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The motility mechanism of certain prokaryotes has long been a mystery, since their motion, known as gliding, involves no external appendages. The physical principles behind gliding still remain poorly understood. Using myxobacteria as an example of such organisms, we identify here the physical principles behind gliding motility and develop a theoretical model that predicts a 2-regime behavior of the gliding speed as a function of the substrate stiffness. Our theory describes the elasto-capillary-hydrodynamic interactions between the membrane of the bacteria, the slime it secretes, and the soft substrate underneath. Defining gliding as the horizontal translation under zero net force, we find the 2-regime behavior is due to 2 distinct mechanisms of motility thrust. On mildly soft substrates, the thrust arises from bacterial shape deformations creating a flow of slime that exerts a pressure along the bacterial length. This pressure in conjunction with the bacterial shape provides the necessary thrust for propulsion. On very soft substrates, however, we show that capillary effects must be considered that lead to the formation of a ridge at the slime-substrate-air interface, thereby creating a thrust in the form of a localized pressure gradient at the bacterial leading edge. To test our theory, we perform experiments with isolated cells on agar substrates of varying stiffness and find the measured gliding speeds in good agreement with the predictions from our elasto-capillary-hydrodynamic model. The mechanisms reported here serve as an important step toward an accurate theory of friction and substrate-mediated interactions between bacteria proliferating in soft media.

MreB is a bacterial actin that is important for cell shape and cell wall biosynthesis in many bacterial species. MreB also plays crucial roles in Myxococcus xanthus gliding motility, but the underlying mechanism remains unknown. Here we tracked the dynamics of single MreB particles in M. xanthus using single-particle tracking photoactivated localization microscopy. We found that a subpopulation of MreB particles moves rapidly along helical trajectories, similar to the movements of the MotAB-like gliding motors. The rapid MreB motion was stalled in the mutants that carried truncated gliding motors. Remarkably, M. xanthus MreB moves one to two orders of magnitude faster than its homologs that move along with the cell wall synthesis machinery in Bacillus subtilis and Escherichia coli, and this rapid movement was not affected by the inhibitors of cell wall biosynthesis. Our results show that in M. xanthus, MreB provides a scaffold for the gliding motors while the gliding machinery drives the movement of MreB filaments, analogous to the interdependent movements of myosin motors and actin in eukaryotic cells.

For many bacteria, motility is essential for survival, growth, virulence, biofilm formation and intra/interspecies interactions. Since natural environments differ, bacteria have evolved remarkable motility systems to adapt, including swimming in aqueous media, and swarming, twitching and gliding on solid and semi-solid surfaces. Although tremendous advances have been achieved in understanding swimming and swarming motilities powered by flagella, and twitching motility powered by Type IV pili, little is known about gliding motility. Bacterial gliders are a heterogeneous group containing diverse bacteria that utilize surface motilities that do not depend on traditional flagella or pili, but are powered by mechanisms that are less well understood. Recently, advances in our understanding of the molecular machineries for several gliding bacteria revealed the roles of modified ion channels, secretion systems and unique machinery for surface movements. These novel mechanisms provide rich source materials for studying the function and evolution of complex microbial nanomachines. In this review, we summarize recent findings made on the gliding mechanisms of the myxobacteria, flavobacteria and mycoplasmas.

Over 30 years ago, GGDEF domain-containing enzymes were shown to be diguanylate cyclases that produce cyclic di-GMP (cdiG), a second messenger that modulates the key bacterial lifestyle transition from a motile to sessile biofilm-forming state. Since then, the ubiquity of genes encoding GGDEF proteins in bacterial genomes has established the dominance of cdiG signaling in bacteria. However, the observation that proteobacteria encode a large number of GGDEF proteins, nearing 1% of coding sequences in some cases, raises the question of why bacteria need so many GGDEF enzymes. In this study, we reveal that a subfamily of GGDEF enzymes synthesizes the asymmetric signaling molecule cyclic AMP-GMP (cAG or 3', 3'-cGAMP). This discovery is unexpected because GGDEF enzymes function as symmetric homodimers, with each monomer binding to one substrate NTP. Detailed analysis of the enzyme from Geobacter sulfurreducens showed it is a dinucleotide cyclase capable of switching the major cyclic dinucleotide (CDN) produced based on ATP-to-GTP ratios. We then establish through bioinformatics and activity assays that hybrid CDN-producing and promiscuous substrate-binding (Hypr) GGDEF enzymes are found in other deltaproteobacteria. Finally, we validated the predictive power of our analysis by showing that cAG is present in surface-grown Myxococcus xanthus. This study reveals that GGDEF enzymes make alternative cyclic dinucleotides to cdiG and expands the role of this widely distributed enzyme family to include regulation of cAG signaling.

