Protease production by immobilized growing cells of Serratia marcescens and Myxococcus xanthus in calcium alginate gel beads

Summary Serratia marcescens and Myxococcus xanthus cells were immobilized in calcium alginate gel beads. Immobilization under various conditions had no effect on the extracellular proteolytic activity of S. marcescens cells. Protease production seemed rather to depend on the free cells in the medium. However, the stability over time of enzyme production was enhanced, as immobilization increased protease production half-life from 5 to 12 days. On the other hand, Myxococcus xanthus produced proteases inside the gel beads which could diffuse into the medium. The proteolytic activity increased as a function of the initial cell content of the beads and of the bead inoculum. Compared to free cells, immobilized cells of Myxococcus xanthus could produce 8 times more proteolytic activity, with a very low free-cell concentration in the medium.     

Production of extracellular native and foreign proteins by immobilized growing cells of Myxococcus xanthus

Summary Several strains of the protein-secreting, Gram negative bacterium Myxococcus xanthus were immobilized in carrageenan beads and the production of extracellular proteins was followed.The extracellular proteolytic activity was enhanced and concentrated in the beads. In contrast, the amount of total protein secreted by the cells was not modified by immobilization, but it was also retained and concentrated in the beads, the more, the harder the gel. The amount of slime produced by the cells did not seem to influence protein retention.Foreign proteins expressed from genes cloned in Myxococcus xanthus chromosome can be secreted into the medium by immobilized recombinant strains. A polygalacturonate lyase, expressed from the pelC gene from Erwinia chrysanthemi was only detectable outside of the beads. The pH 2.5 acid phosphatase expressed from the appA gene from Escherichia coli was secreted by immobilized cells at the same rate than did the free cells. It was predominantly found in the medium outside of the beads which represented a first purification and facilitated the continuous production of this protein by immobilized recombinant cells packed in a reactor.     

Plasmid-mediated UV-protection in Myxococcus xanthus

Summary Plasmid R46 was successfully transferred from Escherichia coli K-12 into Myxococcus xanthus strain MD-1 but not into M. xanthus strain XK. Plasmid R68.45 was transferred from E. coli K-12 into both strains of M. xanthus. The effects of these plasmids on survival of M. xanthus after ultraviolet (UV)-254 nm irradiation, the ability of M. xanthus to reactivate irradiated myxophages, and Weigle reactivation of UV-irradiated myxophages by M. xanthus were studied. Plasmid R46 had no effect on UV survival of M. xanthus, but increased the host's ability to reactivate irradiated myxophages. Plasmid R68.45 protected M. xanthus strains MD-1 and XK against the lethal effects of UV irradiation and also increased the host's ability to reactivate irradiated myxophages.     

Elucidation of the mechanism involved in the regulation of protease production by immobilizedMyxococcus xanthus cells

Summary The mechanism involved in the positive effect of immobilization on protease production byMyxococcus xanthus was investigated. The results have shown that this phenomenon was not related to the difficulty encountered by the potential repressors to diffuse through the gel beads. The positive effect of immobilization on protease synthesis is the result of a different physiological state of the cells due to the stress caused by immobilization.     

Structural instability and stabilization of IncP-1 plasmids integrated into the chromosome of Myxococcus xanthus

IncP-1 plasmids are self transmissible to Myxococcus xanthus and maintained integrated into the host chromosome where they are liable to structural instability: deletions can span through the integrated plasmid; the frequency of these events depends on the recipient strain and on the localization of the insertion on the chromosome, but not on the structure of the plasmid.It is possible to isolate stabilized insertion, even from one of the most unstable strains, by growing immobilized cells in carrageenan beads in continuous nonselective culture. The remaining resistant cells are stabilized. Both the structural instability and the possible stabilization of the insertion can be useful when IncP-1 plasmids are used as cloning vectors in Myxococcus xanthus.     

