The Bactofilin Cytoskeleton Protein BacM of Myxococcus xanthus Forms an Extended β-Sheet Structure Likely Mediated by Hydrophobic Interactions

Bactofilins are novel cytoskeleton proteins that are widespread in Gram-negative bacteria. Myxococcus xanthus, an important predatory soil bacterium, possesses four bactofilins of which one, BacM (Mxan_7475) plays an important role in cell shape maintenance. Electron and fluorescence light microscopy, as well as studies using over-expressed, purified BacM, indicate that this protein polymerizes in vivo and in vitro into ~3 nm wide filaments that further associate into higher ordered fibers of about 10 nm. Here we use a multipronged approach combining secondary structure determination, molecular modeling, biochemistry, and genetics to identify and characterize critical molecular elements that enable BacM to polymerize. Our results indicate that the bactofilin-determining domain DUF583 folds into an extended β-sheet structure, and we hypothesize a left-handed β-helix with polymerization into 3 nm filaments primarily via patches of hydrophobic amino acid residues. These patches form the interface allowing head-to-tail polymerization during filament formation. Biochemical analyses of these processes show that folding and polymerization occur across a wide variety of conditions and even in the presence of chaotropic agents such as one molar urea. Together, these data suggest that bactofilins are comprised of a structure unique to cytoskeleton proteins, which enables robust polymerization.     

A Clp/Hsp100 chaperone functions in Myxococcus xanthus sporulation and self-organization

The Clp/Hsp100 proteins are chaperones that play a role in protein degradation and reactivation. In bacteria, they exhibit a high degree of pleiotropy, affecting both individual and multicellular phenotypes. In this article, we present the first characterization of a Clp/Hsp100 homolog in Myxococcus xanthus (MXAN_4832 gene locus). Deletion of MXAN_4832 causes defects in both swarming and aggregation related to cell motility and the production of fibrils, which are an important component of the extracellular matrix of a swarm. The deletion also affects the formation of myxospores during development, causing them to become sensitive to heat. The protein product of MXAN_4832 can act as a chaperone in vitro, providing biochemical evidence in support of our hypothesis that MXAN_4832 is a functional Clp/Hsp100 homolog. There are a total of 12 Clp/Hsp100 homologs in M. xanthus, including MXAN_4832, and, based on its mutational and biochemical characterization, they may well represent an important group.     

Natural competence in strains of Actinobacillus pleuropneumoniae

The fatty acid (FA) profiles of myxobacteria contain FA species with double bonds at the Δ⁵ and Δ¹¹ positions, the latter being rather unusual among bacteria. Despite this knowledge, the mechanism for introduction of these double bonds has never been described before in myxobacteria. Searches for candidate genes in the genome of the model organism Myxococcus xanthus revealed 16 genes, which have been annotated as FA desaturases. However, due to redundant substrate specificity, functional analyses of these enzymes by construction of inactivation mutants did not lead to the identification of their function or substrate specificity. Therefore, we elucidated the regioselectivity of the desaturation reactions by heterologous expression of eight desaturases from M. xanthus in Pseudomonas putida and thus could prove five of them to be indeed active as desaturases, with three (MXAN_1742, MXAN_3495 and MXAN_5461) and two (MXAN_0317 and MXAN_6306) acting as Δ⁵ and Δ¹¹ desaturases, respectively. This is the first report about the heterologous expression and regioselectivity of FA desaturases in myxobacteria.     