Gliding motility in Myxococcus xanthus is powered by flagella stator homologs that move in helical trajectories using proton motive force. The Frz chemosensory pathway regulates the cell polarity axis through MglA, a Ras family GTPase; however, little is known about how MglA establishes the polarity of gliding, because the gliding motors move simultaneously in opposite directions. Here we examined the localization and dynamics of MglA and gliding motors in high spatial and time resolution. We determined that MglA localizes not only at the cell poles, but also along the cell bodies, forming a decreasing concentration gradient toward the lagging cell pole. MglA directly interacts with the motor protein AglR, and the spatial distribution of AglR reversals is positively correlated with the MglA gradient. Thus, the motors moving toward lagging cell poles are less likely to reverse, generating stronger forward propulsion. MglB, the GTPase-activating protein of MglA, regulates motor reversal by maintaining the MglA gradient. Our results suggest a mechanism whereby bacteria use Ras family proteins to modulate cellular polarity.

Many bacteria glide smoothly on surfaces, despite having no discernable propulsive organelles on their surface. Recent experiments with Myxococcus xanthus and Flavobacterium johnsoniae show that both of these distantly related bacterial species glide using proteins that move in helical tracks, albeit with significantly different motility mechanisms. Both species utilize proton-motive force for movement. Although the motors that power gliding in M. xanthus have been identified, the F. johnsoniae motors remain to be discovered.

Many bacteria move on solid surfaces using gliding motility, without involvement of flagella or pili. Gliding of Myxococcus xanthus is powered by a proton channel homologous to the stators in the bacterial flagellar motor. Instead of being fixed in place and driving the rotation of a circular protein track like the flagellar basal body, the gliding machinery of M. xanthus travels the length of the cell along helical trajectories, while mechanically engaging with the substrate. Such movement entails a different molecular mechanism to generate propulsion on the cell. In this perspective, we will discuss the similarities and differences between the M. xanthus gliding machinery and bacterial flagellar motor, and use biophysical principles to generate hypotheses about the operating mechanism, efficiency, sensitivity to control, and mechanosensing of M. xanthus gliding. [ABSTRACT FROM AUTHOR]

Summary: 3′,3′‐cyclic GMP‐AMP (cGAMP) is the third cyclic dinucleotide (CDN) to be discovered in bacteria. No activators of cGAMP signaling have yet been identified, and the signaling pathways for cGAMP have been inferred to display a narrow distribution based upon the characterized synthases, DncV and Hypr GGDEFs. Here, we report that the ubiquitous second messenger cyclic AMP (cAMP) is an activator of the Hypr GGDEF enzyme GacB from Myxococcus xanthus. Furthermore, we show that GacB is inhibited directly by cyclic di‐GMP, which provides evidence for cross‐regulation between different CDN pathways. Finally, we reveal that the HD‐GYP enzyme PmxA is a cGAMP‐specific phosphodiesterase (GAP) that promotes resistance to osmotic stress in M. xanthus. A signature amino acid change in PmxA was found to reprogram substrate specificity and was applied to predict the presence of non‐canonical HD‐GYP phosphodiesterases in many bacterial species, including phyla previously not known to utilize cGAMP signaling. [ABSTRACT FROM AUTHOR]

Bacterial gliding motility is the smooth movement of cells on solid surfaces unaided by flagella or pili. Many diverse groups of bacteria exhibit gliding, but the mechanism of gliding motility has remained a mystery since it was first observed more than a century ago. Recent studies on the motility of Myxococcus xanthus, a soil myxobacterium, suggest a likely mechanism for gliding in this organism. About forty M. xanthus genes were shown to be involved in gliding motility, and some of their protein products were labeled and localized within cells. These studies suggest that gliding motility in M. xanthus involves large multiprotein structural complexes, regulatory proteins, and cytoskeletal filaments. In this review, we summarize recent experiments that provide the basis for this emerging view of M. xanthus motility. We also discuss alternative models for gliding. [ABSTRACT FROM AUTHOR]

