Xanthus Strain Research Articles

Cell behaviors underlying Myxococcus xanthus aggregate dispersal.

The soil bacterium Myxococcus xanthus is a model organism with a set of diverse behaviors. These behaviors include the starvation-induced multicellular development program, in which cells move collectively to assemble multicellular aggregates. After initial aggregates have formed, some will disperse, with smaller aggregates having a higher chance of dispersal. Initial aggregation is driven by two changes in cell behavior: cells slow down inside of aggregates and bias their motion by reversing direction less frequently when moving toward aggregates. However, the cell behaviors that drive dispersal are unknown. Here, we use fluorescent microscopy to quantify changes in cell behavior after initial aggregates have formed. We observe that after initial aggregate formation, cells adjust the bias in reversal timings by initiating reversals more rapidly when approaching unstable aggregates. Using agent-based modeling, we then show dispersal is predominantly generated by this change in bias, which is strong enough to overcome slowdown inside aggregates. Notably, the change in reversal bias is correlated with the nearest aggregate size, connecting cellular activity to previously observed correlations between aggregate size and fate. To determine if this connection is consistent across strains, we analyze a second M. xanthus strain with reduced levels of dispersal. We find that far fewer cells near smaller aggregates modified their bias. This implies that aggregate dispersal is under genetic control, providing a foundation for further investigations into the role it plays in the life cycle of M. xanthus.IMPORTANCEUnderstanding the processes behind bacterial biofilm formation, maintenance, and dispersal is essential for addressing their effects on health and ecology. Within these multicellular communities, various cues can trigger differentiation into distinct cell types, allowing cells to adapt to their specific local environment. The soil bacterium Myxococcus xanthus forms biofilms in response to starvation, marked by cells aggregating into mounds. Some aggregates persist as spore-filled fruiting bodies, while others disperse after initial formation for unknown reasons. Here, we use a combination of cell tracking analysis and computational simulations to identify behaviors at the cellular level that contribute to aggregate dispersal. Our results suggest that cells in aggregates actively determine whether to disperse or persist and undergo a transition to sporulation based on a self-produced cue related to the aggregate size. Identifying these cues is an important step in understanding and potentially manipulating bacterial cell-fate decisions.

De Novo Assembly and Annotation of the Complete Genome Sequence of Myxococcus xanthus DZ2.

ABSTRACTWe report the assembly and annotation of a high-quality genome sequence for Myxococcus xanthus strain DZ2 (GenBank accession number CP080538), created using a combination of short reads generated using DNBSEQ technology (BGI Genomics) and long high-fidelity (HiFi) reads generated using Pacific Biosciences (PacBio) technology.

Complete Genome Assembly of Myxococcus xanthus Strain DZ2 Using Long High-Fidelity (HiFi) Reads Generated with PacBio Technology.

ABSTRACTMyxococcus xanthus is a Gram-negative social bacterium belonging to the order Myxococcales of the class Deltaproteobacteria. It is a facultative social predator found in soils across the globe and is thought to be crucial for the microbial ecosystem. Here, we report a complete high-quality reference genome of the M. xanthus strain DZ2.

Biocontrol mechanism of Myxococcus xanthus B25-I-1 against Phytophthora infestans

Phytophthora infestans is the pathogen causing potato late blight, one of the most serious diseases of potato. Myxobacteria have become a valuable biological control resource due to their preponderant abilities to produce various secondary metabolites with novel structure and remarkable biological activity. In this study, Myxococcus xanthus strain B25-I-1, which exhibited strong antagonistic activity against P. infestans, was isolated from soil sample and identified by 16S rRNA sequence analysis. The strain exhibited antagonistic activity against several species of fungus and bacteria. Analysis of the biocontrol mechanism showed that the active extract produced by strain B25-I-1 had strong inhibitory effects on mycelium and the asexual and sexual reproductive structures of P. infestans. Furthermore, these active extract decreased the content of soluble proteins and activity of the protective enzymes (PPO, POD, PAL, and SOD), increased the oxidative damage and the permeability of the cell membrane in P. infestans. All of these mechanisms might be the biocontrol mechanism of B25-I-1 against P. infestans. The active extract of strain B25-I-1 was separated by TLC and HPLC, and the components with antibiotic activity were detected by HPLC-MS. It was found that the antagonistic components of B25-I-1 contained methyl (2R)-2-azido-3-hydroxyl-2-methylpropanoate and N-(3-Amino-2-hydroxypropyl)-N-methylsulfuric diamide. The active extract significantly inhibited the infection on detached potato leaves by P. infestans, and these substances did not cause damage to the potato leaves. In conclusion, M. xanthus B25-I-1 produced active extract against P. infestans and might potentially be a candidate to develop into biological pesticides for the control of potato late blight. This study adds to the literature on the isolation and identification of active extracts from myxobacteria, and B25-I-1 in particular, for cures or treatments to potato late blight.

