ABSTRACT Positioning of the division site is precisely regulated. In Myxococcus xanthus, the PomX/Y/Z assembly associates with and translocates across the nucleoid in a PomZ ATPase-dependent manner to position the cytokinetic FtsZ ring at midcell. Unlike other systems incorporating a ParA/MinD ATPase, the PomX/Y/Z system incorporates two ATPase-activating proteins (AAPs), PomX and PomY, that stimulate PomZ activity. While PomX AAP activity, similarly to that of other characterized AAPs of ParA/MinD ATPases, resides in a short positively charged N-terminal peptide (PomXNPEP), the mechanism of PomY-mediated PomZ activation remained unknown. We map PomY AAP activity to its C-terminal residues 496–682 (PomY496-682) and show that this activity is important for division site positioning and the initiation of division. Structural modeling suggests a bipartite PomY496-682 architecture, comprising a globular domain and an intrinsically disordered region containing the short α5 helix. However, this modeling suggests an interaction only between α5 and the PomZ dimer. The suggested α5 binding site partially overlaps with that of the prototype AAP ParB on the ParA dimer, but α5 lacks a positively charged residue critical for ParB AAP activity. Structural modeling suggests that PomXNPEP binds the PomZ dimer at a site distinct from that of α5 and that overlaps with that of the prototype AAP MinE on the MinD dimer. These findings reveal a non-canonical AAP mechanism of PomY, while PomXNPEP likely functions analogously to MinE, indicating that PomX and PomY act in concert through distinct but complementary mechanisms to stimulate PomZ ATPase activity, thereby efficiently driving the positioning of the cytokinetic FtsZ ring.IMPORTANCEIn bacteria, the regulatory systems that position the division site often incorporate a ParA/MinD ATPase. These ATPases also function in the positioning of numerous other macromolecular complexes in bacteria. Characteristically, these ATPases depend on stimulation of their ATPase activity by a single cognate ATPase-activating protein (AAP). However, the Myxococcus xanthus cell division regulatory PomX/Y/Z system stands out by incorporating two AAPs, PomX and PomY, that activate the PomZ ATPase. Similar to other AAPs, PomX uses a short, positively charged N-terminal peptide (PomXNPEP) to stimulate PomZ activity. We map PomY AAP activity to a C-terminal region spanning residues 496–682 (PomY496-682). Structural modeling using AlphaFold 3 suggests that the AAP activity of PomY496-682 and PomXNPEP may involve two distinct, non-overlapping binding sites at the dimer interface of ATP-bound PomZ. Altogether, these results provide insights into the molecular mechanism of activation by two AAPs of a ParA/MinD ATPase.

Exported polysaccharides play crucial functions in bacteria. Polysaccharide biosynthesis in the ubiquitous Wzx/Wzy- and ABC-transporter-dependent pathways starts with the transfer of a sugar-1-phosphate from a nucleotide-sugar donor to undecaprenyl phosphate, a reaction catalyzed by a phosphoglycosyl transferase (PGT). Both reaction substrates are limited and shared among multiple glycoconjugate pathways, raising the question of how bacteria regulate these pathways. In Myxococcus xanthus, EpsZ, which belongs to the family of large monotopic PGTs (monoPGTs), starts the Wzx/Wzy-dependent exopolysaccharide (EPS) biosynthesis. The Dif chemosensory system regulates EPS biosynthesis by an unknown mechanism via the phosphorylated single-domain response regulator EpsW (EpsW~P). Here, we show that EpsW~P stimulates EPS biosynthesis at the post-translational level. Moreover, MiniTurbo-based proximity labeling experiments suggest that EpsW~P interacts directly with EpsZ. Additionally, heterologous expression of these two proteins in Salmonella enterica demonstrates that EpsW stimulates EpsZ enzymatic activity. S. enterica WbaP, the prototype large monoPGT, forms a functional homodimer, with dimerization involving a distinct cytoplasmic β-hairpin. However, AlphaFold-based structural modeling shows that EpsZ lacks this β-hairpin, suggesting an alternative mechanism for dimerization. Structural modeling of an EpsZ-EpsW heterocomplex suggests that EpsW~P, by direct interaction, promotes the formation of the stable EpsZ dimer. These findings suggest a new model for the regulation of polysaccharide biosynthesis in which EpsW~P allosterically facilitates the formation of the active, dimeric conformation of EpsZ, thereby activating EPS biosynthesis at its initial step. Genomics and structural modeling suggest that the regulation of large monoPGTs by a single-domain response regulator is widespread in myxobacteria and potentially beyond.IMPORTANCEBacteria produce various polysaccharides with important biological functions and biotechnological applications. Polysaccharide synthesis is energy-costly and requires substrates that are in limited supply, raising the question of how bacteria regulate these pathways. Here, we explored the regulation of exopolysaccharide biosynthesis in Myxococcus xanthus. We demonstrate that the phosphorylated single-domain response regulator EpsW activates exopolysaccharide biosynthesis at the post-translational level by stimulating the activity of the phosphoglycosyl transferase EpsZ. By interacting with EpsZ, phosphorylated EpsW facilitates the formation of the active, dimeric conformation of EpsZ, thereby activating exopolysaccharide biosynthesis at its initial step. We propose that this previously unrecognized regulatory mechanism is broadly conserved, not only in myxobacteria but also beyond.

