Exopolysaccharides promote Myxococcus xanthus social motility by inhibiting cellular reversals

The biofilm‐forming bacterium Myxococcus xanthus moves on surfaces as structured swarms utilizing type IV pili‐dependent social (S) motility. In contrast to isolated cells that reverse their moving direction frequently, individual cells within swarms rarely reverse. The regulatory mechanisms that inhibit cellular reversal and promote the formation of swarms are not well understood. Here we show that exopolysaccharides (EPS), the major extracellular components of M. xanthus swarms, inhibit cellular reversal in a concentration‐dependent manner. Thus, individual wild‐type cells reverse less frequently in swarms due to high local EPS concentrations. In contrast, cells defective in EPS production hyper‐reverse their moving direction and show severe defects in S‐motility. Surprisingly, S‐motility and wild‐type reversal frequency are restored in double mutants that are defective in both EPS production and the Frz chemosensory system, indicating that EPS regulates cellular reversal in parallel to the Frz pathway. Here we clarify that besides functioning as the structural scaffold in biofilms, EPS is a self‐produced signal that coordinates the group motion of the social bacterium M. xanthus.     

Recording Multicellular Behavior in Myxococcus xanthus Biofilms using Time-lapse Microcinematography

A swarm of the δ-proteobacterium Myxococcus xanthus contains millions of cells that act as a collective, coordinating movement through a series of signals to create complex, dynamic patterns as a response to environmental cues. These patterns are self-organizing and emergent; they cannot be predicted by observing the behavior of the individual cells. Using a time-lapse microcinematography tracking assay, we identified a distinct emergent pattern in M. xanthus called chemotaxis, defined as the directed movement of a swarm up a nutrient gradient toward its source ¹.In order to efficiently characterize chemotaxis via time-lapse microcinematography, we developed a highly modifiable plate complex (Figure 1) and constructed a cluster of 8 microscopes (Figure 2), each capable of capturing time-lapse videos. The assay is rigorous enough to allow consistent replication of quantifiable data, and the resulting videos allow us to observe and track subtle changes in swarm behavior. Once captured, the videos are transferred to an analysis/storage computer with enough memory to process and store thousands of videos. The flexibility of this setup has proven useful to several members of the M. xanthus community.Download video file.^{(133M, mp4)}     

Extracellular fibrils and contact-mediated cell interactions in Myxococcus xanthus.

Contact-mediated cell-cell interactions play an important role in the social life-style of Myxococcus xanthus. Previous investigations have demonstrated that fimbriae (also referred to as pili) and extracellular fibrils are involved in these social interactions (L. J. Shimkets, Microbiol. Rev. 54:473-501, 1990). We have used the relatively new technique of low-voltage scanning electron microscopy (an ultra-high-resolution scanning technique that allows for the nanometer resolution of biological materials) to observe the topological details of cell-cell interactions in M. xanthus. Our observations indicated that the fibrils (which measure approximately 30 nm in diameter) are produced most extensively by cells that are in close contact with each other and are aberrantly produced by the cohesion-deficient dsp mutants. Immunogold analysis identified an antigen which is located exclusively on the extracellular fibrils. Western blots (immunoblots) of this antigen (designated FA-1 for fibrillar antigen 1) indicated that it is composed of several immunoreactive bands (molecular size range, 90 to 14 kDa), all of which are sensitive to protease digestion. A technique for fibril isolation was developed by using FA-1 as a fibril-specific marker. Low-voltage scanning electron microscope observations of swarming cells demonstrated that the expression of fibrils is differentially regulated between adventurous (individual) and socially (group) motile cells. The differential expression of fibrils suggests the existence of a mechanism for the regulation of fibril biosynthesis that functions within the overall system governing social interactions in M. xanthus.     

Intracellular and Extracellular Nucleotides and Related Compounds During the Development of Myxococcus xanthus

Changes in nucleotide pools and extracellular nucleotides during the developmental cycle of the myxobacterium Myxococcus xanthus were determined using a high-pressure liquid chromatography nucleotide analyzer. A general increase in all nucleotide pools occurred during the morphological phase of glycerol conversion of vegetative cells to myxospores. The levels of the nucleoside triphosphate pools remained high as the myxospore matured and throughout subsequent germination. Oxidized nicotinamide adenine dinucleotide levels were elevated in the dormant myxospore and then declined during germination. The adenylate energy charge value was 0.85 +/- 0.02 for vegetative cells, germinating myxospores, and 6-h-old myxospores. It was interesting that the value for the so-called dormant myxospore was the same as that characteristic of physiologically active cells. The germinating myxospores excreted large quantities of uracil along with lesser quantities of purine nucleoside monophosphates. Although the source of the extracellular uracil cannot be determined from these experiments, it may have been derived from a shift in base ratios accompanying an assumed ribonucleic acid turnover during germination.     