Proteome Analyses of Soil Bacteria Grown in the Presence of Potato Suberin, a Recalcitrant Biopolymer.

Suberin is a complex lipidic plant polymer found in various tissues including the potato periderm. The biological degradation of suberin is attributed to fungi. Soil samples from a potato field were used to inoculate a culture medium containing suberin as the carbon source, and a metaproteomic approach was used to identify bacteria that developed in the presence of suberin over a 60-d incubation period. The normalized spectral counts of predicted extracellular proteins produced by the soil bacterial community markedly decreased from day 5 to day 20 and then slowly increased, revealing a succession of bacteria. The population of fast-growing pseudomonads declined and was replaced by species with the ability to develop in the presence of suberin. The recalcitrance of suberin was demonstrated by the emergence of auxotrophic bacteria such as Oscillatoria on the last days of the assay. Nevertheless, two putative lipases from Rhodanobacter thiooxydans (I4WGM2) and Myxococcus xanthus (Q1CWS1) were detected in the culture supernatants, suggesting that at least some bacterial species degrade suberin. When grown in suberin-containing medium, R. thiooxydans strain LCS2 and M. xanthus strain DK 1622 both produced three lipases, including I4WGM2 and Q1CWS1. These strains also produced other proteins linked to lipid metabolism, including fatty acid and lipid transporters and β-oxidation enzymes, suggesting that they participate in the degradation of suberin. However, only the R. thiooxydans strain appeared to retrieve sufficient carbon and energy from this recalcitrant polymer in order to maintain its population over an extended period of time.

Evidence that a chaperone–usher-like pathway of Myxococcus xanthus functions in spore coat formation

Many bacteria use the chaperone-usher (CU) secretion pathway to assemble on their surfaces typical or atypical fimbrial organelles. Four consecutive genes of Myxococcus xanthus DK1622, MXAN3885-3882, were predicted to constitute an operon encoding a CU-like system involved in the assembly of the spore coat; however, experimental evidence supporting this hypothesis was lacking. In this study, co-transcription of MXAN3885-3883 was verified, and we found that this operon was expressed 12-15 h after initiation of M. xanthus development under conditions of stringent starvation. The MXAN3885 protein, which is highly homologous to, but expressed earlier than, the spore coat protein U of another M. xanthus strain, DZF1, was present mainly on the outer surface of myxospores. Inactivation of MXAN3883, encoding a putative outer membrane usher, inhibited assembly of MXAN3885 protein on spore surfaces and caused certain morphological alterations in the spore coat. Hence, the CU-like pathway in M. xanthus indeed functions in spore coat biogenesis. Based on chaperone amino acid sequence comparisons, our analysis suggests that the structural basis of the M. xanthus CU-like pathway for spore coat assembly may be different from that of most surface structures assembled by classical CU systems.

The structure of the carbohydrate backbone of the LPS from Myxococcus xanthus strain DK1622

Gram-negative rod shaped bacterium Myxococcus xanthus DK1622 produces a smooth-type LPS. The structure of the polysaccharide O-chain and the core-lipid A region of the LPS has been determined by chemical and spectroscopic methods. The O-chain was built up of disaccharide repeating units having the following structure: → 6 ) - α - d-Glcp- ( 1 → 4 ) - α - d-GalpNAc 6 OMe ∗ - ( 1 → with partially methylated GalNAc residue. The core region consisted of a phosphorylated hexasaccharide, containing one Kdo residue, unsubstituted at O-4, and no heptose residues. The lipid A component consisted of β-GlcN-(1→6)-α-GlcN1P disaccharide, N-acylated with 13-methyl-C14-3OH ( iso-C15-3OH), C16-3OH, and 15-methyl-C16-3OH ( iso-C17-3OH) acids. The lipid portion contained O-linked iso-C16 acid.