Developmental changes in cell wall structure of phloem fibres of the bamboo Dendrocalamus asper

BACKGROUND AND AIMS: Bamboo culms have excellent physical and mechanical properties, which mainly depend on their fibre content and anatomical structure. One of the features which is known to contribute to the high tensile strength in bamboo is the multilayered structure of the fibre cell wall. The aim of this study was to characterize the development of the layered structure in fibre cell walls of developing and maturing culms of Dendrocalamus asper. METHODS: Cell wall development patterns were investigated in phloem fibre caps of vascular bundles in the inner culm wall areas of Dendrocalamus asper of three different age classes (<6 months old, 1 year old, 3 years old). A combination of light microscopy and image analysis techniques were employed to measure cell wall thickness and to determine number of cell wall layers, as well as to describe the layering structure of fibre walls. Two-dimensional maps showing the distribution pattern of fibres according to the number of cell wall layers were produced. KEY RESULTS: The cell walls of fibres in phloem fibre caps located in the inner part of the culm wall of D. asper developed rapidly during the first year of growth. Six different fibre types could be distinguished based upon their cell wall layering and all were already present in the young, 1-year-old culm. In the mature stage (3 years of age) the multilayering was independent of the cell wall thickness and even the thinner-walled fibres could have a large number of wall layers. The multilayered nature of cell wall structure varied considerably between individual cells and was not exclusively related to the cell wall thickness. Nevertheless, fibres at the periphery of the fibre bundles and immediately adjacent to the phloem elements exhibited a consistent and high degree of layering in their cell walls. CONCLUSIONS: The multilayered structure of fibre cell walls was formed mainly during the first year of growth by the deposition of new wall layers of variable thickness, resulting in a high degree of heterogeneity in the layering patterns amongst individual fibres. A degree of 'order' in the distribution of multilayered fibres within the caps does exist, however, with multilayered cell walls common in fibres adjacent to phloem elements and around the edge of the fibre cap. These findings confirm the observations, primarily in Phyllostachys viridi-glaucescens. The layering structure was not found to be specifically related to the thickness of the cell wall.     

Short-term effects of nitrogen availability on wood formation and fibre properties in hybrid poplar

The application of nitrogen-containing fertilisers is one approach used to increase growth rates and productivity of forest tree plantations. However, the effects of nitrogen fertilisation on wood properties have not been systematically assessed. The aim of this work was to document the impacts of nitrogen fertilisation on wood formation and secondary xylem fibre properties. We used three fertilisation treatments in which the level of ammonium nitrate was adjusted to 0, 1 and 10 mM in a complete nutrient solution applied daily over a period of 28 days in standardised greenhouse experiments with clonal material of Populus trichocarpa (Torr and Gray) × deltoides (Bartr. ex Marsh). We showed that there was a short-term and repeatable response in which xylem fibre morphology and secondary cell wall structure adapt to a shift in N availability. Under high-nitrogen exposure, xylem fibres were 17% wider and 18% shorter compared to the adequate nitrogen treatment. A very significant thickening of the fibre cell walls was also observed throughout the stem of trees receiving the high-N treatment. It appeared that cell wall structure was greatly affected by the high-N treatment as fibres developed a modified inner cell wall layer. Histological observations indicated that the internal cell wall layer was enriched in cellulose and chemical determinations showed that wood contained more holocellulose. Together, these results indicate that the response of poplar to nitrogen availability may involve marked effects on secondary xylem formation.     

黄色粘球菌基于复杂社会性细胞行为的生物被膜

黄色粘球菌具有多样化的细胞行为,具备典型多细胞水平的社会性特征。其形成的生物被膜是目前认知的最为复杂的单种群细菌生物被膜之一。黄色粘球菌的社会性细胞行为主导了其生物被膜形成过程中的关键环节,包括固体介质表面的细胞运动、群体细胞的捕食、亲缘细胞的识别、子实体的发育、粘孢子的分化以及细胞程序性死亡等行为过程。文中将介绍相关领域的研究进展。     

Identification of the Wzx flippase,Wzy polymerase and sugar-modifying enzymes for spore coat polysaccharide biosynthesis in Myxococcus xanthus

