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PubMed1988

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.

Bacterial Proteins↗

PubMed1989

Gliding motility in Myxococcus xanthus: mgl locus, RNA, and predicted protein products.

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.

Amino Acid Sequence↗

PubMed1989

Movement of multiple DNA units between Myxococcus xanthus cells.

Myxococcus xanthus YS produces particles (Mx alpha particles) that transmit genetic information between cells. Mx alpha particles might be viruses, although no host able to sustain lytic growth of Mx alpha has been discovered. The particles could be detected by their ability to transduce a Tn5 transposon tag to recipient bacteria. DNA from purified particles hybridized to a limited number of DNA restriction fragments of strain YS, suggesting that Mx alpha particles contain only specific DNA sequences. A set of Tn5 insertions residing in the transducible region provided genetic markers for cloning cellular DNA packaged by Mx alpha. A map of this region showed that transducible DNA comprised multiple units of approximately 80 kilobases each. Individual units share DNA homology but are divergent in the location of restriction sites. Other wild-type isolates of Myxococcus species contained DNA sequences with homology to Mx alpha DNA, indicating that Mx alpha DNA is widespread in nature. Experiments on the transfer of Mx alpha DNA in strain YS suggested that DNA transfer is enhanced during the developmental cycle.

Blotting, Southern↗

PubMed1966

Light-induced lysis and carotenogenesis in Myxococcus xanthus.

Burchard, Robert P. (University of Minnesota, Minneapolis), and Martin Dworkin. Light-induced lysis and carotenogenesis in Myxococcus xanthus. J. Bacteriol. 91:535-545. 1966.-Myxococcus xanthus, grown vegetatively in the light, developed an orange carotenoid after the cells entered stationary phase of growth; pigment content increased with age. Cells grown in the dark did not develop carotenoid and could be photolysed by relatively low-intensity light only during stationary phase; rate of photolysis increased with age. Photolysis adhered to the reciprocity law, was temperature-independent and oxygen-dependent, and required the presence of nonspecific, monovalent cations; it was inhibited by one of several divalent cations. Logarithmic-phase cells were photosensitized by 100,000 x g pellet preparations of sonic-treated stationary-phase cells grown in the light and dark. A porphyrin with a Soret band at 408 mmu was isolated from photosensitive cells; logarithmic-phase cells contained about 1/16 the amount of porphyrin of stationary-phase cells. The purified material had spectral and chemical properties of protoporphyrin IX and photosensitized logarithmic-phase cells. Its spectrum was similar to the action spectrum for photolysis. We concluded that protoporphyrin IX is the natural endogenous photosensitizer. Carotenogenesis was stimulated by light in the blue-violet region of the visible spectrum and was inhibited by diphenylamine, resulting in photosensitivity of the cells. Photoprotection by carotenoid was lost in the cold. A mutant which synthesized carotenoid in the light and dark was photosensitive only after growth in diphenylamine. The ecological significance of these phenomena is discussed.

Amines↗

PubMed1966

Induction of cellular morphogenesis in Myxococcus xanthus. I. General description.

Dworkin, Martin (University of Minnesota, Minneapolis), and William Sadler. Induction of cellular morphogenesis in Myxococcus xanthus. I. General description. J. Bacteriol. 91:1516-1519. 1966.-The details of a process for converting vegetative rods of Myxococcus xanthus to microcysts rapidly (120 min), quantitatively, and synchronously are presented. The conversion is induced by 0.5 m glycerol. On the basis of a number of morphological and physiological parameters, the microcysts thus produced are identical with those produced within fruiting bodies. Microcyst formation requires Mg(++) or Ca(++) and aeration, and is considerably stimulated by the presence of the growth medium. The process is reversible, by the removal of the inducer, and will take place only with exponentially growing cells. Mutants unable to form microcysts have been isolated. These are also deficient in their ability to form fruiting bodies.

Bacteria↗

PubMed1966

Induction of cellular morphogenesis in Myxococcus xanthus. II. Macromolecular synthesis and mechanism of inducer action.

Sadler, William (University of Minnesota, Minneapolis), and Martin Dworkin. Induction of cellular morphogenesis in Myxococcus xanthus. II. Macromolecular synthesis and mechanism of inducer action. J. Bacteriol. 91:1520-1525. 1966.-Net changes in ribonucleic acid (RNA), deoxyribonucleic acid (DNA), and protein syntheses in cells of Myxococcus xanthus during induced, synchronous conversion to microcysts are described. The net synthesis of all three macromolecules was temporarily halted for a brief period during the initiation of shape change. Synthesis then resumed and leveled off when refractile microcysts began to appear. The conversion was completely sensitive, throughout the process, to low concentrations of chloramphenicol and actinomycin D. The uptake of amino acids and uracil was linear throughout the conversion, suggesting that the plateaus in rates of net synthesis of protein and RNA represented a period of rapid turnover. The most effective inducers of microcyst formation were fully saturated aliphatic compounds containing 2 to 4 carbon atoms and at least one primary or secondary alcohol group. Studies with labeled inducer indicated that the inducer need not be taken up by the cells to be effective, and probably interacts with some peripheral structure of the cell. The possibility that induction involves an alteration of a membrane-DNA complex is discussed.