Detailed three-dimensional analysis of structural features of Myxococcus xanthus fruiting bodies using confocal laser scanning microscopy

Myxococcus xanthus is a motile soil bacterium with complex social behaviors. Upon starvation, a developmental program is initiated that results in cellular aggregation and fruiting body formation. This process requires the exopolysaccharide (EPS) component of the extracellular matrix. With prolonged starvation, a part of the cells within a fruiting body die, while the other cells differentiate into spores. Extensive genetic and biochemical information has been generated that elucidates this interesting developmental process. Little is known, however, about the detailed three-dimensional structural features of native fruiting bodies or the EPS and distribution of live/dead cells (spores) within these structures. In this study, changes in the three-dimensional architecture of fruiting bodies and the distribution of the extracellular matrix within the fruiting bodies during the developmental process were investigated using a gfp-expressing M. xanthus strain and carbohydrate-specific lectins or monoclonal antibodies in combination with confocal laser scanning microscopy. The extracellular matrix was found to form a scaffold within the fruiting body structure. Furthermore, using a bacterial viability staining assay, the distribution of live/dead cells within fruiting bodies was examined at different times. The majority of live cells were found to localize at the outer layer of a mature fruiting body, with dead cells underneath.

Production and characterization of the milk-clotting protease of Myxococcus xanthus strain 422.

The cheese industry is seeking novel sources of enzymes for cheese production. Microbial rennets have several advantages over animal rennets. (1) They are easy to generate and purify and do not rely on the availability of animal material. (2) The production of microbial clotting enzymes may be improved by biotechnological techniques. In this work, the biochemical characterization of a novel milk-clotting extracellular enzyme from Myxococcus xanthus strain 422 and a preliminary evaluation of its cheese-producing ability are reported. Strain 422 was selected from four M. xanthus strains as the best producer of extracellular milk-clotting activity, based on both its enzyme yield and specific milk-clotting activity, which also afforded lower titration values than enzymes from the three other M. xanthus strains. The active milk-clotting enzyme from M. xanthus strain 422 is a true milk-clotting enzyme with a molecular mass of 40 kDa and a pI of 5.0. Highest milk-clotting activity was at pH 6 and 37 degrees C. The enzyme was completely inactivated by heating for 12 min at 65 degrees C. The crude enzyme preparation was resolved by anion-exchange chromatography into two active fractions that were tested in cheese production assays of compositional (dry matter, fat content, fat content/dry-matter ratio, and moisture-non-fat content) and physicochemical properties (firmness, tensile strength, pH and Aw) of the milk curds obtained. Purified protein fraction II exhibited a significantly higher milk-clotting ability than either protein fraction I or a total protein extract, underlining the potential usefulness of M. xanthus strain 422 as a source of rennet for cheese production.

A new cytotoxic epothilone from modified polyketide synthases heterologously expressed in Myxococcus xanthus.

A new epothilone, 10,11-didehydroepothilone D (5), was isolated from a strain of the heterologous host Myxococcus xanthus genetically engineered to produce epothilone D (4). The structure of 5 was determined from NMR and MS data. The epothilone polyketide synthase was further modified in a recombinant M. xanthus strain to produce 5 as the major epothilone-related metabolite. The cytotoxicity of 5 against a panel of tumor cell lines, including several with multidrug resistance, and its effect on tubulin polymerization were comparable to epothilone D (4).

Starvation yields a drastic decrease in outer-membrane permeability to a periplasmic foreign protein in Myxococcus xanthus

A recombinant Myxococcus xanthus strain was constructed that constitutively produces two proteins from Escherichia coli, the cytoplasmic beta-galactosidase and the periplasmic pH 2.5 acid phosphatase (AppA protein). We have previously shown that during vegetative growth, AppA protein is partly accumulated in the periplasm of M. xanthus and partly released into the medium. We demonstrate here that during starvation-induced development, release of periplasmic AppA protein to the medium did not occur over a period of 20 h. This was coincident with, but not caused by, the arrest of the synthesis of the foreign proteins. We have shown that this lack of secretion could be attributed to starvation per se and did not depend on the ability of the cells to undergo development. Our findings suggest that protein secretion which occurs during the first hours of starvation-induced development might therefore take place via a different route from that which occurs in vegetative cells.

