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.     

A unique repetitive DNA sequence in the Myxococcus xanthus genome.

We found a novel type of repetitive DNA sequence in the Myxococcus xanthus genome. The first repetitive sequence is located in the spacer region between the ops and tps genes. We cloned five other repetitive sequences using the first repetitive sequence as a probe and determined their nucleotide sequences. Comparison of these sequences revealed that the repetitive sequences consist of a 87-bp core sequence and that some clones share additional homology on their flanking regions.     

A broad-host-range cloning vector transposable to various replicons

A system is described for the stable insertion of cloned DNA sequences into the chromosomes of Gram-negative bacteria. Two broad-host-range plasmids form the basis of the system: one (the "carrier") contains a transposable DNA sequence into which foreign DNA can be cloned; the second (the "helper") provides transposition functions in trans. Both plasmids can be readily transferred between Gram-negative bacteria by conjugation. Instability of the carrier allows enrichment for the products of transposition to the chromosome of the new host, following which the insertion can be stabilised by elimination of the helper. The system was successfully tested in Escherichia coli, Methylophilus methylotrophus and Pseudomonas aeruginosa, and the insertions were stable in each case (less than 0.02% loss per generation).     

Physical characterization of the genome of the Myxococcus xanthus bacteriophage MX-8

Summary We have constructed a restriction map for the genome of bacteriophage MX-8 from Myxococcus xanthus using the enzymes PvuII, MboI, and EcoRI. The phage genome size, as determined by restriction analysis, is 51.7±0.6 Kb. Double digestions, redigestions of isolated fragments, and crossed-contact hybridization of partial digestion products show that the restriction map is circular. Restriction analysis and Southern hybridization show that the phage DNA molecules are packaged sequentially from a concatemer starting from a specific site which we have mapped. The DNA molecules have an average terminal redundancy of approximately 8% and are circularly permuted over at least 40% of the genome.     

Novel change in the carbohydrate portion of Myxococcus xanthus lipopolysaccharide during development.

We have examined the alterations in lipopolysaccharide during aggregation and early development in Myxococcus xanthus. The lipopolysaccharide was isolated and characterized from cells developing on agar during glycerol induction and vegetative growth. A methylated amino sugar was identified as 6-O-methylgalactosamine by gas-liquid chromatography-mass spectrometry. This novel sugar was enriched in cells developing on agar.     

Molecular cloning, sequence analysis, and characterization of a penicillin-resistant DD-carboxypeptidase of Myxococcus xanthus.

We have cloned a gene, pdcA, from the genomic library of Myxococcus xanthus with an oligonucleotide probe representing conserved regions of penicillin-resistant DD-carboxypeptidases. The amino- and carboxy-terminal halves of the predicted pdcA gene product showed significant sequence similarity to N-acetylmuramoyl-L-alanine amidase and penicillin-resistant DD-carboxypeptidase, respectively. The pdcA gene was expressed in Escherichia coli, and the characteristics of the gene product were similar to those of DD-carboxypeptidase (VanY) of vancomycin-resistant enterococci. No apparent changes in cell growth, sporulation, or germination were observed in pdcA deletion mutants.     

Synthesis and Assembly of a Novel Glycan Layer in Myxococcus xanthus Spores

Myxococcus xanthus is a Gram-negative deltaproteobacterium that has evolved the ability to differentiate into metabolically quiescent spores that are resistant to heat and desiccation. An essential feature of the differentiation processes is the assembly of a rigid, cell wall-like spore coat on the surface of the outer membrane. In this study, we characterize the spore coat composition and describe the machinery necessary for secretion of spore coat material and its subsequent assembly into a stress-bearing matrix. Chemical analyses of isolated spore coat material indicate that the spore coat consists primarily of short 1–4- and 1–3-linked GalNAc polymers that lack significant glycosidic branching and may be connected by glycine peptides. We show that 1–4-linked glucose (Glc) is likely a minor component of the spore coat with the majority of the Glc arising from contamination with extracellular polysaccharides, O-antigen, or storage compounds. Neither of these structures is required for the formation of resistant spores. Our analyses indicate the GalNAc/Glc polymer and glycine are exported by the ExoA-I system, a Wzy-like polysaccharide synthesis and export machinery. Arrangement of the capsular-like polysaccharides into a rigid spore coat requires the NfsA–H proteins, members of which reside in either the cytoplasmic membrane (NfsD, -E, and -G) or outer membrane (NfsA, -B, and -C). The Nfs proteins function together to modulate the chain length of the surface polysaccharides, which is apparently necessary for their assembly into a stress-bearing matrix.     

