An extracellular matrix-associated zinc metalloprotease is required for dilauroyl phosphatidylethanolamine chemotactic excitation in Myxococcus xanthus

An extracellular matrix connects bacteria that live in organized assemblages called biofilms. While the role of the matrix in the regulation of cell behavior has not been extensively examined in bacteria, we suggest that, like mammalian cells, the matrix facilitates cell-cell interactions involved with regulation of cohesion, motility, and sensory transduction. The extracellular matrix of the soil bacterium Myxococcus xanthus is essential for biofilm formation and fruiting body development. The matrix material is extruded as long, thin fibrils that mediate adhesion to surfaces, cohesion to other cells, and excitation by the chemoattractant dilauroyl phosphatidylethanolamine. We report the identification of a putative matrix-associated zinc metalloprotease called FibA (fibril protein A). Western blotting with FibA-specific monoclonal antibody 2105 suggests extensive proteolytic processing of FibA during assembly into fibrils, consistent with the autoprocessing observed with other members of the M4 metalloprotease family. Disruption of fibA had no obvious effect on the structure of the fibrils and did not inhibit cell cohesion, excitation by dioleoyl phosphatidylethanolamine, or activity of the A- or S-motility motors. However, the cells lost the ability to respond to dilauroyl phosphatidylethanolamine and to form well-spaced fruiting bodies, though substantial aggregation was observed. Chemotactic excitation of the fibA mutant was restored by incubation with purified wild-type fibrils. The results suggest that this metalloprotease is involved in sensory transduction.     

Short spacing between the Shine-Dalgarno sequence and P codon destabilizes codon-anticodon pairing in the P site to promote +1 programmed frameshifting

Myxococcus xanthus is a Gram‐negative bacterium capable of complex developmental processes involving vegetative swarming and fruiting body formation. Social (S‐) gliding motility, one of the two motility systems used by M. xanthus, requires at least two cell surface structures: type IV pili (TFP) and extracellular polysaccharides (EPS). Extended TFP that are composed of thousands of copies of PilA retract upon binding to EPS and thereby pull the cell forward. TFP also act as external sensor to regulate EPS production. In this study, we generated a random PilA mutant library and identified one derivative, SW1066, which completely failed to undergo developmental processes. Detailed characterization revealed that SW1066 produced very little EPS but wild‐type amounts of PilA. These mutated PilA subunits, however, are unable to assemble into functional TFP despite their ability to localize to the membrane. By preventing the mutated PilA of SW1066 to translocate from the cytoplasm to the membrane, fruiting body formation and EPS production were restored to the levels observed in mutant strains lacking PilA. This apparent connection between PilA membrane accumulation and reduction in surface EPS implies that specific cellular PilA localization are required to maintain the EPS level necessary to sustain normal S‐motility in M. xanthus.     

Direct visualization of the interaction between pilin and exopolysaccharides of Myxococcus xanthus with eGFP-fused PilA protein

Type IV pili (TFP) and exopolysaccharides (EPS) are important components for social behaviors in Myxococcus xanthus, including gliding motility and fruiting body formation. Although specific interactions between TFP and EPS have been proposed, there have as yet been no direct observations of these interactions under native conditions. In this study, we found that a truncated PilA protein (PilACt) containing only the C-terminal domain (amino acids 32-208) is sufficient for EPS binding in vitro. Furthermore, an enhanced green fluorescent protein (eGFP) and PilACt fusion protein were constructed and used to label the native EPS in M. xanthus. Under confocal laser scanning microscope, the eGFP-PilACt-bound fruiting bodies, trail structures and biofilms exhibited similar patterns as the wheat germ agglutinin lectin-labeled EPS structures. This study showed that eGFP-PilACt fusion protein was able efficiently to label the EPS of M. xanthus, providing evidence for the first time of the direct interaction between the PilA protein and EPS under native conditions.     

Pheromone produced by the myxobacterium Stigmatella aurantiaca.

An extracellular, diffusible signaling molecule (pheromone) was produced by Stigmatella aurantiaca during fruiting body formation. The pheromone decreased the aggregation period in both the light and the dark and substituted for light in stimulating the maturation of aggregates into fruiting bodies. The cells were more sensitive to lower concentrations of pheromone in the light than in the dark, possibly explaining the stimulation of aggregation and fruiting body formation by light. The pheromone also interacted cooperatively with GMP to shorten the aggregation period. The pheromone behaved chemically as a low-molecular-weight lipid.     