Cloning and nucleotide sequence of the Myxococcus xanthus lon gene: indispensability of lon for vegetative growth.

The lon gene of Escherichia coli is known to encode protease La, an ATP-dependent protease associated with cellular protein degradation. A lon gene homolog from Myxococcus xanthus, a soil bacterium which differentiates to form fruiting bodies upon nutrient starvation, was cloned and characterized by use of the lon gene of E. coli as a probe. The nucleotide sequence of the M. xanthus lon gene was determined. It contains an open reading frame that encodes a 92-kDa protein consisting of 817 amino acid residues. The deduced amino acid sequence of the M. xanthus lon gene product showed 60 and 56% identity with those of the E. coli and Bacillus brevis lon gene products, respectively. Analysis of an M. xanthus strain carrying a lon-lacZ operon fusion suggested that the lon gene is similarly expressed during vegetative growth and development in M. xanthus. In contrast to that of E. coli, the M. xanthus lon gene was shown to be essential for cell growth, since a null mutant could not be isolated.

Development-specific sigma-factor essential for late-stage differentiation of Myxococcus xanthus.

The gene for a developmentally expressed sigma-factor, sigB, has been isolated from Myxococcus xanthus by use of the sigA gene (formerly rpoD) of the vegetative sigma-factor as a probe. The sequence of sigB has been determined, and an open reading frame of 193 amino acid residues (Mr = 21,551) was identified. The amino-terminal region of SigB contains 69 residues, of which 35 are identical (50% identity) to the region of SigA required for core RNA polymerase binding and initiation of RNA polymerization. SigB also possesses many features commonly found in other prokaryotic sigma-factors. Analysis of an M. xanthus strain carrying a sigB-lacZ fusion gene revealed that sigB is expressed from a middle to late stage of differentiation corresponding to the period from the onset of sporulation to late development. A sigB deletion mutant displayed normal mound formation and sporulation; however, production of the ops gene product in myxospores of the delta sigB strain was shown to be blocked. Myxospores from the sigB deletion strain also exhibited severe defects in stability and viability during late development. Our data indicate that sigB encodes a sigma-factor essential for the maturation of myxospores at a late stage of M. xanthus differentiation. Our results also suggest that differentiation of M. xanthus is regulated by development-specific sigma-factors.

Characterization of calcium-binding sites in development-specific protein S of Myxococcus xanthus using site-specific mutagenesis.

Protein S, the most abundant protein synthesized during development of the Gram-negative bacterium Myxococcus xanthus, assembles on the surface of the spores. It can be dissociated from the spores using divalent metal chelators and will reassemble on the spores in the presence of calcium. The amino acid sequence of protein S contains regions which have homology to the calcium-binding sites of calmodulin. Protein S was found to bind 2 mol of calcium/mol of protein with Kd values of 27 and 76 microM. Using oligonucleotide-directed site-specific mutagenesis, the gene coding for protein S was changed in each of two regions of homology to calmodulin (Ser40----Arg,Ser129----Arg), and a double mutant was also constructed. Each mutant gene was then transduced into the genome of a M. xanthus strain from which the wild-type genes had been deleted. All three mutants produced protein S normally during development. One of the mutants (Ser129----Arg) had normal amounts of protein S on its spores, whereas the other (Ser40----Arg) bound much less and the double mutant had virtually none. Analysis of the calcium binding affinities of the purified proteins showed that [Arg40]protein S and [Arg40, Arg129]protein S did not bind detectable quantities of calcium, whereas [Arg129]protein S bound less calcium than the wild-type protein and with a reduced affinity.

Saframycin Mx1, a new natural saframycin isolated from a myxobacterium.

A new natural saframycin was discovered in the culture broth of the myxobacterium, Myxococcus xanthus strain Mx x48. The fermentation and isolation of the antibiotic are described. The name, saframycin Mx1, is proposed. The compound appears to interact with cellular DNA.

