黄色粘球菌的运动机制及其多细胞发育调控的研究进展

粘细菌是原核生物中的“高等生物”,具有特殊的运动能力以及类似真核生物的复杂的多细胞发育生活史,其多细胞发育过程的调控一直是粘细菌研究的热点。近年来,许多关于粘细菌研究的新理论、新学说不断涌现,给予粘细菌研究极大的启发。本文综述了模式菌株黄色粘球菌的运动类型、运动机制以及运动的调控系统,并对其多细胞发育过程的信号传递调控模式进行初步阐释,为更深一步的研究粘细菌复杂的生命调控过程奠定基础。     

Gliding Motility Mutants of Myxococcus xanthus

Two gliding motility mutants of Myxococcus xanthus are described. The semimotile mutant (SM) originated by high-frequency segregation from the motile FB(t) strain. Segregation was enhanced by acridine dye treatment. SM cells glide only when apposed to other cells in a swarm. The nonmotile strain (NM) originated by mutation from SM. NM cells neither glide individually nor cooperatively. FB(t), SM, and NM are indistinguishable with respect to fine structure, vegetative growth rate, glycerol-induced microcyst formation, spheroplasting, bacteriophage sensitivity, and responses to light. The motility mutants are more resistant to penicillin and more sensitive to actinomycin D than is the gliding wild type. The NM mutant is also a morphogenetic mutant; it is unable to form fruiting bodies.     

Motility in Myxococcus xanthus and its role in developmental aggregation

The Frz signal transduction system of Myxococcus xanthus was originally thought to be a simple variation of the well-characterized Che system of the enteric bacteria. Recently, however, many additional Frz proteins, along with alternative signal transduction systems, have been discovered. Together these signal transduction pathways coordinate cell-cell behavior, permitting the complex interactions required for developmental aggregation and fruiting body formation.     

Regulation of directed motility in Myxococcus xanthus

Myxococcus xanthus is a Gram-negative bacterium that exhibits a complex life cycle. During vegetative growth, cells move as large swarms. However, when starved, cells aggregate into fruiting bodies and sporulate. Both vegetative swarming and developmental aggregation require gliding motility, which involves the slow movement of cells on a solid surface in the absence of flagella. The frequency of cell reversals controls the direction of movement and is regulated by the frz genes, which encode the 'frizzy' signal-transduction proteins. These proteins contain domains which bear striking similarities to the major chemotaxis proteins of the enteric bacteria: CheA, CheY, CheW, CheR, CheB and Tar. However, significant differences exist between the Myxococcus Frz proteins and the enteric Che/MCP proteins. For example, the Frz system contains three CheY-like response-regulator domains: one is present on FrzE, which also contains a CheA-like domain, and two are present on FrzZ, which is a novel protein required for attractant, but not for repellent, responses. The identification of multiple CheY homologues in this system indicates a more complex regulatory pathway than that found in the enteric bacteria. While responses to repellent stimuli appear to follow the enteric paradigm, responses to attractants during vegetative swarming and development are more complex and may involve self-generated autoattractants. The Frz signal-transduction system regulates directed motility in M. xanthus and is essential for controlling both fruiting-body development and vegetative swarming.     

PlpA,a PilZ‐like protein,regulates directed motility of the bacterium Myxococcus xanthus

The rod‐shaped bacterium Myxococcus xanthus moves on surfaces along its long cell axis and reverses its moving direction regularly. Current models propose that the asymmetric localization of a Ras‐like GTPase, MglA, to leading cell poles determines the moving direction of cells. However, cells are still motile in the mutants where MglA localizes symmetrically, suggesting the existence of additional regulators that control moving direction. In this study, we identified PlpA, a P ilZ‐l ike p rotein that regulates the direction of motility. PlpA and MglA localize into opposite asymmetric patterns. Deletion of the plpA gene abolishes the asymmetry of MglA localization, increases the frequency of cellular reversals and leads to severe defects in cell motility. By tracking the movements of single motor particles, we demonstrated that PlpA and MglA co‐regulated the direction of gliding motility through direct interactions with the gliding motor. PlpA inhibits the reversal of individual gliding motors while MglA promotes motor reversal. By counteracting MglA near lagging cell poles, PlpA reinforces the polarity axis of MglA and thus stabilizes the direction of motility.     

