The differential expression of PilY1 proteins by the HsfBA phosphorelay allows twitching motility in the absence of exopolysaccharides.

Type Four Pili (T4P) are extracellular appendages mediating several bacterial functions such as motility, biofilm formation and infection. The ability to adhere to substrates is essential for all these functions. In Myxococcus xanthus, during twitching motility, the binding of polar T4P to exopolysaccharides (EPS), induces pilus retraction and the forward cell movement. EPS are produced, secreted and weakly associated to the M. xanthus cell surface or deposited on the substrate. In this study, a genetic screen allowed us to identify two factors involved in EPS-independent T4P-dependent twitching motility: the PilY1.1 protein and the HsfBA phosphorelay. Transcriptomic analyses show that HsfBA differentially regulates the expression of PilY1 proteins and that the down-regulation of pilY1.1 together with the accumulation of its homologue pilY1.3, allows twitching motility in the absence of EPS. The genetic and bioinformatic dissection of the PilY1.1 domains shows that PilY1.1 might be a bi-functional protein with a role in priming T4P extension mediated by its conserved N-terminal domain and roles in EPS-dependent motility mediated by an N-terminal DUF4114 domain activated upon binding to Ca2+. We speculate that the differential transcriptional regulation of PilY1 homologs by HsfBA in response to unknown signals, might allow accessorizing T4P tips with different modules allowing twitching motility in the presence of alternative substrates and environmental conditions.

Defining Two Chemosensory Arrays in Shewanella oneidensis.

Shewanella oneidensis has 2 functional chemosensory systems named Che1 and Che3, and 27 chemoreceptors. Che3 is dedicated to chemotaxis while Che1 could be involved in RpoS post-translational regulation. In this study, we have shown that two chemoreceptors Aer2so and McpAso, genetically related to the Che1 system, form distinct core-signaling units and signal to Che1 and Che3, respectively. Moreover, we observed that Aer2so is a cytoplasmic dynamic chemoreceptor that, when in complex with CheA1 and CheW1, localizes at the two poles and the centre of the cells. Altogether, the results obtained indicate that Che1 and Che3 systems are interconnected by these two chemoreceptors allowing a global response for bacterial survival.

The polar Ras-like GTPase MglA activates type IV pilus via SgmX to enable twitching motility in Myxococcus xanthus.

Type IV pili (Tfp) are highly conserved macromolecular structures that fulfill diverse cellular functions, such as adhesion to host cells, the import of extracellular DNA, kin recognition, and cell motility (twitching). Outstandingly, twitching motility enables a poorly understood process by which highly coordinated groups of hundreds of cells move in cooperative manner, providing a basis for multicellular behaviors, such as biofilm formation. In the social bacteria Myxococcus xanthus, we know that twitching motility is under the dependence of the small GTPase MglA, but the underlying molecular mechanisms remain elusive. Here we show that MglA complexed to GTP recruits a newly characterized Tfp regulator, termed SgmX, to activate Tfp machines at the bacterial cell pole. This mechanism also ensures spatial regulation of Tfp, explaining how MglA switching provokes directional reversals. This discovery paves the way to elucidate how polar Tfp machines are regulated to coordinate multicellular movements, a conserved feature in twitching bacteria.

Modulation of bacterial multicellularity via spatio-specific polysaccharide secretion.

The development of multicellularity is a key evolutionary transition allowing for differentiation of physiological functions across a cell population that confers survival benefits; among unicellular bacteria, this can lead to complex developmental behaviors and the formation of higher-order community structures. Herein, we demonstrate that in the social δ-proteobacterium Myxococcus xanthus, the secretion of a novel biosurfactant polysaccharide (BPS) is spatially modulated within communities, mediating swarm migration as well as the formation of multicellular swarm biofilms and fruiting bodies. BPS is a type IV pilus (T4P)-inhibited acidic polymer built of randomly acetylated ß-linked tetrasaccharide repeats. Both BPS and exopolysaccharide (EPS) are produced by dedicated Wzx/Wzy-dependent polysaccharide-assembly pathways distinct from that responsible for spore-coat assembly. While EPS is preferentially produced at the lower-density swarm periphery, BPS production is favored in the higher-density swarm interior; this is consistent with the former being known to stimulate T4P retraction needed for community expansion and a function for the latter in promoting initial cell dispersal. Together, these data reveal the central role of secreted polysaccharides in the intricate behaviors coordinating bacterial multicellularity.

How an unusual chemosensory system forms arrays on the bacterial nucleoid.

