Beiyan Nan

Bacterial motility complexes require the actin-like protein, MreB and the Ras homologue, MglA.

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.

Myxobacteria gliding motility requires cytoskeleton rotation powered by proton motive force

Myxococcus xanthus is a Gram-negative bacterium that glides over surfaces without the aid of flagella. Two motility systems are used for locomotion: social-motility, powered by the retraction of type IV pili, and adventurous (A)-motility, powered by unknown mechanism(s). We have shown that AgmU, an A-motility protein, is part of a multiprotein complex that spans the inner membrane and periplasm of M. xanthus. In this paper, we present evidence that periplasmic AgmU decorates a looped continuous helix that rotates clockwise as cells glide forward, reversing its rotation when cells reverse polarity. Inhibitor studies showed that the AgmU helix rotation is driven by proton motive force (PMF) and depends on actin-like MreB cytoskeletal filaments. The AgmU motility complex was found to interact with MotAB homologs. Our data are consistent with a mechanochemical model in which PMF-driven motors, similar to bacterial flagella stator complexes, run along an endless looped helical track, driving rotation of the track; deformation of the cell surface by the AgmU-associated proteins creates pressure waves in the slime, pushing cells forward.

Uncovering the Mystery of Gliding Motility in the Myxobacteria

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.

A multi-protein complex from Myxococcus xanthus required for bacterial gliding motility

Myxococcus xanthus moves by gliding motility powered by Type IV pili (S‐motility) and a second motility system, A‐motility, whose mechanism remains elusive despite the identification of ∼40 A‐motility genes. In this study, we used biochemistry and cell biology analyses to identify multi‐protein complexes associated with A‐motility. Previously, we showed that the N‐terminal domain of FrzCD, the receptor for the frizzy chemosensory pathway, interacts with two A‐motility proteins, AglZ and AgmU. Here we characterized AgmU, a protein that localized to both the periplasm and cytoplasm. On firm surfaces, AgmU‐mCherry colocalized with AglZ as distributed clusters that remained fixed with respect to the substratum as cells moved forward. Cluster formation was favoured by hard surfaces where A‐motility is favoured. In contrast, AgmU‐mCherry clusters were not observed on soft agar surfaces or when cells were in large groups, conditions that favour S‐motility. Using glutathione‐S‐transferase affinity chromatography, AgmU was found to interact either directly or indirectly with multiple A‐motility proteins including AglZ, AglT, AgmK, AgmX, AglW and CglB. These proteins, important for the correct localization of AgmU and AglZ, appear to be organized as a motility complex, spanning the cytoplasm, inner membrane and the periplasm. Identification of this complex may be important for uncovering the mechanism of A‐motility.

Flagella stator homologs function as motors for myxobacterial gliding motility by moving in helical trajectories

Significance Gliding is a form of enigmatic bacterial surface motility that does not use visible external structures such as flagella or pili. This study characterizes the single-molecule dynamics of the Myxococcus xanthus gliding motor protein AglR, a homolog of the Escherichia coli flagella stator protein MotA. However, the Myxococcus motors, unlike flagella stators, lack peptidoglycan-binding domains. With photoactivatable localization microscopy (PALM), we found that these motor proteins move actively within the cell membrane and generate torque by accumulating in clusters that exert force on the gliding surface. Our model unifies gliding and swimming with conserved power-generating modules. Many bacterial species use gliding motility in natural habitats because external flagella function poorly on hard surfaces. However, the mechanism(s) of gliding remain elusive because surface motility structures are not apparent. Here, we characterized the dynamics of the Myxococcus xanthus gliding motor protein AglR, a homolog of the Escherichia coli flagella stator protein MotA. We observed that AglR decorated a helical structure, and the AglR helices rotated when cells were suspended in liquid or when cells moved on agar surfaces. With photoactivatable localization microscopy, we found that single molecules of AglR, unlike MotA/MotB, can move laterally within the membrane in helical trajectories. AglR slowed down transiently at gliding surfaces, accumulating in clusters. Our work shows that the untethered gliding motors of M. xanthus, by moving within the membrane, can transform helical motion into linear driving forces that push against the surface.