Repair of DNA damage is essential for genome integrity. DNA damage elicits a DNA damage response (DDR) that includes error-free and error-prone, i.e., mutagenic, repair. The SOS response is a widely conserved system in bacteria that regulates the DDR and depends on the recombinase RecA and the transcriptional repressor LexA. However, RecA/LexA-independent DDRs have been identified in several bacterial species. Here, using a whole-cell, label-free quantitative proteomics approach, we map the proteomic response in Myxococcus xanthus to mitomycin C treatment and the lack of LexA. In doing so, we demonstrate a LexA-independent proteomic DDR in M. xanthus. Using a candidate approach, we identify DNA damage-induced protein A (DdiA), a transcriptional regulator of the Xenobiotic Response Element (XRE) family, and demonstrate that it is involved in regulating the abundance of a subset of the LexA-independent DDR proteins. ddiA is expressed heterogeneously in a subpopulation of cells in the absence of exogenous genotoxic stress and reversibly induced population wide in response to such stress. DdiA, indirectly or directly, activates the expression of dnaE2, which encodes the DnaE2 error-prone DNA polymerase, and inhibits the expression of recX, which encodes RecX, a negative regulator of RecA. Accordingly, the ΔddiA mutant not only has a lower mutation frequency than the wild type but also a fitness defect, suggesting that DdiA mediates a trade-off between fitness and mutagenesis. We speculate that the DdiA-dependent response is tailored to counter replication stress, thereby preventing the induction of the complete RecA/LexA-dependent DDR in the absence of exogenous genotoxic stress.IMPORTANCEDNA damage repair is essential for genome integrity and depends on the DNA damage response (DDR). While the RecA/LexA-dependent SOS response is widely conserved in bacteria, there are also RecA/LexA-independent DDRs. Here, we identify the DNA damage-induced transcriptional regulator DdiA in Myxococcus xanthus and demonstrate that it regulates part of a LexA-independent DDR. DdiA activates the expression of dnaE2, which encodes the DnaE2 error-prone DNA polymerase, and inhibits the expression of recX, which encodes RecX, a negative regulator of RecA. Because the ΔddiA mutant has a lower mutation frequency than the wild type but also a fitness defect, we suggest that DdiA mediates a trade-off between fitness and mutagenesis, and the DdiA-dependent DDR is specifically tailored to counter replication stress.

From the humblest of beginnings (i.e. a pile of dry cow dung) over 80 years ago, the Gram-negative bacterium Myxococcus xanthus has emerged as a premier model system for studying diverse fields of bacteriology, including multicellular development, sporulation, motility, cell-envelope biogenesis, spatiotemporal regulation, signaling, photoreception, kin recognition, social evolution, and predation. As the flagship representative of myxobacteria found in varied terrestrial and aquatic environments, M. xanthus research has evolved into a collaborative global effort, as reflected by the contributions to this article. In celebration of the upcoming 50th anniversary of the International Conference on the Biology of Myxobacteria, this review highlights the historical and ongoing contributions of M. xanthus as a multifaceted model bacterium.