Micron, a microsatellite-targeting transposable element in the rice genome

We have isolated a new family of mobile elements, Micron, which occur within microsatellites dispersed throughout the rice (Oryza sativa) genome. The first of these segments, Micron 001, was found in a microsatellite consisting of a (TA)n sequence upstream of the rice phytochrome A (phyA) gene. PCR analysis of related rice species suggests that Micron 001 integrated into this microsatellite locus prior to the divergence of the two wild species O. rufipogon and O. barthii from a common ancestor. Micron elements are short (393-bp), possess subterminal inverted repeats and the single strands have the potential to form stable secondary structures via several internal repeats. Aside from the absence of terminal inverted repeats, these characteristics resemble those of MITEs (Miniature Inverted-Repeat Transposable Elements). We estimate that 100-200 copies of Micron-related sequences are present in the rice nuclear genome, while the chloroplast and mitochondrial genomes lack this sequence. Nineteen homologs of Micron 001 exhibited extremely high nucleotide sequence conservation (greater than 90%), suggesting a recent spread of Micron elements within the genus Oryza. Surprisingly, nucleotide sequence alignments showed that all of the Micron elements are flanked on both sides by microsatellite sequence consisting mainly of (TA)n. Twenty-three elements were mapped to seven separate chromosomes. Therefore Micron elements form a family of dispersed, highly conserved repeats. This is the first report of a transposable element that targets microsatellite loci.     

Isolation of a surface glycoprotein from Myxococcus xanthus.

The isolation of a glycoprotein from vegetative cells of Myxococcus xanthus is reported. The protein, abbreviated VGP, was first identified during a survey of surface proteins as a major protein that could be radioiodinated in vegetative, but not developing, cells (P.Y. Maeba, J. Bacteriol. 155:1033-1041, 1983). The protein was extracted from membranes with Triton X-100 and subsequently purified by DEAE-cellulose chromatography, chromatofocusing, and gel filtration. The protein has an Mr of approximately 74,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and an isoelectric point of 3.2 to 3.3. The carbohydrate moiety which made up approximately 13.5% of the weight of the VGP comprised primarily neutral sugars and smaller amounts of hexosamines and uronic acids. The amino acid content revealed no unusual features, but analysis by the method of Barrantes (F. Barrantes, Biochem. Biophys. Res. Commun. 62:407-414, 1975) indicated it is likely a peripheral membrane protein. The protein makes up approximately 1% of the total cell protein and is a prominent surface structure. Because glycoproteins have been implicated in cellular interactions in a number of systems, the VGP may play an important role in the social behavior exhibited by M. xanthus.     

Nutrition of Myxococcus xanthus, a fruiting myxobacterium.

The minimal requirements for vegetative growth of Myxococcus xanthus have been sought. Isoleucine, leucine, and valine were required, and vitamin B12 was needed for the synthesis of methionine. Pyruvate was an excellent energy source and an efficient source of cellular carbon. Acetate, aspartate, glutamate, and most tricarboxylic acid cycle intermediates could also be utilized, but were less efficient sources of carbon and energy than was pyruvate. Many mono- and disaccharides were tested, but, in agreement with earlier results, none served as carbon-energy sources. A minimal medium (A1) has been devised that includes the essential amino acids and vitamin B12, with pyruvate and aspartate as carbon-energy sources. In this medium, M. xanthus could propagate indefinitely, and on it vegetative cells formed colonies with greater than 75% efficiency; hence, it is likely that no organic cofactors other than those present in A1 are required in more than trace amounts.     