Mode of insertion of the broad-host-range plasmid RP4 and its derivatives into the chromosome of Myxococcus xanthus

The mode of insertion of the broad-host-range plasmid RP4 into the chromosome of Myxococcus xanthus strain DZ1 has been analyzed. The plasmid integrated in numerous sites of the chromosome and generated insertional mutations. There is a hot spot of integration located between 31.5 and 34.5 kb clockwise from the EcoRI site of the plasmid. In the absence of this segment the insertion can, however, take place, but much less efficiently. The presence of transposable elements on the plasmid decreases severely the insertion frequency. Once integrated, RP4 could be transferred back to Escherichia coli, either by precise excision or with a segment of the Myxococcus chromosome. The role of site-specific recombination in RP4 integration is discussed.

Genetic analysis of Myxococcus xanthus and isolation of gene replacements after transduction under conditions of limited homology.

Genetic analysis of Myxococcus xanthus is greatly facilitated by the ability to introduce cloned DNA into M. xanthus to generate gene replacement and merodiploid strains. However, gene replacement strains are difficult to obtain when the region(s) of homology between the cloned DNA and the M. xanthus chromosome is limited (less than 1 kilobase). We found that gene replacements can be obtained at an increased frequency by a two-step procedure involving the use of bacteriophage P1 to isolate merodiploid strains followed by generalized transduction to another M. xanthus strain by using phage Mx4.

Adsorption of antibiotic TA to dental hard tissues.

Antibiotic TA (TA) is a wide-spectrum, bactericidal antibiotic produced by Myxococcus xanthus strain TA. It was previously demonstrated that TA binds tightly to soft tissues while retaining its bactericidal activity in the bound form. The present study was undertaken to investigate TA adsorption to dental hard tissues. Slabs of dental tissues that had been cut from periodontally-involved extracted human teeth were treated with TA and then washed in saline with shaking (saline being replaced every 15 minutes). After 30, 60, and 120 minutes of washing, 45, 39, and 27% of the input TA activities were retained on the slabs, respectively. The tooth-bound TA was released slowly into the aqueous medium in active form. Similar experiments with beta-lactam antibiotics resulted in no significant adhesion to the slabs. The study demonstrates that TA binds tightly to dental tissues while retaining its bactericidal properties.

Gene expression during development of Myxococcus xanthus: Analysis of the genes for protein S

Protein S is an abundant spore coat protein produced during fruiting body formation (development) of the bacterium Myxococcus xanthus. We have cloned the DNA which codes for protein S and have found that this DNA hybridizes to three protein S RNA species from developmental cells but does not hybridize to RNA from vegetative cells. The half-life of protein S RNA was found to be unusually long, about 38 minutes, which, at least in part, accounts for the high level of protein S synthesis observed during development. Hybridization of restriction fragments from cloned M. xanthus DNA to the developmental RNAs enabled us to show that M. xanthus has two directly repeated genes for protein S (gene 1 and gene 2) which are separated by about 10 3 base-pairs on the bacterial chromosome. To study the expression of the protein S genes in M. xanthus, eight M. xanthus strains were isolated with Tn5 insertions at various positions in the DNA which codes for protein S. The strains which contained insertions in gene 1 or between gene 1 and gene 2 synthesized all three protein S RNA species and exhibited normal levels of protein S on spores. In contrast, M. xanthus strains with insertions in gene 2 had no detectable protein S on spores and lacked protein S RNA. Thus, gene 2 is responsible for most if not all of the production of protein S during M. xanthus development. M. xanthus strains containing insertions in gene 1, gene 2 or both genes, were found to aggregate and sporulate normally even though strains bearing insertions in gene 2 contained no detectable protein S. We examined the expression of gene 1 in more detail by constructing a fusion between the lacZ gene of Escherichia coli and the N-terminal portion of protein S gene 1 of M. xanthus. The expression of β-galactosidase activity in an M. xanthus strain containing the gene fusion was shown to be under developmental control. This result suggests that gene 1 is also expressed during development although apparently at a much lower level than gene 2.

A macrocyclic antibiotic M-230B produced by Myxococcus xanthus. Isolation and characterization.

Myxococcus xanthus strain M516E produced at least three related antibiotics against Gram-positive and Gram-negative bacteria. From physico-chemical properties, a main component was identical to myxovirescin A and a second component, designated M-230B was found to be an antibiotic which is closely related to myxovirescin A. The structure of M-230B was determined from its physico-chemical properties, especially from 13C NMR spectrum as compared with that of myxovirescin A. The addition of alcohol, such as isobutyl alcohol, to the culture medium markedly stimulated production of the antibiotics.