ABSTRACT Bacteria synthesize chemically diverse capsular and secreted polysaccharides that function in many physiological processes and are widely used in industrial applications. In the ubiquitous Wzx/Wzy-dependent biosynthetic pathways for these polysaccharides, the polysaccharide co-polymerase (PCP) facilitates the polymerization of repeat units in the periplasm, and in Gram-negative bacteria, also polysaccharide translocation across the outer membrane. These PCPs belong to the PCP-2 family, are integral inner membrane proteins with extended periplasmic domains, and functionally depend on alternating between different oligomeric states. The oligomeric state is determined by a cognate cytoplasmic bacterial tyrosine kinase (BYK), which is either part of the PCP or a stand-alone protein. Interestingly, BYK-like proteins, which lack key catalytic residues and/or the phosphorylated Tyr residues, have been described. In Myxococcus xanthu s, the exopolysaccharide (EPS) is synthesized and exported via the Wzx/Wzy-dependent EPS pathway in which EpsV serves as the PCP. Here, we confirm that EpsV lacks the BYK domain. Using phylogenomics, experiments, and computational structural biology, we identify EcpK as important for EPS biosynthesis and show that it structurally resembles canonical BYKs but lacks residues important for catalysis and Tyr phosphorylation. Using proteomic analyses, two-hybrid assays, and structural modeling, we demonstrate that EcpK directly interacts with EpsV. Based on these findings, we suggest that EcpK is a BY pseudokinase and functions as a scaffold, which by direct protein-protein interactions, rather than by Tyr phosphorylation, facilitates EpsV function. EcpK and EpsV homologs are present in other bacteria, suggesting broad conservation of this mechanism and establishing a phosphorylation-independent PCP-2 subfamily. IMPORTANCE Bacteria produce a variety of polysaccharides with important biological functions. In Wzx/Wzy-dependent pathways for the biosynthesis of secreted and capsular polysaccharides in Gram-negative bacteria, the polysaccharide co-polymerase (PCP) is a key protein that facilitates repeat unit polymerization and polysaccharide translocation across the outer membrane. PCP function depends on assembly/disassembly cycles that are determined by the phosphorylation/dephosphorylation cycles of an associated bacterial tyrosine kinase (BYK). Here, we identify the BY pseudokinase EcpK as essential for exopolysaccharide biosynthesis in Myxococcus xanthus . Based on experiments and computational structural biology, we suggest that EcpK is a scaffold protein, guiding the assembly/disassembly cycles of the partner PCP via binding/unbinding cycles independently of Tyr phosphorylation/dephosphorylation cycles. We suggest that this novel mechanism is broadly conserved.

Repair of DNA damage is essential for genome integrity. DNA damage elicits a DNA damage response (DDR) that includes error-free and error-prone, i.e. mutagenic, repair. The SOS response is a widely conserved system in bacteria that regulates the DDR and depends on the recombinase RecA and the transcriptional repressor LexA. However, RecA/LexA-independent DDRs have been identified in several bacterial species. Here, using a whole-cell, label-free quantitative proteomics approach, we map the proteomic response in Myxococcus xanthus to mitomycin C treatment and the lack of LexA. In doing so, we confirm a LexA-independent DDR in M. xanthus. Using a candidate approach, we identify DdiA, a transcriptional regulator of the XRE family, and demonstrate that it regulates a subset of the LexA-independent DDR genes. ddiA is expressed heterogeneously in a subpopulation of cells in the absence of exogenous genotoxic stress and reversibly induced population-wide in response to such stress. DdiA, indirectly or directly, activates the expression of dnaE2, which encodes the DnaE2 error-prone DNA polymerase, and inhibits the expression of recX, which encodes RecX, a negative regulator of RecA. Accordingly, the ΔddiA mutant has a lower mutation frequency than the wild-type but also a fitness defect, suggesting that DdiA mediates a trade-off between fitness and mutagenesis. We speculate that the DdiA-dependent response is tailored to counter replication stress, thereby preventing the induction of the complete RecA/LexA-dependent DDR in the absence of exogenous genotoxic stress. ### Competing Interest Statement The authors have declared no competing interest.