Proteins associated with the Myxococcus xanthus extracellular matrix

Fruiting body formation of Myxococcus xanthus, like biofilm formation of many other organisms, involves the production of an extracellular matrix (ECM). While the polysaccharide component has been studied, the protein component has been largely unexplored. Proteins associated with the ECM were solubilized from purified ECM by boiling with sodium dodecyl sulfate and were identified by liquid chromatography-tandem mass spectrometry of tryptic fragments. The ECM is enriched in proteins of novel function; putative functions were assigned for only 5 of the 21 proteins. Thirteen putative ECM proteins had lipoprotein secretion signals. The genes for many ECM proteins were disrupted in the wild-type (WT), fibA, and pilA backgrounds. Disruption of the MXAN4860 gene had no effect in the WT or fibA background but in the pilA background resulted in a 24-h delay in aggregation and sporulation compared to its parent. The results of this study show that the M. xanthus ECM proteome is diverse and novel.     

Physical map of the Myxococcus xanthus chromosome.

The genome of Myxococcus xanthus, which is 9,454 kbp, is one of the largest bacterial genomes. The organization of the DNA and the distribution of genes encoding social and developmental behaviors were examined by using pulsed field gel electrophoresis. Intact genomic DNA was digested with AseI into 16 restriction fragments, which were separated by contour-clamped homogeneous electric field electrophoresis, purified, and radiolabeled. Each AseI fragment was hybridized to SpeI-digested DNA and to an M. xanthus genomic library contained in yeast artificial chromosomes. Some SpeI restriction fragments and yeast artificial chromosome clones contained AseI sites and hybridized with two different AseI restriction fragments, providing evidence for the juxtaposition of these AseI restriction fragments in the chromosome. The deduced AseI physical map is circular, suggesting that this bacterium contains a single, circular chromosome. Transposable elements shown by transduction to be in or near genes of interest were located on specific AseI restriction fragments by restriction analysis and Southern hybridization. Most AseI restriction fragments contained genes involved in social and developmental behaviors.     

Discovery of the autonomously replicating plasmid pMF1 from Myxococcus fulvus and development of a gene cloning system in Myxococcus xanthus

Myxobacteria are very important due to their unique characteristics, such as multicellular social behavior and the production of diverse and novel bioactive secondary metabolites. However, the lack of autonomously replicating plasmids has hindered genetic manipulation of myxobacteria for decades. To determine whether indigenous plasmids are present, we screened about 150 myxobacterial strains, and a circular plasmid designated pMF1 was isolated from Myxococcus fulvus 124B02. Sequence analysis showed that this plasmid was 18,634 bp long and had a G+C content of 68.7%. Twenty-three open reading frames were found in the plasmid, and 14 of them were not homologous to any known sequence. Plasmids containing the gene designated pMF1.14, which encodes a large unknown protein, were shown to transform Myxococcus xanthus DZ1 and DK1622 at high frequencies ( approximately 10(5) CFU/microg DNA), suggesting that the locus is responsible for the autonomous replication of pMF1. Shuttle vectors were constructed for both M. xanthus and Escherichia coli. The pilA gene, which is essential for pilus formation and social motility in M. xanthus, was cloned into the shuttle vectors and introduced into the pilA-deficient mutant DK10410. The transformants subsequently exhibited the ability to form pili and social motility. Autonomously replicating plasmid pMF1 provides a new tool for genetic manipulation in Myxococcus.     

Mutants of Myxococcus xanthus insensitive to glycerol-induced myxospore formation.