Alcohols↗

PubMed1966

Lytic enzyme produced by Myxococcus xanthus.

Hart, Beth A. (Cornell University, Ithaca, N.Y.), and Stanley A. Zahler. Lytic enzyme produced by Myxococcus xanthus. J. Bacteriol. 92:1632-1637. 1966.-Strain FBa of Myxococcus xanthus releases into its culture medium an enzyme capable of lysing Micrococcus lysodeikticus cells and of releasing N-acetyl amino sugars from their cell walls. The lysin is stable at pH values near neutrality and at temperatures below 50 C. It lyses a number of bacterial species sensitive to egg-white lysozyme, and fails to lyse lysozyme-resistant species. Sensitivity of M. lysodeikticus cell walls to FBa lysin and to lysozyme is changed in a similar manner by addition and removal of O-acetyl groups. We describe methods for obtaining 190-fold purification of the lysin from culture filtrates, using acetone and ammonium sulfate precipitations and filtration through diethylaminoethyl cellulose. We also describe a gel filtration method for separating the lysin from a protease which is also produced by strain FBa.

Bacteria↗

PubMed1976

The isolation and characterization of gliding motility mutants of Myxococcus xanthus.

Nonmotile and motility-altered mutants of Myxococcus xanthus have been obtained by the use of chemical mutagens, ultraviolet irradiation, and a procedure for selective spontaneous mutants. As judged by their behaviour on a variety of growth media, in both plate and slide culture, the mutants were divided into four groups. One group contains mutants which are truly nonmotile. Myxococcus xanthus NM, previously described as a nonmotile mutant, may be similar to type 3 mutants (described in text).

Hycanthone↗

PubMed1979

The function of fimbriae in Myxococcus xanthus. I. Purification and properties of M. xanthus fimbriae.

Myxococcus xanthus fimbriae have been purified and characterized as part of a study of the function of fimbriae in this prokaryote. Myxococcus xanthus produced two types of fimbriae, termed flaccid (F) and rigid (R) on the basis of electron microscopy. F and R fimbriae differed slightly in their response to pH and freeze-thaw regimes but were similar in their resistance to hydrolytic enzymes, amino acid composition, molecular weight, carbohydrate content, and antigenic determinants. Although the precise relationship between F and R fimbriae is unknown, the possibility is considered that F fimbriae might represent a "contracted" form of the R type. Studies designed to determine fimbriae function in M. xanthus are described in an accompanying report.

Amino Acids↗

PubMed1975

[Myxobacteria of the Myxococcus family as indirect indicators of fecal matter in surface water. 1. Communication (author's transl)].

The fruiting-body-forming Myxobacteria of the Myxococcus order are coprophilic, i.e., they accumulate in biotopes that contain faecal substances. Therefore, a special detection of Myxococcus in water, based on the membrane-filter method, has been worked-out. Field studies, undertaken in the region of a mechanico-biological clarifying plant, above a certain stretch of a stream (the Regnitz) laden with waste-water, and on the Bodensee (=Lake Constance) have revealed a clear correlation between the load of faecal substances in such waters and their content of Myxococci. In this way, then, these germs can be used as indirect bacterial indicators of faecal substances.

Agar↗

PubMed1994

Territorial interactions between two Myxococcus Species.

It is unusual to find fruiting bodies of different myxobacteria occupying the same territory on natural samples. We were thus interested in determining whether myxobacteria establish territorial dominance and, if so, what the mechanism of that interaction is. We had previously observed that vegetative swarms of Myxococcus xanthus and Stigmatella aurantiaca placed close to each other on an agar surface initially merged but eventually separated. Further studies indicated that these two species also formed separate fruiting bodies when mixed together on developmental agar (unpublished observation). We examined the interactions between two more closely related myxobacteria, M. xanthus and M. virescens, in greater detail. When mixtures of a kanamycin-resistant strain of M. xanthus and a kanamycin-sensitive strain of M. virescens were placed together under developmental conditions, the cells sorted themselves out and established separate fruiting body territories. In addition, differential viable counts of a mixture of the two species during development indicated that each strain was producing an extracellular component that inhibited the growth and development of the other. Nevertheless, finally, M. virescens invariably outcompeted M. xanthus at all input ratios of M. xanthus/M. virescens tested. This is consistent with the observation that M. virescens is by far the more commonly encountered of the two species. The properties of the inhibitory substance from M. virescens are consistent with the possibility that it is a bacteriocin. Our working hypothesis is that the bacteriocin plays a role in the establishment of myxobacterial territoriality. If so, this is an example of an ecological function of bacteriocins.