In most bacteria, cell division depends on the tubulin-homolog FtsZ that polymerizes in a GTP-dependent manner to form the cytokinetic Z-ring at the future division site. Subsequently, the Z-ring recruits, directly or indirectly, all other proteins of the divisome complex that executes cytokinesis. A critical step in this process is the precise positioning of the Z-ring at the future division site. While the divisome proteins are generally conserved, the regulatory systems that position the Z-ring are more diverse. However, these systems have in common that they modulate FtsZ polymerization. In Myxococcus, PomX, PomY, and PomZ form precisely one MDa-sized, nonstoichiometric, nucleoid-associated assembly that spatiotemporally guides Z-ring formation. Here, using cryo-correlative light and electron microscopy together with in situ cryoelectron tomography, we determine the PomXYZ assembly’s architecture at close-to-live conditions. PomX forms a porous meshwork of randomly intertwined filaments. Templated by this meshwork, the phase-separating PomY protein forms a biomolecular condensate that compacts and bends the PomX filaments, resulting in the formation of a selective PomXYZ co-condensate that is associated to the nucleoid by PomZ. These studies reveal a hitherto undescribed supramolecular structure and provide a framework for understanding how a nonstoichiometric co-condensate forms, maintains number control, and nucleates GTP-dependent FtsZ polymerization to precisely regulate cell division.

Type IVa pili (T4aP) are widespread and enable bacteria to translocate across surfaces. T4aP engage in cycles of extension, surface adhesion, and retraction, thereby pulling cells forward. Accordingly, the number and localization of T4aP are critical to efficient translocation. Here, we address how T4aP formation is regulated in Myxococcus xanthus, which translocates with a well-defined leading and lagging cell pole using T4aP at the leading pole. This localization is orchestrated by the small GTPase MglA and its downstream effector SgmX that both localize at the leading pole and recruit the PilB extension ATPase to the T4aP machinery at this pole. Here, we identify the previously uncharacterized protein SopA and show that it interacts directly with SgmX, localizes at the leading pole, stimulates polar localization of PilB, and is important for T4aP formation. We corroborate that MglA also recruits FrzS to the leading pole, and FrzS stimulates SgmX recruitment. In addition, FrzS and SgmX separately recruit SopA. Precise quantification of T4aP-formation and T4aP-dependent motility in various mutants supports a model whereby the main pathway for stimulating T4aP formation is the MglA/SgmX pathway. FrzS stimulates this pathway by recruiting SgmX and SopA. SopA stimulates the MglA/SgmX pathway by stimulating the function of SgmX, likely by promoting the SgmX-dependent recruitment of PilB to the T4aP machinery. The architecture of the MglA/SgmX/FrzS/SopA protein interaction network for orchestrating T4aP formation allows for combinatorial regulation of T4aP levels at the leading cell pole resulting in discrete levels of T4aP-dependent motility.

Phenotypic heterogeneity in bacteria can result from stochastic processes or deterministic programs. The deterministic programs often involve the versatile second messenger c-di-GMP, and give rise to daughter cells with different c-di-GMP levels by deploying c-di-GMP metabolizing enzymes asymmetrically during cell division. By contrast, less is known about how phenotypic heterogeneity is kept to a minimum. Here, we identify a deterministic c-di-GMP-dependent program that is hardwired into the cell cycle of Myxococcus xanthus to minimize phenotypic heterogeneity and guarantee the formation of phenotypically similar daughter cells during division. Cells lacking the diguanylate cyclase DmxA have an aberrant motility behaviour. DmxA is recruited to the cell division site and its activity is switched on during cytokinesis, resulting in a transient increase in the c-di-GMP concentration. During cytokinesis, this c-di-GMP burst ensures the symmetric incorporation and allocation of structural motility proteins and motility regulators at the new cell poles of the two daughters, thereby generating phenotypically similar daughters with correct motility behaviours. Thus, our findings suggest a general c-di-GMP-dependent mechanism for minimizing phenotypic heterogeneity, and demonstrate that bacteria can ensure the formation of dissimilar or similar daughter cells by deploying c-di-GMP metabolizing enzymes to distinct subcellular locations.