Chemotaxis plays a role in the social behaviour of Myxococcus xanthus

Myxococcus xanthus is a Gram-negative bacterium that glides on a solid surface and displays a wide range of social behaviour including microbial development. The frz genes are homologues to the chemotaxis genes of Escherichia coli and Salmonella typhimurium and have been shown to be involved in microbial development. However, chemotaxis has never been clearly demonstrated in Myxococcus. In this study, we showed that M. xanthus exhibited tactic movements to many chemicals when they were subjected to steep and stable chemical gradients. M. xanthus was observed to spread into areas with abundant nutrients like yeast extract or Casitone and avoid areas with no nutrients or repellents (short-chain alcohols or DMSO. Responses to attractants and repellents were additive. Movement towards attractants or away from repellents required the frz genes and was correlated with methylation or demethylation of FrzCD, a methyl-accepting taxis protein. Furthermore, the frz genes were found to be required for both fruiting body formation during starvation and swarming in nutrient-rich medium. In wild-type strains, cells near the colony edge were observed to swarm towards the surrounding growth medium and to contain highly methylated FrzCD; cells near the colony centre contained mainly demethylated FrzCD and showed directed movement towards the colony edge. FrzCD was also found to be methylated during the aggregation stage of fruiting body formation on agar but largely demethylated in cells shaken in liquid starvation media. An frzf mutant failed to exhibit directed cell movements and no longer showed modification of FrzCD under these conditions. These observations suggest that M. xanthus does show chemotactic movements, that these movements require the frz genes, and that chemotaxis plays a very important role in the social behaviour of this organism.     

Isolation and characterization of new bacteriophages for Myxococcus xanthus

Bacteriophages for Myxococcus xanthus of similar morphology to phage Mx4 were isolated from cultures of a variety of myxobacterial species. Phages similar to Mx1 and Mx8 were obtained by infecting M. xanthus with one of the phages of the Mx4 group that had been treated with either UV light or a chemical mutagen.The DNA molecules from the phages were characterized by electron microscopy. One phage, Mx113, contains an unusual type of terminal redundancy revealed by examination of denatured and re-annealed DNA.Several of the phages of the Mx4 group and the other two new phages, Mx113 and Mx811, were found capable of transducing genetic markers in M. xanthus.One phage, Mx416, was characterized in more detail. It establishes true lysogens in M. xanthus; the phage plaques on both a non-motile mutant and also on a wild-type host although it is restricted in the latter.We dedicate this paper to Professor Dr. Hans Kühlwein in the year of his retirement and in recognition of his many contributions to the study of Myxobacteria     

Effects of exopolysaccharide production on liquid vegetative growth,stress survival,and stationary phase recovery in <Emphasis Type="Italic">Myxococcus xanthus</Emphasis>

Exopolysaccharide (EPS) of Myxococcus xanthus is a well-regulated cell surface component. In addition to its known functions for social motility and fruiting body formation on solid surfaces, EPS has also been proposed to play a role in multi-cellular clumping in liquid medium, though this phenomenon has not been well studied. In this report, we confirmed that M. xanthus clumps formed in liquid were correlated with EPS levels and demonstrated that the EPS encased cell clumps exhibited biofilm-like structures. The clumps protected the cells at physiologically relevant EPS concentrations, while cells lacking EPS exhibited significant reduction in long-term viability and resistance to stressful conditions. However, excess EPS production was counterproductive to vegetative growth and viable cell recovery declined in extended late stationary phase as cells became trapped in the matrix of clumps. Therefore, optimal EPS production by M. xanthus is important for normal physiological functions in liquid.     

Structural and Functional Characterization of Acinetobacter baumannii Nucleoside Diphosphate Kinase

近年来,鲍曼不动杆菌(Acinetobacter baumannii)在医院里越来越受到人们的关注,尤其是在重症监护病房(ICUs)．它以强大的多重耐药性(multiresistance)而闻名．核苷二磷酸激酶(nucleoside diphosphate kinase,NDK)是一种进化上非常保守的酶,它能催化核苷之间磷酸基团的转移．我们解析了鲍曼不动杆菌NDK野生型和C端氨基酸残基Arg141-Thr142-Arg143(RTR)截短突变体的结构．通过和黄色黏菌(Myxococcus xanthus)NDK的三维结构进行比较,推断鲍曼不动杆菌NDK的催化机制和黄色黏菌类似．通过激酶活性实验和圆二色谱实验,发现鲍曼不动杆菌NDK E28A突变体二级结构发生了改变,从而导致蛋白催化活性降低,说明Glu28是鲍曼不动杆菌NDK结构中非常关键的氨基酸残基．鲍曼不动杆菌NDK C端RTR截短突变体显示出催化活性极大的降低,这可能与C端RTR残基介导的二体间相互作用有关．虽然RTR截短突变体中的Lys33伸向了和野生型中不同的方向,和Val15产生相互作用弥补了一部分因为RTR截短丢失的相互作用,维持了RTR截短突变体和野生型类似的结构．但是,Lys33产生的相互作用依然太弱,不足以维持蛋白在催化的动态过程中整体结构的高效转换．我们解析的鲍曼不动杆菌NDK晶体高分辨率结构将有助于科学家设计针对鲍曼不动杆菌的药物．     