Bacteriocins↗

PubMed1999

Fibrils as extracellular appendages of bacteria: their role in contact-mediated cell-cell interactions in Myxococcus xanthus.

Social behavior in the myxobacterium Myxococcus xanthus involves epicellular, peritrichous appendages called fibrils. These are polysaccharide organelles containing a set of tightly adhering proteins. It is proposed that cell-cell contact is perceived by the fibrils and is mediated by the action of a fibrillar ADP-ribosyl transferase. Fibrils or fibril-like organelles have also been found on a variety of other gram-negative bacteria and at least one archaeon, and may mediate cell-cell contact between the bacteria themselves or between the bacteria and their eukaryotic host cells.

Animals↗

PubMed2002

Optimizing the heterologous production of epothilone D in Myxococcus xanthus.

The heterologous production of epothilone D in Myxococcus xanthus was improved by 140-fold from an initial titer of 0.16 mg/L with the incorporation of an adsorber resin, the identification of a suitable carbon source, and the implementation of a fed-batch process. To reduce the degradation of epothilone D in the basal medium, XAD-16 (20 g/L) was added to stabilize the secreted product. This greatly facilitated its recovery and enhanced the yield by three-fold. The potential of using oils as a carbon source for cell growth and product formation was also evaluated. From a screen of various oils, methyl oleate was shown to have the greatest impact. At the optimal concentration of 7 mL/L in a batch process, the maximum cell density was increased from 0.4 g dry cell weight (DCW)/L to 2 g DCW/L. Product yield, however, depended on the presence of trace elements in the production medium. With an exogenous supplement of trace metals to the basal medium, the peak epothilone D titer was enhanced eight-fold. This finding demonstrates the significant role of metal ions in cell metabolism and in epothilone biosynthesis. To further increase the product yield, a continuous fed-batch process was used to promote a higher cell density and to maintain an extended production period. The optimized fed-batch cultures consistently yielded a cell density of 7 g DCW/L and an average production titer of 23 mg/L.

Drug Stability↗

PubMed2005

Fermentation, purification, formulation, and pharmacological evaluation of a prolyl endopeptidase from Myxococcus xanthus: implications for Celiac Sprue therapy.

Celiac Sprue is a multi-factorial disease characterized by an inflammatory response to ingested wheat gluten and similar proteins in rye and barley. Proline-rich gluten peptides from wheat, rye, and barley are relatively resistant to gastrointestinal digestion, and therefore persist in the intestinal lumen to elicit immunopathology in genetically susceptible individuals. In this study, we characterize the in vitro gluten detoxifying properties of a therapeutically promising prolyl endopeptidase from Myxococcus xanthus (MX PEP), and describe the development of a prototypical enteric-coated capsule containing a pharmacologically useful dose of this enzyme. A high-cell density fed-batch fermentation process was developed for overproduction of recombinant MX PEP in E. coli, yielding 0.25-0.4 g/L purified protein. A simple, scalable purification and lyophilization procedure was established that yields >95% pure, highly active and stable enzyme as a dry powder. The dry powder was blended with excipients and encapsulated in a hard gelatin capsule. The resulting capsule was enteric coated using Eudragit L30-D55 polymer coat, which provided sufficient resistance to gastric conditions (> 1 h in 0.01 M HCl, pH 2 with pepsin) and rapid release under duodenal conditions (15-30 min release in pH 6.0 in the presence of trypsin and chymotrypsin). In conjunction with pancreatic enzymes, MX PEP breaks down whole gluten into a product mixture that is virtually indistinguishable from that generated by the Flavobacterium meningosepticum (FM) PEP as judged by chromatographic assays. Competitive studies involving selected immunogenic peptides mixed with whole gluten reveal that both PEPs have a wide range of substrate specificity. Our results support further in vitro and in vivo evaluation of the MX PEP capsule as an oral therapeutic agent for Celiac Sprue patients.

Celiac Disease↗

PubMed2004

Biosynthesis of volatiles by the myxobacterium Myxococcus xanthus.