Type IVa pili (T4aP) are ubiquitous cell surface filaments important for surface motility, adhesion to surfaces, DNA uptake, biofilm formation, and virulence. T4aP are built from thousands of copies of the major pilin subunit and tipped by a complex composed of minor pilins and in some systems also the PilY1 adhesin. While major pilins of structurally characterized T4aP have lengths of <165 residues, the major pilin PilA of Myxococcus xanthus is unusually large with 208 residues. All major pilins have a conserved N-terminal domain and a variable C-terminal domain, and the additional residues of PilA are due to a larger C-terminal domain. We solved the structure of the M. xanthus T4aP (T4aP Mx ) at a resolution of 3.0 Å using cryo-EM. The T4aP Mx follows the structural blueprint of other T4aP with the pilus core comprised of the interacting N-terminal α1-helices, while the globular domains decorate the T4aP surface. The atomic model of PilA built into this map shows that the large C-terminal domain has more extensive intersubunit contacts than major pilins in other T4aP. As expected from these greater contacts, the bending and axial stiffness of the T4aP Mx is significantly higher than that of other T4aP and supports T4aP-dependent motility on surfaces of different stiffnesses. Notably, T4aP Mx variants with interrupted intersubunit interfaces had decreased bending stiffness, pilus length, and strongly reduced motility. These observations support an evolutionary scenario whereby the large major pilin enables the formation of a rigid T4aP that expands the environmental conditions in which the T4aP system functions.

During cell migration, front-rear polarity is spatiotemporally regulated; however, the underlying design of regulatory interactions varies. In rod-shaped Myxococcus xanthus cells, a spatial toggle switch dynamically regulates front-rear polarity. The polarity module establishes front-rear polarity by guaranteeing front pole-localization of the small GTPase MglA. Conversely, the Frz chemosensory system, by acting on the polarity module, causes polarity inversions. MglA localization depends on the RomR/RomX GEF and MglB/RomY GAP complexes that localize asymmetrically to the poles by unknown mechanisms. Here, we show that RomR and the MglB and MglC roadblock domain proteins generate a positive feedback by forming a RomR/MglC/MglB complex, thereby establishing the rear pole with high GAP activity that is non-permissive to MglA. MglA at the front engages in negative feedback that breaks the RomR/MglC/MglB positive feedback allosterically, thus ensuring low GAP activity at this pole. These findings unravel the design principles of a system for switchable front-rear polarity.

1 HudsonAlpha Institute for Biotechnology, Huntsville, Alabama, United States of America, 2 Department of Genetics, Stanford University School of Medicine, Stanford, California, United States of America, 3 School of Biomolecular and Biomedical Science, Conway Institute, University College Dublin, Dublin, Ireland, 4 Department of Biology and the Integrative Program for Biological and Genome Sciences, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, United States of America, 5 Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, Michigan, United States of America, 6 Department of Ecophysiology, Max Planck Institute for Terrestrial Microbiology, Marburg, Germany, 7 Environmental Genomics, Christian-Albrechts University of Kiel, Kiel, Germany, 8 Max Planck Institute for Evolutionary Biology, Plön, Germany

Cell division is spatiotemporally precisely regulated, but the underlying mechanisms are incompletely understood. In the social bacterium Myxococcus xanthus, the PomX/PomY/PomZ proteins form a single megadalton-sized complex that directly positions and stimulates cytokinetic ring formation by the tubulin homolog FtsZ. Here, we study the structure and mechanism of this complex in vitro and in vivo. We demonstrate that PomY forms liquid-like biomolecular condensates by phase separation, while PomX self-assembles into filaments generating a single large cellular structure. The PomX structure enriches PomY, thereby guaranteeing the formation of precisely one PomY condensate per cell through surface-assisted condensation. In vitro, PomY condensates selectively enrich FtsZ and nucleate GTP-dependent FtsZ polymerization and bundle FtsZ filaments, suggesting a cell division site positioning mechanism in which the single PomY condensate enriches FtsZ to guide FtsZ-ring formation and division. This mechanism shares features with microtubule nucleation by biomolecular condensates in eukaryotes, supporting this mechanism's ancient origin.