Mutants of Myxococcus xanthus FBt unable to form myxospores in response to 0.5 M glycerol arise spontaneously with a frequency of 1--3 X 10(-5). These mutants are designated glc. Ultraviolet mutagenesis increases the frequency to a maximum of 7% of the survivors. The reversion frequency following ultraviolet irradiation of spontaneous glc mutants is less than 10(-3). Of four glc mutants examined, none form myxospores in response to the alternative inducers, ethylene glycol and dimethyl sulphoxide. One glc mutant is induced by 1.5 M glycerol; strain FBt responds to this glycerol concentration with low efficiency myxospore formation. Strain FBt and glc mutants all produce myxospores with low efficiency in response to phenyl ethanol. Of 117 glc mutants tested, 109 form fruiting bodies containing mature myxospores; thus, mutations to the glc phenotype do not normally block myxospore formation within the fruiting cycle of the organism.     

Cell surface properties correlated with cohesion in Myxococcus xanthus.

The gliding behavior of Myxococcus xanthus cells is controlled by two multigene systems, A and S, which encode information for adventurous and social behaviors, respectively. The S system can be genetically disrupted through mutation, such as a dsp mutation, or phenotypically disrupted by treating cells with the diazo dye Congo red (Arnold and Shimkets, J. Bacteriol. 170:5765-5770, 1988). One of the functions controlled by the S system is cell agglutination. Immediately after the induction of agglutination, wild-type cells begin to form aggregates, and within 30 min the cells are packed side-to-side in clumps containing thousands of cells. Changes in the cohesive properties of S+ cells are correlated with changes in the topology of the cell surface observed by electron microscopy. Two types of cell-associated appendages were observed on wild-type cells: thin filaments (ca. 5 nm in diameter), which have been called fimbriae or pili, at one cell pole, and thick, flaccid filaments (ca. 50 nm in diameter), referred to as fibrils, at both the sides and tips of cells. Cohesion was correlated with the secretion of the thick fibrils, which coat the cell surface and form an extracellular matrix in which the cells are interconnected. Several lines of evidence suggest that these thick fibrils are involved in cohesion. First, Dsp cells were unable to agglutinate or secrete this extracellular material. Second, wild-type cells which were treated with Congo red neither agglutinated nor secreted the extracellular fibrils. Finally, removal of the Congo red from wild-type cells restored cohesion and also restored production of the thick fibrils. Attempts to estimate the efficiency with which two cells cohered following collision suggested that under optimal conditions, one in three collisions resulted in stable contact. The collision efficiency decreased linearly as the cell density increased, suggesting a cell density-dependent regulation of cohesion. Some aspects of gliding behavior can be explained in terms of an inducer and an inhibitor of S motility.     

Genetic identification and cloning of a gene required for developmental cell interactions in Myxococcus xanthus

Developmental mutants of Myxococcus xanthus have been previously described which appear to be defective in required cell-cell interactions. These mutants fall into four phenotypic classes, Asg, Bsg, Csg, and Dsg, each of which is unable to differentiate into spores but can be rescued by extracellular complementation by wild-type cells or by mutants of a different class. We report the identification of one of the loci in which mutations result in a Bsg phenotype. The cloned locus was contained on a 12-kilobase EcoRI fragment and then localized by subcloning and a combination of in vitro and transposon mutagenesis. All mutations in this locus behave as a single complementation group, which we designate bsgA (formerly ssbA). Each of the bsgA mutations results in a nonsporulating phenotype, which can be rescued by extracellular complementation. Furthermore, we report that the bsgA mutants have a distinctive interaction with wild-type cells when vegetatively growing, swarming colonies converge.     

Tyrosine phosphorylation in Myxococcus xanthus, a multicellular prokaryote.

Tyrosine phosphorylation is an extremely rare event in prokaryotes, occurring almost exclusively in multicellular eukaryotes. We have identified, for the first time, by the use of antiphosphotyrosine monoclonal antibody and Western blot (immunoblot) analysis, two tyrosine-phosphorylated membrane proteins in the multicellular prokaryote Myxococcus xanthus. The pattern of tyrosine phosphorylation was shown to change during development, indicating a possible role for this regulatory modification during two stages of development, i.e., aggregation and sporulation. Furthermore, the altered pattern of tyrosine phosphorylation observed in a variety of signaling mutants was shown to differ from that observed in the wild type, suggesting further the possible involvement of tyrosine phosphorylation during the development program.