Phosphorylation and Dephosphorylation among Dif Chemosensory Proteins Essential for Exopolysaccharide Regulation in Myxococcus xanthus

Myxococcus xanthus social gliding motility, which is powered by type IV pili, requires the presence of exopolysaccharides (EPS) on the cell surface. The Dif chemosensory system is essential for the regulation of EPS production. It was demonstrated previously that DifA (methyl-accepting chemotaxis protein [MCP]-like), DifC (CheW-like), and DifE (CheA-like) stimulate whereas DifD (CheY-like) and DifG (CheC-like) inhibit EPS production. DifD was found not to function downstream of DifE in EPS regulation, as a difD difE double mutant phenocopied the difE single mutant. It has been proposed that DifA, DifC, and DifE form a ternary signaling complex that positively regulates EPS production through the kinase activity of DifE. DifD was proposed as a phosphate sink of phosphorylated DifE (DifE∼P), while DifG would augment the function of DifD as a phosphatase of phosphorylated DifD (DifD∼P). Here we report in vitro phosphorylation studies with all the Dif chemosensory proteins that were expressed and purified from Escherichia coli. DifE was demonstrated to be an autokinase. Consistent with the formation of a DifA-DifC-DifE complex, DifA and DifC together, but not individually, were found to influence DifE autophosphorylation. DifD, which did not inhibit DifE autophosphorylation directly, was found to accept phosphate from autophosphorylated DifE. While DifD∼P has an unusually long half-life for dephosphorylation in vitro, DifG efficiently dephosphorylated DifD∼P as a phosphatase. These results support a model where DifE complexes with DifA and DifC to regulate EPS production through phosphorylation of a downstream target, while DifD and DifG function synergistically to divert phosphates away from DifE∼P.The proper regulation of bacterial motility is critical for the survival of bacteria in their natural environment. One such form of regulation is bacterial chemotaxis, which enables organisms to move toward more favorable niches and away from hazardous ones. Chemotaxis regulation in flagellated swimming bacteria has been well studied in model organisms such as Escherichia coli and Bacillus subtilis (2, 36). In general, environmental changes are detected and transduced to the cytoplasmic side of the cell by a transmembrane ternary signaling complex composed of methyl-accepting chemotaxis proteins (MCPs), CheW, and CheA. Typically, MCPs anchor the complex to the membrane through their two transmembrane (TM) domains. Chemical changes in the environment are detected by the periplasmic domain of an MCP, resulting in conformational changes in the conserved cytoplasmic signaling domain. These changes can modulate the activity of the CheA kinase via interactions with CheW in the signaling complex. The response regulator CheY, another essential component of the bacterial chemotaxis pathway, is a substrate of the CheA kinase that accepts a phosphate from autophosphorylated CheA. Phosphorylated CheY (CheY∼P) interacts with the flagellar motor complex to effect bacterial swimming behavior. Although the dephosphorylation of CheY∼P can occur spontaneously, it is accelerated by phosphatases such as CheZ in E. coli and CheC as well as FliY in B. subtilis. The dephosphorylation of CheY∼P is critical for chemotaxis since it is one of the mechanisms for the desensitization of a stimulated chemotaxis pathway. This basic architecture of chemotaxis pathways is generally conserved in all the flagellated swimming bacteria examined to date (31, 36).Myxococcus xanthus is a gliding Gram-negative bacterium that encodes eight chemosensory systems based on the genome sequence (18, 54). This bacterium, which develops fruiting bodies under nutrient deprivation (25), is motile on surfaces by adventurous (A) and social (S) gliding motility (22). While A motility enables the movement of a cell that is well separated from others, S motility is functional only when cells are in close proximity. S motility is analogous to bacterial twitching in that both are powered by retraction of the type 4 pilus (Tfp) (24, 30, 38). M. xanthus S motility additionally requires exopolysaccharides (EPS) to function (29). For S motility, EPS on one cell is thought to provide the anchor and trigger for the retraction of Tfp from a neighboring cell, thus explaining the proximity requirement. Chemotaxis regulation in M. xanthus has also been investigated extensively (27, 54). One of the surprises from these investigations was that among the eight chemosensory systems, only Frz signal transduction plays a primary role in chemotaxis regulation and mutants in other systems have no or only specific defects in chemotaxis under certain experimental conditions.The M. xanthus Dif chemosensory system, while also important for tactic responses to certain species of phosphatidylethanolamine (PE) (8), plays a primary role in the regulation of EPS production (7, 53). difA, difC, and difE mutants produce no detectable levels of EPS, whereas difD and difG mutants overproduce EPS (3, 7, 53). Mutations in difD and difG have additive effects on EPS production but failed to suppress mutations in difE (6). Additional analysis, including the use of yeast two- and three-hybrid (Y2H and Y3H, respectively) systems, led to a working model for the regulation of EPS by the Dif system (6, 52). DifA (MCP-like), DifC (CheW-like), and DifE (CheA-like) were projected to form a ternary signaling complex as do the MCPs, CheW, and CheA in bacterial chemotaxis. DifE is proposed to be an autokinase whose activity is modulated by DifA in combination with DifC (6, 52). The output of the signaling complex is the phosphorylation of an unidentified downstream component by DifE. DifD (CheY-like) and DifG (CheC-like), negative regulators of EPS production, are proposed to be ancillary modulators of the output of DifE by partially diverting phosphate from the DifE kinase and thus away from its downstream target(s) (6). That is, DifD may accept phosphate from autophosphorylated DifE (DifE∼P) and DifG may function as a phosphatase to accelerate the autodephosphorylation of phosphorylated DifD (DifD∼P). Phosphorylation and dephosphorylation events, which are obviously critical to this model, had not been examined prior to this present report.In this study, we used purified Dif proteins expressed in E. coli to examine the autophosphorylation, phosphotransfer, and dephosphorylation properties of the Dif proteins in vitro. Our results provide strong evidence for most of the proposed biochemical and physical interactions among the Dif proteins. Necessary modifications of the model based on the results here are also discussed.     