Eukaryotic-like protein serine/threonine kinases in Myxococcus xanthus,a developmental bacterium exhibiting social behavior

Myxococcus xanthus, a gram-negative bacterium exhibits a spectacular life cycle and social behavior. Its developmental cycle and multicellular morphogenesis resemble those of eukaryotic slime molds such as Dictyostelium discoideum. On the basis of this resemblance, we explored the existence of eukaryotic-like protein serine/threonine kinases which are known to play important roles in signal transduction during development of D. discoideum. It was indeed found that M. xanthus contains a large family of protein serine/threonine kinases related to the eukaryotic enzymes. This is the first unambiguous demonstration of eukaryotic-like protein serine/threonine kinases in the prokaryotes. © 1993 Wiley-Liss, Inc.     

Silver Sorption to Myxococcus xanthus Biomass

This paper deals with silver sorption to Myxococcus xanthus biomass. The dry biomass of this microorganism is shown to be a good sorbent for the recovery of silver present at low solution concentrations. Between initial silver concentrations of 2 and 0.05 mM, the percentage of accumulation ranges from 8.12% to 75% of the total silver present in the solution. Transmission electron microscopy study of M. xanthus wet biomass after silver accumulation shows the sorption within the extracellular polysaccharide, on the cell wall, and in the cytoplasm. The presence of silver deposits in the cytoplasm indicates that at least two mechanisms are involved in silver sorption by this bacterium biomass. First, silver was bound to the cell surface and extracellular polysaccharide, and second, a silver intracellular deposition process took place. The higher amount of silver deposits in the extracellular polysaccharide, present abundantly in M. xanthus cells, explains the capacity of this bacterium to bind silver efficiently. The results obtained indicate that the removal of silver by M. xanthus from the diluted solutions could be used in recycling this valuable metal. One interesting observation of this investigation is the crystalline form, possibly as chlorargyrite, in which the silver deposits are found in the M. xanthus cells.     

Bacillus licheniformis escapes from Myxococcus xanthus predation by deactivating myxovirescin A through enzymatic glucosylation

Myxococcus xanthus kills susceptible bacteria using myxovirescin A (TA) during predation. However, whether prey cells in nature can escape M. xanthus by developing resistance to TA is unknown. We observed that many field-isolated Bacillus licheniformis strains could survive encounters with M. xanthus, which was correlated to their TA resistance. A TA glycoside was identified in the broth of predation-resistant B. licheniformis J32 co-cultured with M. xanthus, and a glycosyltransferase gene (yjiC) was up-regulated in J32 after the addition of TA. Hetero-expressed YjiC-modified TA to a TA glucoside (TA-Gluc) by conjugating a glucose moiety to the C-21 hydroxyl group, and the resulting compound was identical to the TA glycoside present in the co-culture broth. TA-Gluc exhibited diminished bactericidal activity due to its weaker binding with LspA, as suggested by in silico docking data. Heterologous expression of the yjiC gene conferred both TA and M. xanthus-predation resistance to the host Escherichia coli cells. Furthermore, under predatory pressure, B. licheniformis Y071 rapidly developed predation resistance by acquiring TA resistance through the overexpression of yjiC and lspA genes. These results suggest that M. xanthus predation resistance in B. licheniformis is due to the TA deactivation by glucosylation, which is induced in a predator-mediated manner.     