The rod-shaped cells of Myxococcus xanthus, a Gram-negative deltaproteobacterium, differentiate to environmentally resistant spores upon starvation or chemical stress. The environmental resistance depends on a spore coat polysaccharide that is synthesised by the ExoA-I proteins, some of which are part of a Wzx/Wzy-dependent pathway for polysaccharide synthesis and export; however, key components of this pathway have remained unidentified. Here, we identify and characterise two additional loci encoding proteins with homology to enzymes involved in polysaccharide synthesis and export, as well as sugar modification and show that six of the proteins encoded by these loci are essential for the formation of environmentally resistant spores. Our data support that MXAN_3260, renamed ExoM and MXAN_3026, renamed ExoJ, are the Wzx flippase and Wzy polymerase, respectively, responsible for translocation and polymerisation of the repeat unit of the spore coat polysaccharide. Moreover, we provide evidence that three glycosyltransferases (MXAN_3027/ExoK, MXAN_3262/ExoO and MXAN_3263/ExoP) and a polysaccharide deacetylase (MXAN_3259/ExoL) are important for formation of the intact spore coat, while ExoE is the polyisoprenyl-phosphate hexose-1-phosphate transferase responsible for initiating repeat unit synthesis, likely by transferring N-acetylgalactosamine-1-P to undecaprenyl-phosphate. Together, our data generate a more complete model of the Exo pathway for spore coat polysaccharide biosynthesis and export.     

Ultrastructure of fibre and parenchyma cell walls during early stages of culm development in Dendrocalamus asper

BACKGROUND AND AIMS: The anatomy of bamboo culms and the multilayered structure of fibre cell walls are known to be the main determinant factors for its physical and mechanical properties. Studies on the bamboo cell wall have focussed mainly on fully elongated and mature fibres. The main aim of this study was to describe the ultrastructure of primary and secondary cell walls in culm tissues of Dendrocalamus asper at different stages of development. METHODS: The development of fibre and parenchyma tissues was classified into four stages based on light microscopy observations made in tissues from juvenile plants. The stages were used as a basis for transmission electron microscopy study on the ultrastructure of the cell wall during the process of primary and early secondary cell wall formation. Macerations and phloroglucinol-HCl staining were employed to investigate fibre cell elongation and fibre cell wall lignification, respectively. KEY RESULTS: The observations indicated that the primary wall is formed by the deposition of two distinct layers during the elongation of the internode and that secondary wall synthesis may begin before the complete cessation of internode and fibre elongation. Elongation was followed by a maturation phase characterized by the deposition of multiple secondary wall layers, which varied in number according to the cell type, location in the culm tissue and stage of shoot development. Lignification of fibre cell walls started at the period prior to the cessation of internode elongation. CONCLUSIONS: The structure of the primary cell wall was comprised of two layers. The fibre secondary cell wall began to be laid down while the cells were still undergoing some elongation, suggesting that it may act to cause the slow-down and eventual cessation of cell elongation.     

Experimentally guided computational model discovers important elements for social behavior in myxobacteria

Identifying essential factors in cellular interactions and organized movement of cells is important in predicting behavioral phenotypes exhibited by many bacterial cells. We chose to study Myxococcus xanthus, a soil bacterium whose individual cell behavior changes while in groups, leading to spontaneous formation of aggregation center during the early stage of fruiting body development. In this paper, we develop a cell-based computational model that solely relies on experimentally determined parameters to investigate minimal elements required to produce the observed social behaviors in M. xanthus. The model verifies previously known essential parameters and identifies one novel parameter, the active turning, which we define as the ability and tendency of a cell to turn to a certain angle without the presence of any obvious external factors. The simulation is able to produce both gliding pattern and spontaneous aggregation center formation as observed in experiments. The model is tested against several known M. xanthus mutants and our modification of parameter values relevant for the individual mutants produces good phenotypic agreements. This outcome indicates the strong predictive potential of our model for the social behaviors of uncharacterized mutants and their expected phenotypes during development.     