Gliding motility in the bacterium Myxococcus xanthus uses two motility engines: S-motility powered by type-IV pili and A-motility powered by uncharacterized motor proteins and focal adhesion complexes. In this paper, we identified MreB, an actin-like protein, and MglA, a small GTPase of the Ras superfamily, as essential for both motility systems. A22, an inhibitor of MreB cytoskeleton assembly, reversibly inhibited S- and A-motility, causing rapid dispersal of S- and A-motility protein clusters, FrzS and AglZ. This suggests that the MreB cytoskeleton is involved in directing the positioning of these proteins. We also found that a ΔmglA motility mutant showed defective localization of AglZ and FrzS clusters. Interestingly, MglA–YFP localization mimicked both FrzS and AglZ patterns and was perturbed by A22 treatment, consistent with results indicating that both MglA and MreB bind to motility complexes. We propose that MglA and the MreB cytoskeleton act together in a pathway to localize motility proteins such as AglZ and FrzS to assemble the A-motility machineries. Interestingly, M. xanthus motility systems, like eukaryotic systems, use an actin-like protein and a small GTPase spatial regulator. [ABSTRACT FROM AUTHOR]

Abstract: C4-dicarboxylates are the major carbon and energy sources during the symbiotic growth of rhizobia. Responses to C4-dicarboxylates depend on typical two-component systems (TCS) consisting of a transmembrane sensor histidine kinase and a cytoplasmic response regulator. The DctB–DctD system is the first identified TCS for C4-dicarboxylates sensing. Direct ligand binding to the sensor domain of DctB is believed to be the first step of the sensing events. In this report, the water-soluble periplasmic sensor domain of Sinorhizobium meliloti DctB (DctBp) was studied, and three crystal structures were solved: the apo protein, a complex with C4 succinate, and a complex with C3 malonate. Different from the two structurally known CitA family of carboxylate sensor proteins CitA and DcuS, the structure of DctBp consists of two tandem Per–Arnt–Sim (PAS) domains and one N-terminal helical region. Only the membrane-distal PAS domain was found to bind the ligands, whereas the proximal PAS domain was empty. Comparison of DctB, CitA, and DcuS suggests a detailed stereochemistry of C4-dicarboxylates ligand perception. The structures of the different ligand binding states of DctBp also revealed a series of conformational changes initiated upon ligand binding and propagated to the N-terminal domain responsible for dimerization, providing insights into understanding the detailed mechanism of the signal transduction of TCS histidine kinases. [Copyright &y& Elsevier]

The Sinorhizobium meliloti C4-dicarboxylate transport (Dct) system is essential for symbiotic nitrogen fixation. The dctA gene, encoding the C4-dicarboxylate permease, is expressed in both free living and symbiotic cells. But in free living cells expression of dctD and dctB is absolutely required for the expression of dctA. In this study, in order to investigate the effect of oxygen concentration on the induction of Dct system, E. coli DH5α strain which carries the plasmid-encoded dctABD operon was used in tube assays. It was found that the specific induction of Dct system occurred only at a certain depth under the surface of M630.6% agar media, suggesting that Dct system could respond to oxygen concentration during succinate-induced expression. Furthermore, when measured at different oxygen concentrations, the highest expression level was observed at oxygen concentration of 2%. Thus, we predict that in addition to dicarboxylates, the induction of Dct system may also regulated by oxygen concentration. [ABSTRACT FROM AUTHOR]

A fundamental question in biology is how cell shapes are genetically encoded and enzymatically generated. Prevalent shapes among walled bacteria include spheres and rods. These shapes are chiefly determined by the peptidoglycan (PG) cell wall. Bacterial division results in two daughter cells, whose shapes are predetermined by the mother. This makes it difficult to explore the origin of cell shapes in healthy bacteria. In this review, we argue that the Gram-negative bacterium Myxococcus xanthus is an ideal model for understanding PG assembly and bacterial morphogenesis, because it forms rods and spheres at different life stages. Rod-shaped vegetative cells of M. xanthus can thoroughly degrade their PG and form spherical spores. As these spores germinate, cells rebuild their PG and reestablish rod shape without preexisting templates. Such a unique sphere-to-rod transition provides a rare opportunity to visualize de novo PG assembly and rod-like morphogenesis in a well-established model organism. [ABSTRACT FROM AUTHOR]