Multicellular development in Myxococcus xanthus is stimulated by predator-prey interactions

Myxococcus xanthus is a predatory bacterium that exhibits complex social behavior. The most pronounced behavior is the aggregation of cells into raised fruiting body structures in which cells differentiate into stress-resistant spores. In the laboratory, monocultures of M. xanthus at a very high density will reproducibly induce hundreds of randomly localized fruiting bodies when exposed to low nutrient availability and a solid surface. In this report, we analyze how M. xanthus fruiting body development proceeds in a coculture with suitable prey. Our analysis indicates that when prey bacteria are provided as a nutrient source, fruiting body aggregation is more organized, such that fruiting bodies form specifically after a step-down or loss of prey availability, whereas a step-up in prey availability inhibits fruiting body formation. This localization of aggregates occurs independently of the basal nutrient levels tested, indicating that starvation is not required for this process. Analysis of early developmental signaling relA and asgD mutants indicates that they are capable of forming fruiting body aggregates in the presence of prey, demonstrating that the stringent response and A-signal production are surprisingly not required for the initiation of fruiting behavior. However, these strains are still defective in differentiating to spores. We conclude that fruiting body formation does not occur exclusively in response to starvation and propose an alternative model in which multicellular development is driven by the interactions between M. xanthus cells and their cognate prey.     

Characterization of developmental autolysis in myxobacterial fruiting body morphogenesis with profiling of amino acids using capillary electrophoresis method

Summary. Capillary electrophoresis equipped with Laser-induced fluorescence (CE-LIF), combining with micro-culture technique was employed to determine extracellular amino acids in single myxobacterial fruiting body morphogenesis. The result showed that in the early aggregation stage, there was a remarkable increase of extracellular amino acids, which was produced by developmentally induced autolysis. The amino acids were then consumed by the vegetative cells in aggregation stage. In the following developmental events, the extracellular amino acids were kept at low level, which indicated that in the stages of fruiting body formation and myxospore development, there was no further cell autolysis. Using this novel method may provide detailed insight into the mechanisms of the developmental phenomena.     

Short-Range Guiding Can Result in the Formation of Circular Aggregates in Myxobacteria Populations

Myxobacteria are social bacteria that upon starvation form multicellular fruiting bodies whose shape in different species can range from simple mounds to elaborate tree-like structures. The formation of fruiting bodies is a result of collective cell movement on a solid surface. In the course of development, groups of flexible rod-shaped cells form streams and move in circular or spiral patterns to form aggregation centers that can become sites of fruiting body formation. The mechanisms of such cell movement patterns are not well understood. It has been suggested that myxobacterial development depends on short-range contact-mediated interactions between individual cells, i.e. cell aggregation does not require long-range signaling in the population. In this study, by means of a computational mass-spring model, we investigate what types of short-range interactions between cells can result in the formation of streams and circular aggregates during myxobacterial development. We consider short-range head-to-tail guiding between individual cells, whereby movement direction of the head of one cell is affected by the nearby presence of the tail of another cell. We demonstrate that stable streams and circular aggregates can arise only when the trailing cell, in addition to being steered by the tail of the leading cell, is able to speed up to catch up with it. It is suggested that necessary head-to-tail interactions between cells can arise from physical adhesion, response to a diffusible substance or slime extruded by cells, or pulling by motility engine pili. Finally, we consider a case of long-range guiding between cells and show that circular aggregates are able to form without cells increasing speed. These findings present a possibility to discriminate between short-range and long-range guiding mechanisms in myxobacteria by experimentally measuring distribution of cell speeds in circular aggregates.     

Cloning of fibA, encoding an immunogenic subunit of the fibril-like surface structure of Peptostreptococcus micros