Bactofilins,a ubiquitous class of cytoskeletal proteins mediating polar localization of a cell wall synthase in Caulobacter crescentus

The cytoskeleton has a key function in the temporal and spatial organization of both prokaryotic and eukaryotic cells. Here, we report the identification of a new class of polymer-forming proteins, termed bactofilins, that are widely conserved among bacteria. In Caulobacter crescentus, two bactofilin paralogues cooperate to form a sheet-like structure lining the cytoplasmic membrane in proximity of the stalked cell pole. These assemblies mediate polar localization of a peptidoglycan synthase involved in stalk morphogenesis, thus complementing the function of the actin-like cytoskeleton and the cell division machinery in the regulation of cell wall biogenesis. In other bacteria, bactofilins can establish rod-shaped filaments or associate with the cell division apparatus, indicating considerable structural and functional flexibility. Bactofilins polymerize spontaneously in the absence of additional cofactors in vitro, forming stable ribbon- or rod-like filament bundles. Our results suggest that these structures have evolved as an alternative to intermediate filaments, serving as versatile molecular scaffolds in a variety of cellular pathways.     

Synthesis and salvage of purines during cellular morphogenesis of Myxococcus xanthus.

Intact cells of Myxococcus xanthus were examined for de novo purine synthesis and salvage utilization. The cellular uptake rates of radioactive glycine (de novo purine precursor), adenine, and guanine were measured, and thin-layer chromatography and radioautography were used to examine cell extracts for de novo synthesized purine nucleotides. Intact vegatative cells, glycerol-induced myxospores, and germinating cells of M. xanthus CW-1 were able to carry out de novo purine and salvage synthesis. Germinating cells and glycerol-induced myxospores were metabolically more active or as active as vegetative cells with respect to purine anabolism. We conclude that M. xanthus is capable of synthesizing purine nucleotides and salvaging purines throughout the glycerol version of its life cycle.     

Host and cell type affect the mode of degradation by Meripilus giganteus

Wood degradation by the white-rot basidiomycete Meripilus giganteus (Pers.: Pers.) Karst. was studied in naturally infected and artificially inoculated wood of beech ( Fagus sylvatica L.) and large-leaved lime ( Tilia platyphyllos Scop.). Semi-thin sections revealed that the secondary walls of most fibres contained internal cavities. Three distinct types of cavity formation, which differed not only between hosts, but also between cell type and location in the annual ring, were identified.
Within discoloured wood of naturally infected beech, the structure of the cavities and their formation by the associated hyphae were reminiscent of a soft-rot. By contrast, cavity formation in artificially inoculated beech and large-leaved lime wood differed from a soft-rot mode of attack as extensive delignification always preceded cavity formation, and neither T-branching, L-bending, nor hyphal growth were found within cell walls. The formation of half-moon shaped cavities in beech wood was present only in tension-wood fibres. From large diameter hyphae, growing within the fibre lumen, numerous fine perforation hyphae extended transversely via helical cracks into the cell wall. Subsequent degradation of cellulose within concentric layers of the tension-wood fibres commenced from the apices of perforation hyphae.
Sections stained with ruthenium red and hydroxlamine-ferric chloride, revealed that M. giganteus preferentially degrades pectin-rich regions of the middle lamellae in xylary ray cells. In large-leaved lime, such regions were uniformly located in the middle lamellae of axial and ray parenchyma. In beech wood, degradation of pectin-rich middle lamellae regions commenced after the delignification of secondary walls and resulted in a conspicuous hollowing of multiseriate xylem rays. Plasticity in wood degradation modes by M. giganteus in large-leaved lime and beech wood reflects variations in cell wall structure and/or prevailing wood conditions.     

Cloning of the gene for myxobacterial hemagglutinin and isolation and analysis of structural gene mutations.

Myxobacterial hemagglutinin (MBHA) is a major developmentally induced protein that accumulates during the period of cellular aggregation in the bacterium Myxococcus xanthus. It has been shown that this lectin is targeted to the cell surface and periplasmic space of developmental cells, suggesting that it may play a role in cell-cell recognition or agglutination. We have cloned the structural gene for MBHA by using synthetic deoxyoligonucleotides containing sequences deduced from the amino acid sequence of MBHA and have used the cloned gene to construct strains of M. xanthus that cannot synthesize MBHA. We found that although the MBHA-deficient strains are delayed in their developmental time course, they are otherwise able to aggregate and sporulate normally. Our results suggest that MBHA may function to increase the efficiency of fruiting-body formation but is not a critical component of cellular aggregation.     