The volatiles emitted from cell cultures of myxobacterium Myxococcus xanthus were collected by use of a closed-loop stripping apparatus (CLSA) and analyzed by GC-MS. Two new natural products, (S)-9-methyldecan-3-ol ((S)-1) and 9-methyldecan-3-one (2), were identified and synthesized, together with other aliphatic ketones and alcohols, and terpenes. Biosynthesis of the two main components (S)-1 and 2 was examined in feeding experiments carried out with the wild-type strain DK1622 and two mutant strains JD300 and DK11017, which are impaired in the degradation pathway from leucine to isovaleryl-SCoA. Isovaleryl-SCoA is used as a starter, followed by chain elongation with two malonate units. Subsequent use of methyl malonate and decarboxylation leads to (S)-1 and 2. Furthermore, 3,3-dimethylacrylic acid (DMAA) can be used by the mutant strain to form isovaleryl-SCoA, which corroborates recent data on the detection of a novel variety of the mevalonate pathway giving rise to isovaleryl-SCoA from HMGCoA.

Acrylates↗

PubMed2006

Self-organized and highly ordered domain structures within swarms of Myxococcus xanthus.

Coordinated group movement (swarming) is a key aspect of Myxococcus xanthus' social behavior. Here we report observation of domain structures formed by multiple cells within large three-dimensional swarming groups grown on amorphous glass substrates, using the atomic force microscope (AFM). Novel analyses revealed that 90% of the wild type swarms displayed some form of preferential cell alignment. In contrast, cells with mutations in the social and adventurous motility systems displayed a distinct lack of cell alignment. Video microscopy observations of domain features of in vivo swarming M. xanthus cells were also consistent with the AFM data. The results presented here reveal that unique domain formation within swarms of wild type cells is a biologically driven process requiring the social and adventurous motility systems and is not a statistical phenomenon or thermodynamic process arising from liquid crystal behavior.

Microscopy, Atomic Force↗

PubMed1993

Eukaryotic-like protein serine/threonine kinases in Myxococcus xanthus, a developmental bacterium exhibiting social behavior.

Myxococcus xanthus, a gram-negative bacterium exhibits a spectacular life cycle and social behavior. Its developmental cycle and multicellular morphogenesis resemble those of eukaryotic slime molds such as Dictyostelium discoideum. On the basis of this resemblance, we explored the existence of eukaryotic-like protein serine/threonine kinases which are known to play important roles in signal transduction during development of D. discoideum. It was indeed found that M. xanthus contains a large family of protein serine/threonine kinases related to the eukaryotic enzymes. This is the first unambiguous demonstration of eukaryotic-like serine/threonine kinases in the prokaryotes.

Amino Acid Sequence↗

PubMed1993

Crystal structure of Myxococcus xanthus nucleoside diphosphate kinase and its interaction with a nucleotide substrate at 2.0 A resolution.

The X-ray crystallographic structure of nucleoside diphosphate (NDP) kinase from Myxococcus xanthus has been determined using multiple isomorphous replacement techniques and refined at 2.0 A resolution to a crystallographic R-factor of 0.17. This is the first report of the structure of an enzymatically active NDP kinase and of the enzyme with a bound nucleotide. The structure has been determined in P4(3)2(1)2 and I222 crystal forms. The enzyme monomer consists of a four-stranded antiparallel beta-sheet. The surfaces of the sheet are partially covered with five helical segments. There are two protein molecules in the asymmetric unit of the tetragonal crystal form. They form a dimer with an extensive interface in which 1092 A2 per monomer is buried. The majority of the contact area in the dimer interface is between hydrophobic or aromatic residues. Two dimers are related by a crystallographic 2-fold axis to yield a tetramer. This tetramer is also present in the orthorhombic crystals; however, in this case, the 222 symmetry is entirely crystallographic. Upon tetramer formation, an additional 473 A2 of solvent-accessible surface area from each monomer becomes buried. The interface between dimers in the tetramer is stabilized by salt bridges. Equilibrium sedimentation studies are consistent with the enzyme being a tetramer in solution. The structure of a complex of adenosine diphosphate (ADP) with the enzyme was determined and reveals that most of the nucleotide interactions with the protein are with the pyrophosphate and ribose groups, while the base has no hydrogen bonds with the protein and interacts only by stacking with the side chain of Phe59. The Mg2+ interacts with the pyrophosphate of the ADP and via a solvent molecule with the side chain of the conserved Asp120 residue. The mode of interaction with the nucleotide is novel, with the nucleotide binding at the side of the beta-sheet. The structures of the nucleotide in crystals grown in the presence or absence of Mg2+ are essentially identical. In addition, the phosphotransfer reaction from adenosine triphosphate (ATP) to the enzyme can occur without Mg2+. This suggests that only the second step of the reaction in which the enzyme transfers the phosphate to a nucleoside diphosphate acceptor is significantly catalyzed by the metal.

Adenosine Diphosphate↗