Cell Division Resets Polarity and Motility for the Bacterium Myxococcus xanthus

Links between cell division and other cellular processes are poorly understood. It is difficult to simultaneously examine division and function in most cell types. Most of the research probing aspects of cell division has experimented with stationary or immobilized cells or distinctly asymmetrical cells. Here we took an alternative approach by examining cell division events within motile groups of cells growing on solid medium by time-lapse microscopy. A total of 558 cell divisions were identified among approximately 12,000 cells. We found an interconnection of division, motility, and polarity in the bacterium Myxococcus xanthus. For every division event, motile cells stop moving to divide. Progeny cells of binary fission subsequently move in opposing directions. This behavior involves M. xanthus Frz proteins that regulate M. xanthus motility reversals but is independent of type IV pilus “S motility.” The inheritance of opposing polarity is correlated with the distribution of the G protein RomR within these dividing cells. The constriction at the point of division limits the intracellular distribution of RomR. Thus, the asymmetric distribution of RomR at the parent cell poles becomes mirrored at new poles initiated at the site of division.     

Regulation of cohesion-dependent cell interactions in Myxococcus xanthus.

Myxococcus xanthus has two nearly independent genetic systems, A and S, which appear to mediate adventurous (single-cell) movement and social (group) movement, respectively. In addition to a notable reduction in group movement, social motility mutants exhibit decreased biofilm formation, cell cohesion, dye binding, fibril production, and fruiting body formation. The stk-1907 allele, containing transposon Tn5 insertion omega DK1907, was introduced into wild-type cells and many social motility mutants. This allele, which was epistatic to most social motility mutations, caused wild-type and most mutant cells to exhibit increased group movement, cell cohesion, dye binding, and production of cell surface fibrils. The presence of the stk-1907 allele in dsp mutants, which almost completely lack cell surface fibrils, did not result in these phenotypic changes; therefore, stk-1907 is hypostatic to dsp mutations. Those mutants which exhibited increased group movement and cell cohesion with the stk-1907 allele also had increased fruiting body formation, but no significant changes in spore production were observed. These results suggest that fibrils may mediate cell cohesion, dye binding, and group movement. Additionally, the results suggest that the dsp locus contains genes involved in subunit synthesis, transport, and/or assembly of fibrils. The wild-type and mutant alleles of stk were cloned and studied in merodiploids. The mutant allele is recessive, suggesting that Tn5 omega DK1907 caused a null mutation in a gene which acts as a negative regulator of fibril synthesis. The stk-1907 allele appears to cause utilization of the A motility system for group movement, possibly because of increased fibril production.     