Although we are currently unaware of its biological function, the fibril-like surface structure is a prominent characteristic of the rough (Rg) genotype of the gram-positive periodontal pathogen Peptostreptococcus micros. The smooth (Sm) type of this species as well as the smooth variant of the Rg type (RgSm) lack these structures on their surface. A fibril-specific serum, as determined by immunogold electron microscopy, was obtained through adsorption of a rabbit anti-Rg type serum with excess bacteria of the RgSm type. This serum recognized a 42-kDa protein, which was subjected to N-terminal sequencing. Both clones of a lambdaTriplEx expression library that were selected by immunoscreening with the fibril-specific serum contained an open reading frame, designated fibA, encoding a 393-amino-acid protein (FibA). The 15-residue N-terminal amino acid sequence of the 42-kDa antigen was present at positions 39 to 53 in FibA; from this we conclude that the mature FibA protein contains 355 amino acids, resulting in a predicted molecular mass of 41,368 Da. The putative 38-residue signal sequence of FibA strongly resembles other gram-positive secretion signal sequences. The C termini of FibA and two open reading frames directly upstream and downstream of fibA exhibited significant sequence homology to the C termini of a group of secreted and surface-located proteins of other gram-positive cocci that are all presumably involved in anchoring of the protein to carbohydrate structures. We conclude that FibA is a secreted and surface-located protein and as such is part of the fibril-like structures.     

Excreted adenosine is a cell density signal for the initiation of fruiting body formation in Myxococcus xanthus

Myxococcus xanthus is a bacterium that forms multicellular fruiting bodies in response to starvation. The initiation of fruiting body formation is cell density dependent, and we suggest that cells measure their cell density by titering the extracellular concentration of excreted adenosine. Our evidence is as follows: (1) At low cell densities fruiting body formation does not occur unless adenosine is added. (2) Norit, a substance that binds purines, inhibits fruiting body formation, and this inhibition is reversed by adenosine. (3) Cells labeled with [¹⁴C]carbonate excrete [¹⁴C]adenosine which is bound by the Norit. Furthermore, [¹⁴C]adenosine is excreted by developing cells at a concentration that will induce fruiting body formation at low cell density. The extracellular adenosine concentration increases with the cell density over a broad range of densities. (4) Hadacidin, an inhibitor of de novo AMP synthesis, inhibits fruiting body formation, and inhibition by hadacidin can be reversed with adenosine. Adenosine also appears to be involved in the aggregation process because the shape and size of the fruiting bodies are sensitive to the external concentration of adenosine.     

The control of fruiting body formation in the ascomycete Sordaria macrospora Auersw. by regulation of hyphal development

The morphological effects of biotin and L-arginine on fruiting body formation of the ascomycete Sordaria macrospora are investigated by scanning electron and light microscopy. Biotin is recognized as an elongation factor and arginine as a branching factor in vegetative and reproductive hyphae. In the absence of exogenous biotin, development is blocked after the ascogonium-core hypha stage of protoperithecial morphogenesis, whereas linear growth of the myceliar front is maintained. The addition of exogenous arginine to a biotin deficient culture induces the formation of numerous side branches even in the older mycelium. Fruiting body formation, however, remains blocked at the protoperithecial stage as before, because of the inability of the side branches to elongate. When biotin and arginine are administered simultaneously, a most vigorous branching and growth are induced in the older mycelium, accompanied by a rapid and maximal formation of fruiting bodies. The results are summarized in a model of the exogenous control of hyphal morphogenesis. The model is designed to explain the relationship between fruiting and hyphal density as well as the edge effect on fruiting body formation.     

The developmental capacity of various stages of a macrocyst-forming strain of the cellular slime mold, Dictyostelium mucoroides.

Vegetative cells of certain strains of Dictyostelium mucoroides form fruiting bodies on an agar surface and macrocysts when placed under saline. This study sought to determine whether the aggregation and pseudoplasmodial stages of fruiting body formation could be induced to form macrocysts when placed under saline. Likewise, different stages in macrocyst formation were put on an agar surface to determine their potential to switch to fruiting body formation. It was found that 78% of the aggregates and 21% of the pseudoplasmodia placed under saline formed macrocysts indicating that as fruiting body development proceeds, there is a restriction of the capability of cells to respond to environmental conditions favoring macrocyst formation. Stages in macrocyst development prior to the formation of precysts always formed fruiting bodies when put on agar. Once precysts had formed, surrounded by their acellular sheath, they always developed as macrocysts on agar. Peripheral cells isolated from precysts and put on agar quickly aggregated; the aggregates became surounded by a sheath and developed as macrocysts. If isolated peripheral cells were allowed to proliferate on the agar surface, the resulting cells aggregated and formed fruiting bodies.     

粘细菌的多细胞形态发生及其分子调控

粘细菌的多细胞形态发生是粘细菌细胞社会性行为的主要表现.包括细胞有序聚集、细胞自溶、子实体发育和粘孢子的分化形成等.粘细菌的形态发生过程涉及复杂的信号系统和调控,与真核生物具有较大的相似性.是研究原核生物细胞分化发育以及生物进化的重要模式材料.     

