and myxospores could be detected. The fatty acid composition of M. xanthus was found to be markedly similar to that of Stigmatella aurantiaca. It is suggested that a fatty acid pattern consisting of a large proportion of iso-branched C₁₅ and C₁₇ acids and a substantial amount of an n-16:1 acid is characteristic of myxobacteria.     

Natural transformation of Myxococcus xanthus

Myxococcus xanthus belongs to the delta class of the proteobacteria and is notable for its complex life-style with social behaviors and relatively large genome. Although previous observations have suggested the existence of horizontal gene transfer in M. xanthus, its ability to take up exogenous DNA via natural transformation has not been experimentally demonstrated. In this study, we achieved natural transformation in M. xanthus using the autonomously replicating myxobacterial plasmid pZJY41 as donor DNA. M. xanthus exopolysaccharide (EPS) was shown to be an extracellular barrier for transformation. Cells deficient in EPS production, e.g., mutant strains carrying ΔdifA or ΔepsA, became naturally transformable. Among the inner barriers to transformation were restriction-modification systems in M. xanthus, which could be partially overcome by methylating DNA in vitro using cell extracts of M. xanthus prior to transformation. In addition, the incubation time of DNA with cells and the presence of divalent magnesium ion affected transformation frequency of M. xanthus. Furthermore, we also observed a potential involvement of the type IV pilus system in the DNA uptake machinery of M. xanthus. The natural transformation was totally eliminated in the ΔpilQ/epsA and Δtgl/epsA mutants, and null mutation of pilB or pilC in an ΔepsA background diminished the transformation rate. Our study, to the best of our knowledge, provides the first example of a naturally transformable species among deltaproteobacteria.     

Cohesion-defective mutants of Myxococcus xanthus

Cohesion of Myxococcus xanthus cells involves interaction of a cell surface cohesin with a component of the extracellular matrix. In this work, two previously isolated cohesion-defective (fbd) mutants were characterized. The fbdA and fbdB genes do not encode the cohesins but are necessary for their production. Both mutants produce type IV pili, suggesting that PilA is not a major cohesin.     

Identification and characterization of five cspA homologous genes from Myxococcus xanthus.

Escherichia coli contains a large CspA family consisting of nine homologues, in which four are cold-shock inducible and one is stationary-phase inducible. Here, we demonstrate that Myxococcus xanthus possesses at least five CspA homologues, CspA to CspE. Hydrophobic residues forming a hydrophobic core, and aromatic residues, which are included in functional motifs RNP-1 and RNP-2 involved in binding to RNA and ssDNA, are well conserved. These facts suggest that M. xanthus CspA homologues have a similar structure and function as E. coli CspA. However, in contrast to the E. coli CspA family, the expression of M. xanthus csp genes as judged by primer extension analysis is not significantly regulated by temperature changes, except for cspB of which expression was reduced to less than 10% upon heat shock at 42 degrees C. Such constitutive expression of the csp genes may be important for M. xanthus, a soil-dwelling bacterium, to survive under conditions of exposure to various environmental changes in nature.     

Microcyst Germination in Myxococcus xanthus

Germination of glycerol-prepared microcysts of Myxococcus xanthus was studied. The sequence of morphological events during germination resembled that of germinating fruiting body-microcysts. The turbidity drop of a culture of germinating microcysts could be described by McCormick's formula derived for germinating Bacillus spores. The rate of uptake of labeled glycine and acetate did not change during germination. Temperature, aeration, and pH optima for germination were the same as for vegetative cell growth. Germination was induced by protein hydrolysates and the individual amino acids glycine, alanine, valine, aspartic acid, and glutamic acid. A number of organic compounds, including sugars, alcohols, aldehydes, ketones, organic acids, and chelating agents, did not induce germination. The inorganic ions HPO(4) (2-), Mg(++), Ca(++), and NH(4) (+) induced germination, although ionic strength was not a factor. Microcysts incubated in distilled water at concentrations greater than about 10(9) cells/ml germinated; supernatant fluid from such suspensions (germination factor) induced germination of less concentrated suspensions. The activity of germination factor was resistant to boiling, but was lost on charring and dialysis. Germination of microcysts and growth of vegetative cells was equally sensitive to a variety of metabolic inhibitors, including penicillin and chloramphenicol. Germination was more resistant than vegetative growth to inhibition by antibiotics of the streptomycin family and by actinomycin D.