Type IV pili (T4P) are ubiquitous bacterial cell surface filaments important for surface motility, adhesion to biotic and abiotic surfaces, DNA uptake, biofilm formation, and virulence. T4P are built from thousands of copies of the major pilin subunit and tipped by a complex composed of minor pilins and in some systems also the PilY1 adhesin. While the major pilins of structurally characterized T4P have lengths of up to 161 residues, the major pilin PilA of Myxococcus xanthus is unusually large with 208 residues. All major pilins have a highly conserved N-terminal domain and a highly variable C-terminal domain, and the additional residues in the M. xanthus PilA are due to a larger C-terminal domain. We solved the structure of the M. xanthus T4P (T4P Mx ) at a resolution of 3.0 Å using cryo-electron microscopy (cryo-EM). The T4P Mx follows the structural blueprint observed in other T4P with the pilus core comprised of the extensively interacting N-terminal α1-helices while the globular domains decorate the T4P surface. The atomic model of PilA built into this map shows that the large C-terminal domain has much more extensive intersubunit contacts than major pilins in other T4P. As expected from these greater contacts, the bending and axial stiffness of the T4P Mx is significantly higher than that of other T4P and supports T4P-dependent motility on surfaces of different stiffnesses. Notably, T4P Mx variants with interrupted intersubunit interfaces had decreased bending stiffness and strongly reduced motility on all surfaces. These observations support an evolutionary scenario whereby the large major pilin enables the formation of a rigid T4P that expands the environmental conditions in which the T4P system functions.

ABSTRACT Type IVa pili (T4aP) are important for bacterial motility, adhesion, biofilm formation, and virulence. This versatility is based on their cycles of extension, adhesion, and retraction. The conserved T4aP machine (T4aPM) drives these cycles; however, the piliation pattern varies between species. To understand how these patterns are established, we focused on the T4aPM in Myxococcus xanthus that assembles following an outside-in pathway, starting with the polar incorporation of the PilQ secretin forming a multimeric T4aP conduit in the outer membrane. We demonstrate that PilQ recruitment to the nascent poles initiates during cytokinesis, but most are recruited to the new poles in the daughters after the completion of cytokinesis. This recruitment depends on the peptidoglycan-binding AMIN domains in PilQ. Moreover, the pilotin Tgl stimulates PilQ multimerization in the outer membrane, is transiently recruited to the nascent and new poles in a PilQ-dependent manner, and dissociates after the completion of secretin assembly. Altogether, our data support a model whereby PilQ polar recruitment and multimerization occur in two steps: the PilQ AMIN domains bind septal and polar peptidoglycan, thereby enabling polar Tgl localization, which then stimulates secretin multimerization in the outer membrane. Using computational analyses, we provide evidence for a conserved mechanism of T4aPM pilotins whereby the pilotin transiently interacts with the unfolded β-lip of the secretin monomer, i.e., the region that eventually inserts into the outer membrane. Finally, we suggest that the presence/absence of AMIN domain(s) in T4aPM secretins contributes to the different T4aPM localization patterns across bacteria. IMPORTANCE Type IVa pili (T4aP) are widespread bacterial cell surface structures with important functions in motility, surface adhesion, biofilm formation, and virulence. Different bacteria have adapted different piliation patterns. To address how these patterns are established, we focused on the bipolar localization of the T4aP machine in the model organism Myxococcus xanthus by studying the localization of the PilQ secretin, the first component of this machine that assembles at the poles. Based on experiments using a combination of fluorescence microscopy, biochemistry, and computational structural analysis, we propose that PilQ, and specifically its AMIN domains, binds septal and polar peptidoglycan, thereby enabling polar Tgl localization, which then stimulates PilQ multimerization in the outer membrane. We also propose that the presence and absence of AMIN domains in T4aP secretins contribute to the different piliation patterns across bacteria.