Release of a cell surface protein during development of Myxococcus xanthus.

VGP is a major cell-surface glycoprotein present in vegetative cells of Myxococcus xanthus. Serological assays indicated that this protein was released from cells and accumulated in the medium during development, i.e., aggregation, fruiting body formation, and myxosporulation. Cells induced to form spores in the absence of aggregation retained VGP, indicating that loss of VGP was associated with developmental aggregation rather than myxosporulation. Anti-VGP antibodies inhibited vegetative cell gliding, suggesting the protein may also be required for motility.     

Role of cell cohesion in Myxococcus xanthus fruiting body formation.

Dsp mutants of Myxococcus xanthus have a complex phenotype with abnormal cell cohesion, social motility, and development. All three defects are the result of a single mutation in the dsp locus, a region of DNA about 14 kilobases long. Cohesion appears to play a central role in social motility, since nonsocial mutants exhibit weak agglutination or, in the case of Dsp cells, no agglutination (L. J. Shimkets, J. Bacteriol. 166:837-841, 1986). However, Dsp cells can be agglutinated by cohesive strains of M. xanthus. This provided the opportunity to examine the role of cohesion during development by comparing the developmental phenotype of Dsp cells with that of Dsp cells mixed with cohesive strains. Dsp mutants were unable to complete any of the developmental behaviors: aggregation, fruiting body formation, developmental autolysis, and sporulation. Contact with cohesive strains seemed to restore some developmental characteristics to the Dsp cells. When allowed to develop with wild-type cells, Dsp cells accumulated in fruiting bodies and underwent developmental autolysis, but did not form a significant portion of the spore population. Igl mutants, which may be similar to the previously described frizzy mutants, are cohesive strains that are unable to form fruiting bodies. Mixing Igl cells with Dsp cells under developmental conditions resulted in fruiting body formation, although the Dsp cells were unable to form significant levels of myxospores. In spite of their inability to sporulate under developmental conditions, Dsp mutants did not appear to be defective in the sporulation process. In fact, they formed normal levels of myxospores in response to the chemical inducer glycerol.     

Biological Activity of the Alternative Promoters of the Dictyostelium discoideum Adenylyl Cyclase A Gene

Amoebae of the Dictyostelium discoideum species form multicellular fruiting bodies upon starvation. Cyclic adenosine monophosphate (cAMP) is used as intercellular signalling molecule in cell-aggregation, cell differentiation and morphogenesis. This molecule is synthesized by three adenylyl cyclases, one of which, ACA, is required for cell aggregation. The gene coding for ACA (acaA) is transcribed from three different promoters that are active at different developmental stages. Promoter 1 is active during cell-aggregation, promoters 2 and 3 are active in prespore and prestalk tip cells at subsequent developmental stages. The biological relevance of acaA expression from each of the promoters has been studied in this article. The acaA gene was expressed in acaA-mutant cells, that do not aggregate, under control of each of the three acaA promoters. acaA expression under promoter 1 control induced cell aggregation although subsequent development was delayed, very small fruiting bodies were formed and cell differentiation genes were expressed at very low levels. Promoter 2-driven acaA expression induced the formation of small aggregates and small fruiting bodies were formed at the same time as in wild-type strains and differentiation genes were also expressed at lower levels. Expression of acaA from promoter 3 induced aggregates and fruiting bodies formation and their size and the expression of differentiation genes were more similar to that of wild-type cells. Expression of acaA from promoters 1 and 2 in AX4 cells also produced smaller structures. In conclusion, the expression of acaA under control of the aggregation-specific Promoter 1 is able to induce cell aggregation in acaA-mutant strains. Expression from promoters 2 and 3 also recovered aggregation and development although promoter 3 induced a more complete recovery of fruiting body formation.     

Spatial organization of Myxococcus xanthus during fruiting body formation

Microcinematography was used to examine fruiting body development of Myxococcus xanthus. Wild-type cells progress through three distinct phases: a quiescent phase with some motility but little aggregation (0 to 8 h), a period of vigorous motility leading to raised fruiting bodies (8 to 16 h), and a period of maturation during which sporulation is initiated (16 to 48 h). Fruiting bodies are extended vertically in a series of tiers, each involving the addition of a cell monolayer on top of the uppermost layer. A pilA (MXAN_5783) mutant produced less extracellular matrix material and thus allowed closer examination of tiered aggregate formation. A csgA (MXAN_1294) mutant exhibited no quiescent phase, aberrant aggregation in phase 2, and disintegration of the fruiting bodies in the third phase.     