Extracellular DNA in Single- and Multiple-Species Unsaturated Biofilms

The extracellular polymeric substances (EPS) of bacterial biofilms form a hydrated barrier between cells and their external environment. Better characterization of EPS could be useful in understanding biofilm physiology. The EPS are chemically complex, changing with both bacterial strain and culture conditions. Previously, we reported that Pseudomonas aeruginosa unsaturated biofilm EPS contains large amounts of extracellular DNA (eDNA) (R. E. Steinberger, A. R. Allen, H. G. Hansma, and P. A. Holden, Microb. Ecol. 43:416-423, 2002). Here, we investigated the compositional similarity of eDNA to cellular DNA, the relative quantity of eDNA, and the terminal restriction fragment length polymorphism (TRFLP) community profile of eDNA in multiple-species biofilms. By randomly amplified polymorphic DNA analysis, cellular DNA and eDNA appear identical for P. aeruginosa biofilms. Significantly more eDNA was produced in P. aeruginosa and Pseudomonas putida biofilms than in Rhodococcus erythropolis or Variovorax paradoxus biofilms. While the amount of eDNA in dual-species biofilms was of the same order of magnitude as that of of single-species biofilms, the amounts were not predictable from single-strain measurements. By the Shannon diversity index and principle components analysis of TRFLP profiles generated from 16S rRNA genes, eDNA of four-species biofilms differed significantly from either cellular or total DNA of the same biofilm. However, total DNA- and cellular DNA-based TRFLP analyses of this biofilm community yielded identical results. We conclude that extracellular DNA production in unsaturated biofilms is species dependent and that the phylogenetic information contained in this DNA pool is quantifiable and distinct from either total or cellular DNA.     

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 Chaperone in the HSP70 Family Controls Production of Extracellular Fibrils in Myxococcus xanthus

Three independent Tn5-lac insertions in the S1 locus of Myxococcus xanthus inactivate the sglK gene, which is nonessential for growth but required for social motility and multicellular development. The sequence of sglK reveals that it encodes a homologue of the chaperone HSP70 (DnaK). The sglK gene is cotranscribed with the upstream grpS gene, which encodes a GrpE homologue. Unlike sglK, grpS is not required for social motility or development. Wild-type M. xanthus is encased in extracellular polysaccharide filaments associated with the multimeric fibrillin protein. Mutations in sglK inhibit cell cohesion, the binding of Congo red, and the synthesis or secretion of fibrillin, indicating that sglK mutants do not make fibrils. The fibR gene, located immediately upstream of the grpS-sglK operon, encodes a product which is predicted to have a sequence similar to those of the repressors of alginate biosynthesis in Pseudomonas aeruginosa and Pseudomonas putida. Inactivation of fibR leads to the overproduction of fibrillin, suggesting that M. xanthus fibril production and Pseudomonas alginate production are regulated in analogous ways. M. xanthus and Pseudomonas exopolysaccharides may play similar roles in a mechanism of social motility conserved in these gram-negative bacteria.     

ADP-ribosylation by the extracellular fibrils of Myxococcus xanthus

The isolated, extracellular fibrils of the myxobacterium, Myxococcus xanthus , are capable of carrying out ADP-ribosylation. The substrate for the ADP-ribosylation is reactive with monoclonal antibody 2105, which has been shown to be directed specifically against the integral fibril proteins. The extracellular fibrils thus contain both the ADP-ribosyl transferase and the substrate for the ribosylation. This process may play a role in the contact-mediated cell–cell interactions that are an important part of the social behaviour of M. xanthus .     

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.     

Morphogenesis in Myxococcus xanthus and Myxococcus virescens (Myxobacterales)

1. Myxococcus xanthus B and M. virescens V2 were compared with a view to establishing the control of their morphogenetic cycles. Both organisms are typical myxococci and on solid media with low concentrations of nutrient they form fruiting bodies, within which vegetative cells convert to myxospores. Ultrathin sections of vegetative M. virescens resembled those of M. xanthus and contained prominent heavily stained bodies, presumed to be polyphosphate granules. Shadowed preparations showed fimbriae associated with M. xanthus but not with M. virescens. 2. M. xanthus B converted to myxospores in liquid medium in response to certain alcohols. M. virescens V2 produced phase-refractile spheres, which were not viable and had an unusual ultrastructure. 3. The distributions of fruiting bodies on solid media containing 0.02% Casitone were recorded for the two species and were compared with a Poisson distribution. Cells responded to differences in cell density in a manner suggestive of a response to a chemotactic attractant. Cells growing vegetatively and also cells forming fruiting bodies produced 3',5'-cyclic adenosine monophosphate (cAMP) as measured by the incorporation of exogeneous [3H] adenosine into cAMP. 4. The significance of these findings for theories of fruiting body formation are discussed.     