A bacteriophage for Myxococcus xanthus: isolation, characterization and relation of infectivity to host morphogenesis

Burchard, Robert P. (University of Minnesota, Minneapolis), and M. Dworkin. A bacteriophage for Myxococcus xanthus: isolation, characterization and relation of infectivity to host morphogenesis. J. Bacteriol. 91:1305-1313. 1966.-A bacteriophage (MX-1) infecting Myxococcus xanthus FB(t) has been isolated from cow dung. The bacteriophage particle is approximately 175 mmu long. A tail about 100 mmu in length is encased in a contractile sheath and terminates in a tail plate. The head is polyhedral with a width of about 75 mmu. The nucleic acid of the bacteriophage is deoxyribonucleic acid and has a guanine plus cytosine content of 55.5%. The bacteriophage requires 10(-3)m Ca(++) and 10(-2)m monovalent cation for optimal adsorption. Grown on vegetative cells of M. xanthus FB(t) at 30 C in 2% Casitone medium, the bacteriophage has a latent period of 120 min and a burst size of approximately 100. Host range studies indicate that three strains of M. xanthus including a morphogenetic mutant are sensitive to the bacteriophage, whereas M. fulvus, Cytophaga, Sporocytophaga myxococcoides, and a fourth strain of M. xanthus are not. Of the two cellular forms characteristic of the Myxococcus life cycle, the bacteriophage infect only the vegetative cells; they do not adsorb to microcysts. Ability to adsorb bacteriophage is lost between 65 and 75 min after initiation of the relatively synchronous conversion of vegetative cells to microcysts. The bacteriophage does not adsorb to spheroplasts. After the appearance of visible morphogenesis and before the loss of bacteriophage receptor sites, addition of bacteriophage results in the formation of microcysts which give rise to infective centers only upon germination. The possibility that the infected microcysts are harboring intact bacteriophages has been eliminated.     

Cellular contractility and substrate elasticity: a numerical investigation of the actin cytoskeleton and cell adhesion

Numerous experimental studies have established that cells can sense the stiffness of underlying substrates and have quantified the effect of substrate stiffness on stress fibre formation, focal adhesion area, cell traction, and cell shape. In order to capture such behaviour, the current study couples a mixed mode thermodynamic and mechanical framework that predicts focal adhesion formation and growth with a material model that predicts stress fibre formation, contractility, and dissociation in a fully 3D implementation. Simulations reveal that SF contractility plays a critical role in the substrate-dependent response of cells. Compliant substrates do not provide sufficient tension for stress fibre persistence, causing dissociation of stress fibres and lower focal adhesion formation. In contrast, cells on stiffer substrates are predicted to contain large amounts of dominant stress fibres. Different levels of cellular contractility representative of different cell phenotypes are found to alter the range of substrate stiffness that cause the most significant changes in stress fibre and focal adhesion formation. Furthermore, stress fibre and focal adhesion formation evolve as a cell spreads on a substrate and leading to the formation of bands of fibres leading from the cell periphery over the nucleus. Inhibiting the formation of FAs during cell spreading is found to limit stress fibre formation. The predictions of this mutually dependent material-interface framework are strongly supported by experimental observations of cells adhered to elastic substrates and offer insight into the inter-dependent biomechanical processes regulating stress fibre and focal adhesion formation.     