AglZ regulates adventurous (A-) motility in Myxococcus xanthus through its interaction with the cytoplasmic receptor, FrzCD

Myxococcus xanthus moves by gliding motility powered by type IV pili (S‐motility) and distributed motor complexes (A‐motility). The Frz chemosensory pathway controls reversals for both motility systems. However, it is unclear how the Frz pathway can communicate with these different systems. In this article, we show that FrzCD, the Frz pathway receptor, interacts with AglZ, a protein associated with A‐motility. Affinity chromatography and cross‐linking experiments showed that the FrzCD–AglZ interaction occurs between the uncharacterized N‐terminal region of FrzCD and the N‐terminal pseudo‐receiver domain of AglZ. Fluorescence microscopy showed AglZ–mCherry and FrzCD–GFP localized in clusters that occupy different positions in cells. To study the role of the Frz system in the regulation of A‐motility, we constructed aglZ frzCD double mutants and aglZ frzCD pilA triple mutants. To our surprise, these mutants, predicted to show no A‐motility (A‐S+) or no motility at all (A‐S‐), respectively, showed restored A‐motility. These results indicate that AglZ modulates a FrzCD activity that inhibits A‐motility. We hypothesize that AglZ–FrzCD interactions are favoured when cells are isolated and moving by A‐motility and inhibited when S‐motility predominates and A‐motility is reduced.

Hybrid promiscuous (Hypr) GGDEF enzymes produce cyclic AMP-GMP (3', 3'-cGAMP)

Significance Cyclic di-GMP (cdiG) is an important bacterial signaling molecule because it regulates motility and affects surface colonization and biofilm formation. It has long been established that cdiG is made by GGDEF enzymes, which are named after five conserved amino acids in the catalytic domain. However, a major group of bacteria, proteobacteria, have a high abundance of these enzymes, which raises the possibility that some of these enzymes have alternate functions. This study details the discovery of GGDEF enzymes that can also make cyclic AMP-GMP (cAG), a recently identified signaling molecule that regulates surface sensing and attachment in pathogenic and environmental bacteria. This provides the first evidence to our knowledge that GGDEF enzymes can make alternative cyclic dinucleotides to cdiG and that cAG is more widespread in proteobacteria. 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.

Novel mechanisms power bacterial gliding motility

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.

The polarity of myxobacterial gliding is regulated by direct interactions between the gliding motors and the Ras homolog MglA

Significance The bacterium Myxococcus xanthus moves on surfaces by directed gliding motility. MglA, a Ras family GTPase, regulates cell polarity in M. xanthus; However, little is known about how MglA establishes cell polarity during gliding, because gliding motors move simultaneously in opposite directions. We found that MglA interacts with the gliding motors directly and is localized in a decreasing intracellular gradient. Furthermore, the motors tend to reverse their moving direction at locations where the activity of MglA is high. Our data suggest that biased reversals along the MglA gradient make the motors moving toward the lagging cell poles less likely to reverse, generating stronger forward propulsion. Thus, bacterial cells, like eukaryotic cells, can use Ras homolog localization to establish cellular polarity. 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.

Exopolysaccharides promote Myxococcus xanthus social motility by inhibiting cellular reversals

The biofilm‐forming bacterium Myxococcus xanthus moves on surfaces as structured swarms utilizing type IV pili‐dependent social (S) motility. In contrast to isolated cells that reverse their moving direction frequently, individual cells within swarms rarely reverse. The regulatory mechanisms that inhibit cellular reversal and promote the formation of swarms are not well understood. Here we show that exopolysaccharides (EPS), the major extracellular components of M. xanthus swarms, inhibit cellular reversal in a concentration‐dependent manner. Thus, individual wild‐type cells reverse less frequently in swarms due to high local EPS concentrations. In contrast, cells defective in EPS production hyper‐reverse their moving direction and show severe defects in S‐motility. Surprisingly, S‐motility and wild‐type reversal frequency are restored in double mutants that are defective in both EPS production and the Frz chemosensory system, indicating that EPS regulates cellular reversal in parallel to the Frz pathway. Here we clarify that besides functioning as the structural scaffold in biofilms, EPS is a self‐produced signal that coordinates the group motion of the social bacterium M. xanthus.