Myxococcus xanthus Viability Depends on GroEL Supplied by Either of Two Genes,but the Paralogs Have Different Functions during Heat Shock,Predation, and Development

Myxococcus xanthus DK1622 contains two paralogous groEL gene loci that possess both different sequences and different organizations within the genome. Deletion of either one of these two genes alone does not affect cell viability. However, deletion of both groEL genes results in cell death unless a complemented groEL1 or groEL2 gene is present. The groEL1 gene was determined to be essential for cell survival under heat shock conditions; a strain with mutant groEL2 caused cells to be more sensitive than the wild-type strain to higher temperatures. Mutants with a single deletion of either groEL1 (MXAN_4895) or groEL2 (MXAN_4467) had a growth curve similar to that of the wild-type strain DK1622 in medium containing hydrolyzed proteins as the substrate. However, when cells were cultured on medium containing either Escherichia coli cells or casein as the substrate, deletion of groEL2, but not groEL1, led to a deficiency in cell predation and macromolecular feeding. Furthermore, groEL1 was found to play an indispensable role in the development and sporulation of cells, but deletion of groEL2 had no visible effects. Our results suggest that, although alternatively required for cell viability, the products of the two groEL genes have divergent functions in the multicellular social life cycle of M. xanthus DK1622.Myxobacteria are characterized among the prokaryotes by their unique social behavior (4, 30, 36). The social behavior of myxobacterial cells is present in each stage of the life cycle; cells glide on solid surfaces in swarms, feed on macromolecules and other microbial cells in groups, and develop multicellular resting structures called fruiting bodies that contain myxospores when food is exhausted (32). Many genes are required to conduct this complicated social lifestyle. Genome sequencing revealed that myxobacteria have the largest bacterial genome and possess many multicopy genes (8, 31). A major duplication occurs with chaperone genes, which are essential for cell functions by assisting protein folding, assembly, transport, and degradation (21, 26), not only in normal cellular processes but also in response to nonpermissive temperatures (their products thus belong to the heat shock protein family) (5, 23). There are several reports related to the functions of chaperones in the sociality of Myxococcus. For example, in Myxococcus xanthus, there appear to be several duplicated genes encoding the chaperone HSP70 protein (DnaK) (8), of which only one, sglK (MXAN_6671), has been studied. SglK was shown to be essential for social motility and multicellular development in M. xanthus DK1622, but it did not respond to temperature changes or heat shock (35, 37).GroEL protein, a major type of chaperonin, is ubiquitously distributed in bacteria (11). Bacterial cells usually contain one copy of the groEL gene within the genome. However, approximately 20% of sequenced bacterial genomes were found to have duplicate or multiple copies of the groEL gene (10, 11). The products of duplicated groEL genes have been reported to play divergent roles in various bacteria. For example, in Mycobacterium smegmatis, which possess duplicate groEL genes, the GroEL2 protein was shown to be essential for cell growth. In contrast, inactivation of the groEL1 gene did not affect the normal growth of cells but prevented the formation of mature biofilms and the biosynthesis of mycolic acid (24). There are three copies of the groEL gene in Rhizobium leguminosarum. The cpn60.1 gene (groEL1) has been shown to be indispensable for the growth of this organism, while the other two paralogous genes, cpn60.2 and cpn60.3 (groEL2 and groEL3), can be deleted without affecting cell viability (28). The three GroEL homologues of R. leguminosarum display distinct properties in vitro (7) and preferentially self-assemble rather than form mixed hetero-oligomeric proteins when coexpressed in E. coli cells (10). Furthermore, Sinorhizobium meliloti has five copies of the groEL gene in its genome. However, only the product encoded by groEL1, which is required for symbiosis, is required for viability (2).In different myxobacteria, there are also two copies of the groEL gene (13), of which little is known with regard to their specific cellular roles, especially in the social life of myxobacterial cells. In this study, the functions of duplicate groEL genes in cell growth, predation feeding, development, and heat shock response were investigated. Our results demonstrate that either copy of the duplicate groEL genes is indispensable for the survival of Myxococcus xanthus DK1622. Furthermore, these studies also suggest that the products of the two groEL genes have played divergent roles in the social life cycle of myxobacterial cells.     

Resource Level Affects Relative Performance of the Two Motility Systems of Myxococcus xanthus