Ecological Variables Affecting Predatory Success in <Emphasis Type="BoldItalic">Myxococcus xanthus</Emphasis>

The feeding efficiency of microbial predators depends on both the availability of various prey species and abiotic variables. Myxococcus xanthus is a bacterial predator that searches for microbial prey by gliding motility, and then kills and lyses its prey with secreted compounds. We manipulated three ecological variables to examine their effects on the predatory performance of M. xanthus to better understand its behavior and how it affects prey populations. Experiments were designed to determine how surface solidity (hard vs soft agar), density of prey patches (1 vs 2 cm grids), and type of prey (Gram-positive Micrococcus luteus vs Gram-negative Escherichia coli) affect predatory swarming and prey killing by M. xanthus. The prey were dispersed in patches on a buffered agar surface. M. xanthus swarms attacked a greater proportion of prey patches when patches were densely arranged on a hard-agar surface, compared with either soft-agar surfaces or low-patch-density arrangements. These ecological variables did not significantly influence the rate of killing of individual prey within a patch, although a few surviving prey were more likely to be recovered on soft agar than on hard agar. These results indicate that M. xanthus quickly kills most nearby E. coli or M. luteus regardless of the surface. However, the ability of M. xanthus to search out patches of these prey is affected by surface hardness, the density of prey patches, and the prey species.     

Autocides and a paracide,antibiotic TA,produced byMyxococcus xanthus

Myxococcus xanthus produces two categories of low molecular weight antibacterial materials, autocides and paracides, that have diametrically opposite host ranges. Low concentrations of autocides lyseM. xanthus, the producing organism, whereas paracides exert their effects on other bacteria. Antibiotic TA (a paracide) kills all growing bacteria tested that have a peptidoglycan cell wall exceptM. xanthus. It is a macrocyclic polyketide with a molecular weight of 623. The two major autocides produced byM. xanthus are phosphatidylethanolamine and a mixture of fatty acids. The modes of action, host ranges and biosynthesis of antibiotic TA and the autocides are presented, and then an attempt is made to explain their role in the complex life cycle ofM. xanthus. In addition, the remarkable adhesion properties of antibiotic TA and a new semisynthetic derivative of it, focusin, are presented.     

Propionyl-CoA carboxylase of Myxococcus xanthus: catalytic properties and function in developing cells

An acyl-coenzyme A carboxylase that carboxylates acetyl-CoA, butyryl-CoA, propionyl-CoA, and succinyl-CoA was purified from Myxococcus xanthus. Since the enzyme showed maximal rates of carboxylation with propionyl-CoA, the enzyme is thought to be propionyl-CoA carboxylase. The apparent K _m values for acetyl-CoA, butyryl-CoA, propionyl-CoA, and succinyl-CoA were found to be 0.2, 0.2, 0.03, and 1.0 mM, respectively. The native enzyme has a molecular mass of 605–615 kDa and is composed of nonidentical subunits (α and β) with molecular masses of 53 and 56 kDa, respectively. The enzyme showed maximal activity at pH 7.0–7.5 and at 25–30°C, and was affected by variation in concentrations of ATP and Mg²⁺. During development of M. xanthus, the propionyl-CoA carboxylase activity increased gradually, with maximum activity observed during the sporulation stage. Previous work has shown that a propionyl-CoA-carboxylase-deficient mutant of M. xanthus reduces levels of long-chain fatty acids. These results suggest that the propionyl-CoA carboxylase is also responsible for the carboxylation of acetyl-CoA to malonyl-CoA used for the synthesis of long-chain fatty acids during development. Received: 24 February 1998 / Accepted: 25 May 1998     

Entrapment and lysis of the cyanobacterium Phormidium luridum by aqueous colonies of Myxococcus xanthus PCO2

A Myxococcus xanthus isolate from a farm drainage ditch, designated strain PCO2, is capable of rapidly inducing lysis of both agar and liquid-grown cultures of the cyanobacterium, Phormidium luridum, var. olivacea. Microscopic studies of the predator-prey interaction demonstrate that lysis of the cyanobacterium occurs within clumps and spherules formed by the cells of M. xanthus PCO2. In the earliest stage, one sees the formation of irregular microclumps of bacteria and cyanobacterial filaments. As these clumps mature, colonies 1 to 6 mm in diameter develops. The center of these densely green colonies contains cyanohacteria in various stages of degradation, while the periphery is almost exclusively a tightly woven mass of myxobacterial cells. Electron microscopy shows that long extrusions from the outer membrane of the M. xanthus PCO2 cells are involved in the formation both of initial clumps and of mature colonial spherules. These extrusions appear to efficiently entangle the cyanobacterial filaments in the culture environment. Predator-to-prey ratios of 1/10, 1/100 and 1/1,000 have resulted in cyanobacterial lysis. Because the entrapment and lysis of P. luridum filaments by M. xanthus PCO2 appears to be independent of any other heterotrophic nutritional requirement, as well as of environmental agitation, this system has potential as a biological control technique for undesirable aquatic cyanobacteria.Abbreviations TEM transmission electron microscopy - SEM scanning electron microscopy - AB algae broth - ABT algae broth plus 0.2% tryptone     