Chemosensory systems are signaling pathways elegantly organized in hexagonal arrays that confer unique functional features to these systems such as signal amplification. Chemosensory arrays adopt different subcellular localizations from one bacterial species to another, yet keeping their supramolecular organization unmodified. In the gliding bacterium Myxococcus xanthus, a cytoplasmic chemosensory system, Frz, forms multiple clusters on the nucleoid through the direct binding of the FrzCD receptor to DNA. A small CheW-like protein, FrzB, might be responsible for the formation of multiple (instead of just one) Frz arrays. In this review, we summarize what is known on Frz array formation on the bacterial chromosome and discuss hypotheses on how FrzB might contribute to the nucleation of multiple clusters. Finally, we will propose some possible biological explanations for this type of localization pattern.

A divergent CheW confers plasticity to nucleoid-associated chemosensory arrays.

Chemosensory systems are highly organized signaling pathways that allow bacteria to adapt to environmental changes. The Frz chemosensory system from M. xanthus possesses two CheW-like proteins, FrzA (the core CheW) and FrzB. We found that FrzB does not interact with FrzE (the cognate CheA) as it lacks the amino acid region responsible for this interaction. FrzB, instead, acts upstream of FrzCD in the regulation of M. xanthus chemotaxis behaviors and activates the Frz pathway by allowing the formation and distribution of multiple chemosensory clusters on the nucleoid. These results, together, show that the lack of the CheA-interacting region in FrzB confers new functions to this small protein.

Cellular targeting and segregation of bacterial chemosensory systems.

The bacterial cytoplasm is not a homogeneous solution of macromolecules, but rather a highly organized and compartmentalized space where the clustering and segregation of macromolecular complexes in certain cell regions confers functional efficiency. Bacterial chemoreceptors represent a versatile model system to study the subcellular localization of macromolecules, as they are present in almost all motile bacterial and archaeal species, where they tend to form highly ordered arrays that occupy distinct positions in cells. The positioning of chemoreceptor clusters, as well as their segregation mechanism on cell division, varies from species to species and probably depends on cells size, environment and speed of movement. In this review, we summarize the current understanding of the architecture and the segregation mechanisms of chemoreceptors in a limited number of bacterial model systems and suggest that the pattern of chemoreceptor distribution is coupled to behavioral life-style of that species.

The nucleoid as a scaffold for the assembly of bacterial signaling complexes.

The FrzCD chemoreceptor from the gliding bacterium Myxococcus xanthus forms cytoplasmic clusters that occupy a large central region of the cell body also occupied by the nucleoid. In this work, we show that FrzCD directly binds to the nucleoid with its N-terminal positively charged tail and recruits active signaling complexes at this location. The FrzCD binding to the nucleoid occur in a DNA-sequence independent manner and leads to the formation of multiple distributed clusters that explore constrained areas. This organization might be required for cooperative interactions between clustered receptors as observed in membrane-bound chemosensory arrays.

Evolution and Design Governing Signal Precision and Amplification in a Bacterial Chemosensory Pathway.

Understanding the principles underlying the plasticity of signal transduction networks is fundamental to decipher the functioning of living cells. In Myxococcus xanthus, a particular chemosensory system (Frz) coordinates the activity of two separate motility systems (the A- and S-motility systems), promoting multicellular development. This unusual structure asks how signal is transduced in a branched signal transduction pathway. Using combined evolution-guided and single cell approaches, we successfully uncoupled the regulations and showed that the A-motility regulation system branched-off an existing signaling system that initially only controlled S-motility. Pathway branching emerged in part following a gene duplication event and changes in the circuit structure increasing the signaling efficiency. In the evolved pathway, the Frz histidine kinase generates a steep biphasic response to increasing external stimulations, which is essential for signal partitioning to the motility systems. We further show that this behavior results from the action of two accessory response regulator proteins that act independently to filter and amplify signals from the upstream kinase. Thus, signal amplification loops may underlie the emergence of new connectivity in signal transduction pathways.

Functional organization of a multimodular bacterial chemosensory apparatus.

Chemosensory systems (CSS) are complex regulatory pathways capable of perceiving external signals and translating them into different cellular behaviors such as motility and development. In the δ-proteobacterium Myxococcus xanthus, chemosensing allows groups of cells to orient themselves and aggregate into specialized multicellular biofilms termed fruiting bodies. M. xanthus contains eight predicted CSS and 21 chemoreceptors. In this work, we systematically deleted genes encoding components of each CSS and chemoreceptors and determined their effects on M. xanthus social behaviors. Then, to understand how the 21 chemoreceptors are distributed among the eight CSS, we examined their phylogenetic distribution, genomic organization and subcellular localization. We found that, in vivo, receptors belonging to the same phylogenetic group colocalize and interact with CSS components of the respective phylogenetic group. Finally, we identified a large chemosensory module formed by three interconnected CSS and multiple chemoreceptors and showed that complex behaviors such as cell group motility and biofilm formation require regulatory apparatus composed of multiple interconnected Che-like systems.

A versatile class of cell surface directional motors gives rise to gliding motility and sporulation in Myxococcus xanthus.