The Myxococcus xanthus developmental program can be delayed by inhibition of DNA replication

Under conditions of nutrient deprivation, Myxococcus xanthus undergoes a developmental process that results in the formation of a fruiting body containing environmentally resistant myxospores. We have shown that myxospores contain two copies of the genome, suggesting that cells must replicate the genome prior to or during development. To further investigate the role of DNA replication in development, a temperature-sensitive dnaB mutant, DnaB^A116V, was isolated from M. xanthus. Unlike what happens in Escherichia coli dnaB mutants, where DNA replication immediately halts upon a shift to a nonpermissive temperature, growth and DNA replication of the M. xanthus mutant ceased after one cell doubling at a nonpermissive temperature, 37°C. We demonstrated that at the nonpermissive temperature the DnaB^A116V mutant arrested as a population of 1n cells, implying that these cells could complete one round of the cell cycle but did not initiate new rounds of DNA replication. In developmental assays, the DnaB^A116V mutant was unable to develop into fruiting bodies and produced fewer myxospores than the wild type at the nonpermissive temperature. However, the mutant was able to undergo development when it was shifted to a permissive temperature, suggesting that cells had the capacity to undergo DNA replication during development and to allow the formation of myxospores.     

Isolation by distance in the spore-forming soil bacterium Myxococcus xanthus

Genetic differentiation between spatially separated populations within a species is commonly observed in plants and animals, but its existence in microbes has long been a contentious issue. Traditionally, many microbial ecologists have reasoned that microbes are not limited by dispersal as a result of their immense numbers and microscopic size. In this view, the absence of barriers to gene flow between populations would prevent differentiation of populations by genetic drift and hinder local adaptation. Myxococcus xanthus is a globally distributed, spore-forming bacterium that offers a robust test for genetic differentiation among populations because sporulation is expected to enhance dispersal. Using multi-locus sequence data, we show here that both diversity and the degree of differentiation between populations increase as a function of distance in M. xanthus. Populations are consistently differentiated at scales exceeding 10(2)-10(3) km, and isolation by distance, the divergence of populations by genetic drift due to limited dispersal, is responsible. Our results provide new insights into how genetic diversity within species of free-living microbes is distributed from centimeter to global scales.     

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.     

Changes in Myxococcus xanthus pigments induced by phosphate and temperature

Pigment levels have been measured in a number of wild strains and colour mutants of Myxococcus xanthus . The non-carotenoid yellow pigment and carotenoid pigment contents changed according to growth temperature and phosphate concentrations in liquid media. The highest content of both types of pigment was observed at 28°C with all strains. A high phosphate concentration depressed pigment content in both wild and mutant strains at all temperatures, except with two colour mutants, where the pigments levels remained unchanged at 28°C and 33°C.     

Light-induced carotenogenesis in Myxococcus xanthus: DNA sequence analysis of the carR region

The carR region encodes a light-inducible promoter, a negative regulator of the promoter and a trans-acting activator that controls the light-inducible Myxococcus xanthus carotenoid biosynthesis regulon. DNA sequence analysis revealed, downstream of the promoter, three translationally coupled genes, carQ, carR and carS. Sequencing of mutations demonstrated that carR encoded the negative regulator and was an integral membrane protein. Mutant construction and sequencing revealed that carS was the trans-acting activator and that carQ was a positive regulator of the promoter. Neither gene encodes proteins with known sequence-specific DNA-binding motifs. The sequence of the light-inducible promoter region, identified by primer extension analysis, showed similarity to the consensus sequence of the Escherichia coli stress response (‘heat-shock’) promoters.     

Protein and lipid methylation by methionine and S-adenosylmethionine in Myxococcus xanthus

Methylation of lipids and proteins has been examined in Myxococcus xanthus using radioactive methionine and S-adenosylmethionine as methyl donors. S-adenosylmethionine is shown to be taken up by these cells and utilized directly. This permits detection of methylation in the presence of protein synthesis. Patterns of methylation obtained using methionine and S-adenosylmethionine during vegetative growth are compared by polyacrylamide gel electrophoresis, and inhibitors of protein synthesis and S-adenosylmethionine synthesis are examined for their effects on methylation. The ability to investigate methylation using exogenous S-adenosylmethionine will be advantageous in studying the role of methylation under conditions of growth and development where ongoing protein synthesis is required.     

Sensory transduction in the gliding bacterium Myxococcus xanthus

Sensory transduction in the gliding bacterium Myxococcus xanthus is mediated by the frz genes. These genes are homologous to the chemotaxis genes of enteric bacteria and control the rate of cell reversal during gliding. Sensory transduction is hypothesized to involve the recognition of substances present in the medium at the cell surface and the subsequent stimulation of a cytoplasmic methyl-accepting protein, FrzCD. Phosphorylation of FrzE is also involved in the sensory transduction pathway. Despite the similarities between the chemotaxis proteins of enteric bacteria and M. xanthus Frz proteins, fundamental differences exist between these different bacteria in terms of the ability of cells to recognize and respond to substances in their environment. The mechanism of directional switching and the nature of the gliding motor remain obscure. It is hoped that the study of the interaction of the Frz proteins will allow greater understanding of these problems.