The adventurous (A) and social (S) motility systems of the microbial predator Myxococcus xanthus show differential swarming performance on distinct surface types. Under standard laboratory conditions, A-motility performs well on hard agar but poorly on soft agar, whereas the inverse pattern is shown by S-motility. These properties may allow M. xanthus to swarm effectively across a greater diversity of natural surfaces than would be possible with one motility system alone. Nonetheless, the range of ecological conditions under which dual motility enhances effective swarming across distinct surfaces and how ecological parameters affect the complementarity of A-motility and S-motility remain unclear. Here we have examined the role of nutrient concentration in determining swarming patterns driven by dual motility on distinct agar surfaces, as well as the relative contributions of A-motility and S-motility to these patterns. Swarm expansion rates of dually motile (A⁺S⁺), solely A-motile (A⁺S⁻), and solely S-motile (A⁻S⁺) strains were compared on hard and soft agar across a wide range of casitone concentrations. At low casitone concentrations (0–0.1%), swarming on soft agar driven by S-motility is very poor, and is significantly slower than swarming on hard agar driven by A-motility. This reverses at high casitone concentration (1–3.2%) such that swarming on soft agar is much faster than swarming on hard agar. This pattern greatly constrained the ability of M. xanthus to encounter patches of prey bacteria on a soft agar surface when nutrient levels between the patches were low. The swarming patterns of a strain that is unable to produce extracellular fibrils indicate that these appendages are responsible for the elevated swarming of S-motility at high resource levels. Together, these data suggest that large contributions by S-motility to predatory swarming in natural soils may be limited to soft, wet, high-nutrient conditions that may be uncommon. Several likely benefits of S-motility to the M. xanthus life cycle are discussed, including synergistic interactions with A-motility across a wide variety of conditions.     

A DnaK Homolog in Myxococcus xanthus Is Involved in Social Motility and Fruiting Body Formation

Myxococcus xanthus is a gram-negative soil bacterium which exhibits a complex life cycle and social behavior. In this study, two developmental mutants of M. xanthus were isolated through Tn5 transposon mutagenesis. The mutants were found to be defective in cellular aggregation as well as in sporulation. Further phenotypic characterization indicated that the mutants were defective in social motility but normal in directed cell movements. Both mutations were cloned by a transposon-tagging method. Sequence analysis indicated that both insertions occurred in the same gene, which encodes a homolog of DnaK. Unlike the dnaK genes in other bacteria, this M. xanthus homolog appears not to be regulated by temperature or heat shock and is constitutively expressed during vegetative growth and under starvation. The defects of the mutants indicate that this DnaK homolog is important for the social motility and development of M. xanthus.     

Regulation of expression of the pilA gene in Myxococcus xanthus.

Type IV pili are required for social gliding motility in Myxococcus xanthus. In this work, the expression of pilin (the pilA gene product) during vegetative growth and fruiting-body development was examined. A polyclonal antibody against the pilA gene product (prepilin) was prepared, along with a pilA-lacZ fusion, and was used to assay expression of pilA in M. xanthus in different mutant backgrounds. pilA expression required the response regulator pilR but was negatively regulated by the putative sensor kinase pilS. pilA expression did not require pilB, pilC, or pilT. pilA was also autoregulated; a mutation which altered an invariant glutamate five residues from the presumed prepilin processing site eliminated this autoregulation, as did a deletion of the pilA gene. Primer extension and S1 nuclease analysis identified a sigma54 promoter upstream of pilA, consistent with the homology of pilR to the NtrC family of response regulators. Expression of pilA was found to be developmentally regulated; however, the timing of this expression pattern was not entirely dependent on pilS or pilR. Finally, pilA expression was induced by high nutrient concentrations, an effect that was also not dependent on pilS or pilR.     

Colony Expansion of Socially Motile Myxococcus xanthus Cells Is Driven by Growth,Motility, and Exopolysaccharide Production

Myxococcus xanthus, a model organism for studies of multicellular behavior in bacteria, moves exclusively on solid surfaces using two distinct but coordinated motility mechanisms. One of these, social (S) motility is powered by the extension and retraction of type IV pili and requires the presence of exopolysaccharides (EPS) produced by neighboring cells. As a result, S motility requires close cell-to-cell proximity and isolated cells do not translocate. Previous studies measuring S motility by observing the colony expansion of cells deposited on agar have shown that the expansion rate increases with initial cell density, but the biophysical mechanisms involved remain largely unknown. To understand the dynamics of S motility-driven colony expansion, we developed a reaction-diffusion model describing the effects of cell density, EPS deposition and nutrient exposure on the expansion rate. Our results show that at steady state the population expands as a traveling wave with a speed determined by the interplay of cell motility and growth, a well-known characteristic of Fisher’s equation. The model explains the density-dependence of the colony expansion by demonstrating the presence of a lag phase–a transient period of very slow expansion with a duration dependent on the initial cell density. We propose that at a low initial density, more time is required for the cells to accumulate enough EPS to activate S-motility resulting in a longer lag period. Furthermore, our model makes the novel prediction that following the lag phase the population expands at a constant rate independent of the cell density. These predictions were confirmed by S motility experiments capturing long-term expansion dynamics.