Pulling together with type IV pili

Type IV pili are an efficient and versatile device for bacterial surface motility. They are widespread among the beta-, gamma-, and delta-proteobacteria and the cyanobacteria. Within that diversity, there is a core of conserved proteins that includes the pilin (PilA), the motors PilB and PilT, and various components of pilus biogenesis and assembly, PilC, PilD, PilM, PilN, PilO, PilP, and PilQ. Progress has been made in understanding the motor and the secretory functions. PilT is a motor protein that catalyzes pilus retraction; PilB may play a similar role in pilus extension. Type IV pili are multifunctional complexes that can act as bacterial virulence factors because pilus-based motility is used to spread pathogens over the surface of a tissue, or to build multicellular structures such as biofilms and fruiting bodies.     

Morphogenetic cell movement in Dictyostelium

Dictyostelium morphogenesis starts with the chemotactic aggregation of starving individual cells. The cells move in response to propagating waves of the chemoattractant cyclic AMP initiated by cells in the aggregation centre. During aggregation the cells begin to differentiate into several types with different signalling and chemotactic properties. These cell types sort out from each other to form an axial pattern in the slug. There is now good evidence that periodic chemotactic signals not only control aggregation, but also later stages of morphogenesis. These signals take the form of target patterns, spirals, multi-armed spirals and scroll waves. I will discuss their role in the control of cell movement during mound and slug formation and in the formation of the fruiting body.     

Developmental aggregation of Myxococcus xanthus requires frgA, an frz-related gene

Myxococcus xanthus is a gram-negative bacterium which has a complex life cycle that includes multicellular fruiting body formation. Frizzy mutants are characterized by the formation of tangled filaments instead of hemispherical fruiting bodies on fruiting agar. Mutations in the frz genes have been shown to cause defects in directed motility, which is essential for both vegetative swarming and fruiting body formation. In this paper, we report the discovery of a new gene, called frgA (for frz-related gene), which confers a subset of the frizzy phenotype when mutated. The frgA null mutant showed reduced swarming and the formation of frizzy aggregates on fruiting agar. However, this mutant still displayed directed motility in a spatial chemotaxis assay, whereas the majority of frz mutants fail to show directed movements in this assay. Furthermore, the frizzy phenotype of the frgA mutant could be complemented extracellularly by wild-type cells or strains carrying non-frz mutations. The phenotype of the frgA mutant is similar to that of the abcA mutant and suggests that both of these mutants could be defective in the production or export of extracellular signals required for fruiting body formation rather than in the sensing of such extracellular signals. The frgA gene encodes a large protein of 883 amino acids which lacks homologues in the databases. The frgA gene is part of an operon which includes two additional genes, frgB and frgC. The frgB gene encodes a putative histidine protein kinase, and the frgC gene encodes a putative response regulator. The frgB and frgC null mutants, however, formed wild-type fruiting bodies.     

The Possible Involvement of Cyclic AMP and Volatile Substance (s) in the Development of a Macrocyst-Forming Strain of Dictyostelium mucoroides

A mutant MF1 previously isolated from Dictyostelium mucoroides -7 (Dm7) formed macrocysts with or without light when plated on agar at high cell dinsities. At lower cell densities, however, the MF1 cells formed only fruiting bodies. This failure to form macrocysts was shown to be due to the subthreshfold concentration of a volatile substance(s) required for macrocyst formation. Although ammonia is a volatile substance produced by both the Dm7 and MF1 cells, no evidence of its involvement in macrocyst formation was obtained. Mixing the Dm7 and MF1 in a one-to-one ratio resulted only in fruiting body formation suggesting that the Dm7 cells produced a factor which allowed MF1 cells to form fruiting bodies. This factor may be cyclic AMP (cAMP) since addition of cAMP to the medium directed development of MF1 cells to fruiting body formation. The effect of cAMP was exhibited most conspicuously when MF1 cells were exposed at the aggregation stage. Based on these results it is suggested that developmental pathway of the D. mucoroides macrocystforming strain Dm7 and its mutant MF1 may be determined by the relative concentrations of the volatile, macrocyst-inducing substance(s) and cAMP at the aggregation stage.