Eukaryotic cells utilize an arsenal of processive transport systems to deliver macromolecules to specific subcellular sites. In prokaryotes, such transport mechanisms have only been shown to mediate gliding motility, a form of microbial surface translocation. Here, we show that the motility function of the Myxococcus xanthus Agl-Glt machinery results from the recent specialization of a versatile class of bacterial transporters. Specifically, we demonstrate that the Agl motility motor is modular and dissociates from the rest of the gliding machinery (the Glt complex) to bind the newly expressed Nfs complex, a close Glt paralogue, during sporulation. Following this association, the Agl system transports Nfs proteins directionally around the spore surface. Since the main spore coat polymer is secreted at discrete sites around the spore surface, its transport by Agl-Nfs ensures its distribution around the spore. Thus, the Agl-Glt/Nfs machineries may constitute a novel class of directional bacterial surface transporters that can be diversified to specific tasks depending on the cognate cargo and machinery-specific accessories.

Cell biology of bacterial sensory modules.

Despite their small size, bacterial cells possess very efficient sensory apparatus that allow them to perceive and respond to the external environment with cell movement. In enteric bacteria, these apparatus are complex lattices of different chemoreceptors working in concert and forming clusters positioned at the cell poles. Since the study of chemotaxis has been expanded to other bacterial species, examples of chemosensory systems regulating functions different than taxis have been described and chemoreceptors localizing in ways divergent from the enteric paradigm have been visualized. The scope of this review is to revise and summarize the architecture of different bacterial chemoreceptors. Then, hypotheses will be proposed on how chemoreceptor distribution in cells is coupled to specific functions and life styles in well-characterized bacterial model systems, such as Escherichia coli, Rhodobacter sphaeroides, Caulobacter crescentus and Myxococcus xanthus.

Gliding motility revisited: how do the myxobacteria move without flagella?

In bacteria, motility is important for a wide variety of biological functions such as virulence, fruiting body formation, and biofilm formation. While most bacteria move by using specialized appendages, usually external or periplasmic flagella, some bacteria use other mechanisms for their movements that are less well characterized. These mechanisms do not always exhibit obvious motility structures. Myxococcus xanthus is a motile bacterium that does not produce flagella but glides slowly over solid surfaces. How M. xanthus moves has remained a puzzle that has challenged microbiologists for over 50 years. Fortunately, recent advances in the analysis of motility mutants, bioinformatics, and protein localization have revealed likely mechanisms for the two M. xanthus motility systems. These results are summarized in this review.

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 approximately 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.

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 DeltamglA 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.

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.

Localization of a bacterial cytoplasmic receptor is dynamic and changes with cell-cell contacts.

Directional motility in the gliding bacterium Myxococcus xanthus requires controlled cell reversals mediated by the Frz chemosensory system. FrzCD, a cytoplasmic chemoreceptor, does not form membrane-bound polar clusters typical for most bacteria, but rather cytoplasmic clusters that appear helically arranged and span the cell length. The distribution of FrzCD in living cells was found to be dynamic: FrzCD was localized in clusters that continuously changed their size, number, and position. The number of FrzCD clusters was correlated with cellular reversal frequency: fewer clusters were observed in hypo-reversing mutants and additional clusters were observed in hyper-reversing mutants. When moving cells made side-to-side contacts, FrzCD clusters in adjacent cells showed transient alignments. These events were frequently followed by one of the interacting cells reversing. These observations suggest that FrzCD detects signals from a cell contact-sensitive signaling system and then re-localizes as it directs reversals to distributed motility engines.

Chemotaxis mediated by NarX-FrzCD chimeras and nonadapting repellent responses in Myxococcus xanthus.

Myxococcus xanthus requires gliding motility for swarming and fruiting body formation. It uses the Frz chemosensory pathway to regulate cell reversals. FrzCD is a cytoplasmic chemoreceptor required for sensing effectors for this pathway. NarX is a transmembrane sensor for nitrate from Escherichia coli. In this study, two NarX-FrzCD chimeras were constructed to investigate M. xanthus chemotaxis: NazD(F) contains the N-terminal sensory module of NarX fused to the C-terminal signalling domain of FrzCD; NazD(R) is similar except that it contains a G51R mutation in the NarX domain known to reverse the signalling output of a NarX-Tar chimera to nitrate. We report that while nitrate had no effect on the wild type, it decreased the reversal frequency of M. xanthus expressing NazD(F) and increased that of M. xanthus expressing NazD(R). These results show that directional motility in M. xanthus can be regulated independently of cellular metabolism and physiology. Surprisingly, the NazD(R) strain failed to adapt to nitrate in temporal assays as did the wild type to known repellents. The lack of temporal adaptation to negative stimuli appears to be a general feature in M. xanthus chemotaxis. Thus, the appearance of biased movements by M. xanthus in repellent gradients is likely due to the inhibition of net translocation by repellents.