Biosorption of uranium by Myxococcus xanthus

This paper deals with uranium biosorption by Myxococcus xanthus biomass in which dry biomass, accumulating up to 2.4 mM of uranium g⁻¹, is demonstrated to be a more efficient biosorbent than wet biomass. For uranium concentrations of 0.1–0.3 mM, between 95.79% and 95.99% of the uranium was taken up from the solution. Dry biomass biosorption was found to be relatively rapid, reaching equilibrium after 5–10 min. In addition, the pH influenced biosorption, pH 4.5 promoting maximum uptake. It was also established that the biosorbed uranium is located on the cellular wall and within the extracellular mucopolysaccharide of this microorganism. Furthermore, using sodium carbonate as a desorbent agent, 80.82% of the biosorbed uranium could be recovered. The results obtained indicate the possible utilization of M. xanthus biomass to solve some problems of the water contaminated by uranium.     

Cyclic nucleotides and development ofMyxococcus xanthus: Analysis of mutants

Approximately 60 developmental mutants ofMyxococcus xanthus M300 were obtained through nitrosoguanidine mutagenesis and placed into three operationally defined categories. Type-I strains exhibited no aggregation or sporulation. Type-II strains were able to aggregate but did not sporulate. A strain classed as a type-III strain was a low-capacity fruiter. Each category displayed defects in cyclic nucleotide behavior that could be predicted from the current model. Most significantly, several aggregationless (type I) mutants lacking cGMP phosphodiesterase aggregated in the presence of externally applied phosphodiesterase. A requirement for cell-cell contact in sporulation has been confirmed. Evidence is presented that suggests the involvement of cAMP phosphodiesterase in sporulation and that sporulation may be a developmental pathway independent of aggregation. These results support a previously published hypothesis of the role of cyclic nucleotides in the development ofM. xanthus.     

Type IV‐pili dependent motility is co‐regulated by PilSR and PilS2R2 two‐component systems via distinct pathways in Myxococcus xanthus

Myxococcus xanthus is an environmental bacterium with two forms of motility. One type, known as social motility, is dependent on extension and retraction of Type‐IV pili (T4P) and production of extracellular polysaccharides (EPS). Several signaling systems have been linked to regulation of T4P‐dependent motility. In particular, expression of the pilin subunit pilA requires the PilSR two‐component signaling system (TCS). A second TCS, PilS2R2, encoded within the same locus that encodes PilSR, has also been linked to M. xanthus T4P‐dependent motility. We demonstrate that PilSR and PilS2R2 regulate M. xanthus T4P‐dependent motility through distinct pathways. Consistent with known roles of PilSR, our results indicate that the primary function of PilSR is to regulate expression of pilA. In contrast, PilS2 and PilR2 have little to no affect on PilA protein levels. However, deletion of pilR2 resulted in a reduction of assembled pili, significant decreases in EPS production and loss of T4P‐dependent motility. Furthermore, the pilR2 mutation led to increased production of outer membrane vesicles (OMV). Collectively, we propose that PilS2R2 is required for proper assembly of T4P and regulation of OMV production, and hypothesize that production of these vesicles is related to M. xanthus motility.     

Genetics of gliding motility and development inMyxococcus xanthus

Successful development in multicellular eukaryotes requires cell-cell communication and the coordinated spatial and temporal movements of cells. The complex array of networks required to bring eukaryotic development to fruition can be modeled by the development of the simpler prokaryoteMyxococcus xanthus. As part of its life cycle,M. xanthus forms multicellular fruiting bodies containing differentiated cells. Analysis of the genes essential forM. xanthus development is possible because strains with mutations that block development can be maintained in the vegetative state. Development inM. xanthus is induced by starvation, and early events in development suggest that signaling, stages have evolved to monitor the metabolic state of the developing cell. In the absence of these signals, which include amino acids, α-keto acids, and other intermediary metabolites, the ability of cells to differentiate into myxospores is impaired. Mutations that block genes controlling gliding, motility disrupt the morphogenesis of fruiting bodies and sporogenesis in surprising ways. In this review, we present data that encourage future genetic and biochemical studies of the relationships between motility, cell-cell signaling, and development inM. xanthus.