3D Non-Woven Polyvinylidene Fluoride Scaffolds: Fibre Cross Section and Texturizing Patterns Have Impact on Growth of Mesenchymal Stromal Cells

Several applications in tissue engineering require transplantation of cells embedded in appropriate biomaterial scaffolds. Such structures may consist of 3D non-woven fibrous materials whereas little is known about the impact of mesh size, pore architecture and fibre morphology on cellular behavior. In this study, we have developed polyvinylidene fluoride (PVDF) non-woven scaffolds with round, trilobal, or snowflake fibre cross section and different fibre crimp patterns (10, 16, or 28 needles per inch). Human mesenchymal stromal cells (MSCs) from adipose tissue were seeded in parallel on these scaffolds and their growth was compared. Initial cell adhesion during the seeding procedure was higher on non-wovens with round fibres than on those with snowflake or trilobal cross sections. All PVDF non-woven fabrics facilitated cell growth over a time course of 15 days. Interestingly, proliferation was significantly higher on non-wovens with round or trilobal fibres as compared to those with snowflake profile. Furthermore, proliferation increased in a wider, less dense network. Scanning electron microscopy (SEM) revealed that the MSCs aligned along the fibres and formed cellular layers spanning over the pores. 3D PVDF non-woven scaffolds support growth of MSCs, however fibre morphology and mesh size are relevant: proliferation is enhanced by round fibre cross sections and in rather wide-meshed scaffolds.     

Actin stress fibre subtypes in mesenchymal-migrating cells

Mesenchymal cell migration is important for embryogenesis and tissue regeneration. In addition, it has been implicated in pathological conditions such as the dissemination of cancer cells. A characteristic of mesenchymal-migrating cells is the presence of actin stress fibres, which are thought to mediate myosin II-based contractility in close cooperation with associated focal adhesions. Myosin II-based contractility regulates various cellular activities, which occur in a spatial and temporal manner to achieve directional cell migration. These myosin II-based activities involve the maturation of integrin-based adhesions, generation of traction forces, establishment of the front-to-back polarity axis, retraction of the trailing edge, extracellular matrix remodelling and mechanotransduction. Growing evidence suggests that actin stress fibre subtypes, namely dorsal stress fibres, transverse arcs and ventral stress fibres, could provide this spatial and temporal myosin II-based activity. Consistent with their functional differences, recent studies have demonstrated that the molecular composition of actin stress fibre subtypes differ significantly. This present review focuses on the current view of the molecular composition of actin stress fibre subtypes and how these fibre subtypes regulate mesenchymal cell migration.     

Identification of the lipopolysaccharide O‐antigen biosynthesis priming enzyme and the O‐antigen ligase in Myxococcus xanthus: critical role of LPS O‐antigen in motility and development

Myxococcus xanthus is a model bacterium to study social behavior. At the cellular level, the different social behaviors of M. xanthus involve extensive cell–cell contacts. Here, we used bioinformatics, genetics, heterologous expression and biochemical experiments to identify and characterize the key enzymes in M. xanthus implicated in O‐antigen and lipopolysaccharide (LPS) biosynthesis and examined the role of LPS O‐antigen in M. xanthus social behaviors. We identified WbaP_Mx (MXAN_2922) as the polyisoprenyl‐phosphate hexose‐1‐phosphate transferase responsible for priming O‐antigen synthesis. In heterologous expression experiments, WbaP_Mx complemented a Salmonella enterica mutant lacking the endogenous WbaP that primes O‐antigen synthesis, indicating that WbaP_Mx transfers galactose‐1‐P to undecaprenyl‐phosphate. We also identified WaaL_Mx (MXAN_2919), as the O‐antigen ligase that joins O‐antigen to lipid A‐core. Our data also support the previous suggestion that Wzm_Mx (MXAN_4622) and Wzt_Mx (MXAN_4623) form the Wzm/Wzt ABC transporter. We show that mutations that block different steps in LPS O‐antigen synthesis can cause pleiotropic phenotypes. Also, using a wbaP_Mx deletion mutant, we revisited the role of LPS O‐antigen and demonstrate that it is important for gliding motility, conditionally important for type IV pili‐dependent motility and required to complete the developmental program leading to the formation of spore‐filled fruiting bodies.