Protein-protein interactions are foundational for many cellular processes. Such interactions are especially challenging to identify if they are transient or depend on environmental conditions. This protocol details steps to identify stable and transient protein interactomes in the bacterium Myxococcus xanthus using biotin ligase miniTurbo-based proximity labeling. We include instructions for opti-mizing the expression of control proteins, in vivo biotin labeling of bacteria grown on a surface or in suspension culture, enrichment of biotinylated proteins, and sample processing for proteomic analysis.For complete details on the use and execution of this protocol, please refer to Branon et al. (2018).1

C-di-GMP is a bacterial second messenger that regulates diverse processes in response to environmental or cellular cues. The nucleoid-associated protein (NAP) CdbA in Myxococcus xanthus binds c-di-GMP and DNA in a mutually exclusive manner in vitro. CdbA is essential for viability, and CdbA depletion causes defects in chromosome organization, leading to a block in cell division and, ultimately, cell death. Most NAPs are not essential; therefore, to explore the paradoxical cdbA essentiality, we isolated suppressor mutations that restored cell viability without CdbA. Most mutations mapped to cdbS, which encodes a stand-alone c-di-GMP binding PilZ domain protein, and caused loss-of-function of cdbS. Cells lacking CdbA and CdbS or only CdbS were fully viable and had no defects in chromosome organization. CdbA depletion caused post-transcriptional upregulation of CdbS accumulation, and this CdbS over-accumulation was sufficient to disrupt chromosome organization and cause cell death. CdbA depletion also caused increased accumulation of CsdK1 and CsdK2, two unusual PilZ-DnaK chaperones. During CdbA depletion, CsdK1 and CsdK2, in turn, enabled the increased accumulation and toxicity of CdbS, likely by stabilizing CdbS. Moreover, we demonstrate that heat stress, possibly involving an increased cellular c-di-GMP concentration, induced the CdbA/CsdK1/CsdK2/CdbS system, causing a CsdK1- and CsdK2-dependent increase in CdbS accumulation. Thereby this system accelerates heat stress-induced chromosome mis-organization and cell death. Collectively, this work describes a unique system that contributes to regulated cell death in M. xanthus and suggests a link between c-di-GMP signaling and regulated cell death in bacteria.

The second messenger c-di-GMP is important during both stages of the nutrient-regulated biphasic life cycle of Myxococcus xanthus with the formation of predatory swarms in the presence of nutrients and spore-filled fruiting bodies in the absence of nutrients. However, different enzymes involved in c-di-GMP synthesis and degradation are important during distinct life cycle stages.

The Ras-like GTPase MglA is a key regulator of front-rear polarity in the rod-shaped Myxococcus xanthus cells. MglA-GTP localizes to the leading cell pole and stimulates assembly of the two machineries for type IV pili-dependent motility and gliding motility. MglA-GTP localization is spatially constrained by its cognate GEF, the RomR/RomX complex, and GAP, the MglB Roadblock-domain protein. Paradoxically, RomR/RomX and MglB localize similarly with low and high concentrations at the leading and lagging poles, respectively. Yet, GEF activity dominates at the leading and GAP activity at the lagging pole by unknown mechanisms. Here, we identify RomY and show that it stimulates MglB GAP activity. The MglB/RomY interaction is low affinity, restricting formation of the bipartite MglB/RomY GAP complex almost exclusively to the lagging pole with the high MglB concentration. Our data support a model wherein RomY, by forming a low-affinity complex with MglB, ensures that the high MglB/RomY GAP activity is confined to the lagging pole where it dominates and outcompetes the GEF activity of the RomR/RomX complex. Thereby, MglA-GTP localization is constrained to the leading pole establishing front-rear polarity.

Type IVa pili (T4aP) are versatile bacterial cell surface structures that undergo extension/adhesion/retraction cycles powered by the cell envelope-spanning T4aP machine. In this machine, a complex composed of four minor pilins and PilY1 primes T4aP extension and is also present at the pilus tip mediating adhesion. Similar to many several other bacteria, Myxococcus xanthus contains multiple minor pilins/PilY1 sets that are incompletely understood. Here, we report that minor pilins and PilY1 (PilY1.1) of cluster_1 form priming and tip complexes contingent on calcium and a noncanonical cytochrome c (TfcP) with an unusual His/Cys heme ligation. We provide evidence that TfcP is unlikely to participate in electron transport and instead stimulates calcium binding by PilY1.1 at low-calcium concentrations, thereby stabilizing PilY1.1 and enabling T4aP function in a broader range of calcium concentrations. These results not only identify a previously undescribed function of cytochromes c but also illustrate how incorporation of an accessory factor expands the environmental range under which the T4aP system functions.