Abstract: Sinorhizobium meliloti DctBD is a well-characterized two-component system. It is believed that DctB senses the concentration of C4-dicarboxylate compounds on the outside of the bacterium and phosphorylates DctD, which in turn activates transcription of the dctA gene, coding for a gene of C4-dicarboxylate permease. The structure and function of the ligand-binding domain of DctB has not been thoroughly investigated. In this study, this domain was produced in E. coli in soluble form, and purified to homogeneity. Crystals were obtained by hanging-drop vapor-diffusion method. The crystals diffracted to 2.3 Å resolution and belonged to P42 space group with unit cell dimensions of a = b = 71.77 Å, c = 227.14 Å. The asymmetric unit contains four molecules with a corresponding V M of 2.4 Å3 Da−1 and a solvent content of 49.1%. [Copyright &y& Elsevier]

Summary Some bacteria glide mysteriously on surfaces without using flagella, pili, or other external appendages. Recent studies suggest how gliding motors in the inner membrane may transduce force to the cell surface. [ABSTRACT FROM AUTHOR]

Mitochondrial membrane biogenesis requires the import of phospholipids; however, the molecular mechanisms underlying this process remain elusive. Recent work has implicated membrane contact sites between the mitochondria, endoplasmic reticulum (ER), and vacuole in phospholipid transport. Utilizing a genetic approach focused on these membrane contact site proteins, we have discovered a 'moonlighting' role of the membrane contact site and vesicular fusion protein, Vps39, in phosphatidylethanolamine (PE) transport to the mitochondria. We show that the deletion of Vps39 prevents ethanolamine-stimulated elevation of mitochondrial PE levels without affecting PE biosynthesis in the ER or its transport to other sub-cellular organelles. The loss of Vps39 did not alter the levels of other mitochondrial phospholipids that are biosynthesized ex situ , implying a PE-specific role of Vps39. The abundance of Vps39 and its recruitment to the mitochondria and the ER is dependent on PE levels in each of these organelles, directly implicating Vps39 in the PE transport process. Deletion of essential subunits of Vps39-containing complexes, vCLAMP and HOPS, did not abrogate ethanolamine-stimulated PE elevation in the mitochondria, suggesting an independent role of Vps39 in intracellular PE trafficking. Our work thus identifies Vps39 as a novel player in ethanolamine-stimulated PE transport to the mitochondria. • Vps39 is required for ethanolamine stimulated PE transport to yeast mitochondria. • The role of Vps39 in PE transport is independent of HOPS and vCLAMP complexes. • Vps39 abundance is reduced in cells lacking mitochondrial PE biosynthetic enzyme. • The Kennedy pathway enzyme for PE synthesis, Ept1, localizes to the ER. [ABSTRACT FROM AUTHOR]

Adjusting motility patterns according to environmental cues is important for bacterial survival. Myxococcus xanthus , a bacterium moving on surfaces by gliding and twitching mechanisms, modulates the reversal frequency of its front-back polarity in response to mechanical cues like substrate stiffness and cell-cell contact. In this study, we propose that M. xanthus 's gliding machinery senses environmental mechanical cues during force generation and modulates cell reversal accordingly. To examine our hypothesis, we expand an existing mathematical model for periodic polarity reversal in M. xanthus , incorporating the experimental data on the intracellular dynamics of the gliding machinery and the interaction between the gliding machinery and a key polarity regulator. The model successfully reproduces the dependence of cell reversal frequency on substrate stiffness observed in M. xanthus gliding. We further propose reversal control networks between the gliding and twitching motility machineries to explain the opposite reversal responses observed in wild type M. xanthus cells that possess both motility mechanisms. These results provide testable predictions for future experimental investigations. In conclusion, our model suggests that the gliding machinery in M. xanthus can function as a mechanosensor, which transduces mechanical cues into a cell reversal signal., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Chen, Topo, Nan and Chen.)

We report the assembly and annotation of a high-quality genome sequence for Myxococcus xanthus strain DZ2 (GenBank accession number CP080538), created using a combination of short reads generated using DNBSEQ technology (BGI Genomics) and long high-fidelity (HiFi) reads generated using Pacific Biosciences (PacBio) technology.

Intraflagellar transport (IFT) is essential for the assembly and maintenance of flagella and cilia. Recent biochemical studies have shown that IFT complex B (IFT-B) is comprised of two subcomplexes, IFT-B1 and IFT-B2. The IFT-B2 subunit IFT57 lies at the interface between IFT-B1 and IFT-B2. Here, using a Chlamydomonas reinhardtii mutant for IFT57, we tested whether IFT57 is required for IFT-B complex assembly by bridging IFT-B1 and IFT-B2 together. In the ift57-1 mutant, levels of IFT57 and other IFT-B proteins were greatly reduced at the whole-cell level. However, strikingly, in the protease-free flagellar compartment, while the level of IFT57 was reduced, the levels of other IFT particle proteins were not concomitantly reduced but were present at the wild-type level. The IFT movement of the IFT57-deficient IFT particles was also unchanged. Moreover, IFT57 depletion disrupted the flagellar waveform, leading to cell swimming defects. Analysis of the mutant flagellar protein composition showed that certain axonemal proteins were altered. Taken together, these findings suggest that IFT57 does not play an essential structural role in the IFT particle complex but rather functions to prevent it from degradation. Additionally, IFT57 is involved in transporting specific motility-related proteins., (© 2017. Published by The Company of Biologists Ltd.)

C(4)-dicarboxylates are the major carbon and energy sources during the symbiotic growth of rhizobia. Responses to C(4)-dicarboxylates depend on typical two-component systems (TCS) consisting of a transmembrane sensor histidine kinase and a cytoplasmic response regulator. The DctB-DctD system is the first identified TCS for C(4)-dicarboxylates sensing. Direct ligand binding to the sensor domain of DctB is believed to be the first step of the sensing events. In this report, the water-soluble periplasmic sensor domain of Sinorhizobium meliloti DctB (DctBp) was studied, and three crystal structures were solved: the apo protein, a complex with C(4) succinate, and a complex with C(3) malonate. Different from the two structurally known CitA family of carboxylate sensor proteins CitA and DcuS, the structure of DctBp consists of two tandem Per-Arnt-Sim (PAS) domains and one N-terminal helical region. Only the membrane-distal PAS domain was found to bind the ligands, whereas the proximal PAS domain was empty. Comparison of DctB, CitA, and DcuS suggests a detailed stereochemistry of C(4)-dicarboxylates ligand perception. The structures of the different ligand binding states of DctBp also revealed a series of conformational changes initiated upon ligand binding and propagated to the N-terminal domain responsible for dimerization, providing insights into understanding the detailed mechanism of the signal transduction of TCS histidine kinases.

Protein biotinylation and lipoylation are post-translational modifications, in which biotin or lipoic acid is covalently attached to specific proteins containing biotin/lipoyl attachment domains. All the currently reported natural proteins containing biotin/lipoyl attachment domains are multidomain proteins and can only be modified by either biotin or lipoic acid in vivo. We have identified a single domain protein with 73 amino acid residues from Bacillus subtilis strain 168, and it can be both biotinylated and lipoylated in Escherichia coli. The protein is therefore named as biotin/lipoyl attachment protein (BLAP). This is the first report that a natural single domain protein exists as both a biotin and lipoic acid receptor. The solution structure of apo-BLAP showed that it adopts a typical fold of biotin/lipoyl attachment domain. The structure of biotinylated BLAP revealed that the biotin moiety is covalently attached to the side chain of Lys(35), and the bicyclic ring of biotin is folded back and immobilized on the protein surface. The biotin moiety immobilization is mainly due to an interaction between the biotin ureido ring and the indole ring of Trp(12). NMR study also indicated that the lipoyl group of the lipoylated BLAP is also immobilized on the protein surface in a similar fashion as the biotin moiety in the biotinylated protein.