Biology subjects

Sogaard-Andersen, L.

Publications and source records attributed to Sogaard-Andersen, L..

At least 19 recordsLinked to original sources

bioRxiv2025

Molecular insights into the dual activation of PomZ, a ParA/MinD P-loop ATPase, by the two ATPase activating proteins PomX and PomY

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 other characterized AAPs of ParA/MinD ATPases, resides in a short positively charged N-terminal peptide (PomXNPEP), the mechanism of PomY-mediated activation of PomZ 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 cell division. Structural modeling suggests a bipartite PomY496-682 architecture, comprising a globular domain and an intrinsically disordered region containing the short 5 helix. However, modeling supported an interaction only between 5 and the PomZ dimer. The 5 binding site partially overlaps with that of the prototype AAP ParB on the ParA dimer but 5 lacks a positively charged residue critical for ParB AAP activity. Modeling supported that PomXNPEP binds the PomZ dimer at a site distinct from that of 5 and which 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 system 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 supports that the AAP activity of PomY496-682 and PomXNPEP involves two distinct, non-overlapping binding sites at the dimer interface of ATP-bound PomZ. Altogether, these results provide insights into the molecular mechanism of the activation by two AAPs of a ParA/MinD ATPase.

microbiology↗

bioRxiv2025

A single-domain response regulator activates exopolysaccharide synthesis by interacting with the initiating phosphoglycosyl transferase

Exported polysaccharides have 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 {beta}-hairpin. However, AlphaFold-based structural modeling shows that EpsZ lacks this {beta}-hairpin, suggesting an alternative mechanism for dimerization. Our structural modeling of a EpsZ-EpsW heterocomplex suggests that EpsW[~]P, by direct interaction, promotes the formation of the stable EpsZ dimer. These findings uncover 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. SignificanceBacteria 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 directly 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.

microbiology↗

bioRxiv2025

DdiA, an XRE family transcriptional regulator, regulates a LexA-independent DNA damage response in Myxococcus xanthus

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 {Delta}ddiA 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. 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 the RecA/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 {Delta}ddiA 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 that the DdiA-dependent DDR is specifically tailored to counter replication stress.

microbiology↗

bioRxiv2024

Identification of EcpK, a bacterial tyrosine pseudokinase important for exopolysaccharide biosynthesis in Myxococcus xanthus

Bacteria synthesize and export chemically diverse 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 are integral inner membrane proteins with extended periplasmic domains, and functionally depend on alternating between different oligomeric states. The oligomeric state, in turn, 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 xanthus, 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 a broad conservation of this mechanism. ImportanceBacteria 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.

microbiology↗

bioRxiv2024

Combinatorial control of type IVa pili formation by the four polarized regulators MglA, SgmX, FrzS and SopA

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 that FrzS stimulates SgmX recruitment. In addition, FrzS and SgmX separately recruit SopA. Precise quantification of T4aP formation and T4aP-dependent motility in various mutants support 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. 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.

microbiology↗

bioRxiv2024

A deterministic, c-di-GMP-dependent genetic program ensures the generation of phenotypically similar, symmetric daughter cells during cytokinesis

Phenotypic heterogeneity in bacteria results from stochastic processes or deterministic genetic programs. These deterministic programs often incorporate the versatile second messenger c-di-GMP, and by deploying c-di-GMP metabolizing enzyme(s) asymmetrically during cell division give rise to daughter cells with different c-di-GMP levels. By contrast, less is known about how phenotypic heterogeneity is kept to a minimum. Here, we identify a deterministic c-di-GMP-dependent genetic 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 switched on during cytokinesis, resulting in a dramatic but 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 mirror-symmetric, phenotypically similar daughters with correct motility behaviours. These findings suggest a general c-di-GMP-dependent mechanism for minimizing phenotypic heterogeneity, and demonstrate that bacteria by deploying c-di-GMP metabolizing enzymes to distinct subcellular locations ensure the formation of dissimilar or similar daughter cells.

microbiology↗

bioRxiv2023

Large pilin subunits provide distinct structural and mechanical properties for the Myxococcus xanthus type IV pilus

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 (T4PMx) at a resolution of 3.0 [A] using cryo-electron microscopy (cryo-EM). The T4PMx follows the structural blueprint observed in other T4P with the pilus core comprised of the extensively interacting N-terminal 1-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 T4PMx is significantly higher than that of other T4P and supports T4P-dependent motility on surfaces of different stiffnesses. Notably, T4PMx 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.

microbiology↗

bioRxiv2023

The mechanism for polar localization of the type IVa pilus machine

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 is recruited to the new poles in the daughters after 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 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 {beta}-lip, i.e. the region that eventually inserts into the outer membrane, of the secretin monomer. Finally, we suggest that the presence/absence of AMIN domain(s) in T4aPM secretins determines the different T4aPM localization patterns across bacteria. ImportanceType 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 M. 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 determine the different piliation patterns across bacteria.

microbiology↗

bioRxiv2023

During heat stress in Myxococcus xanthus, the CdbS PilZ domain protein, along with two PilZ-DnaK chaperones, perturbs chromosome organization and accelerates cell death

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, stabilized CdbS, thereby enabling its increased accumulation and toxicity. Moreover, we demonstrate that heat stress, possibly involving an increased cellular c-di-GMP concentration, induces the CdbA/CsdK1/CsdK2/CdbA 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. Author summaryThe nucleotide-based second messenger c-di-GMP in bacteria controls numerous processes in response to environmental or cellular cues. Typically, these processes are related to lifestyle transitions between motile and sessile behaviors. However, c-di-GMP also regulates other processes. In Myxococcus xanthus, CdbA is a DNA-binding and nucleoid-associated protein that helps to organize the large chromosome. CdbA binds c-di-GMP and DNA in a mutually exclusive manner. While other nucleoid-associated proteins are not essential, CdbA is essential. Here, we show that the crucial function of CdbA is to maintain the level of the c-di-GMP-binding PilZ-domain protein CdbS appropriately low. The CdbS level is not only increased upon depletion of CdbA but also in response to heat stress. Under both conditions, the increased CdbS level perturbs chromosome organization and ultimately causes cell death. The CdbA/CdbS system represents 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.

microbiology↗

bioRxiv2022

Molecular basis and design principles of a system for switchable front-rear polarity and directional migration

During cell migration, front-rear polarity is spatiotemporally regulated; however, the underlying design of regulatory interactions vary. 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 inhibits 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.

microbiology↗

bioRxiv2022

A phase-separated biomolecular condensate nucleates polymerization of the tubulin homolog FtsZ to spatiotemporally regulate bacterial cell division

Cell division is spatiotemporally precisely regulated, but the underlying mechanisms are incompletely understood. In the social, predatory bacterium Myxococcus xanthus, the PomX/PomY/PomZ proteins form a single large megadalton-sized complex that directly positions and stimulates cytokinetic ring formation by the tubulin homolog FtsZ. Here, we studied 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, suggesting a novel cell division site positioning mechanism in which the single PomY condensate enriches FtsZ to guide FtsZ-ring formation and division. PomY-nucleated FtsZ polymerization shares features with microtubule nucleation by biomolecular condensates in eukaryotes, supporting this mechanisms ancient origin.

cell biology↗

bioRxiv2022

Evidence for a widespread third system for bacterial polysaccharide export across the outer membrane comprising a composite OPX/β-barrel translocon

In Gram-negative bacteria, secreted polysaccharides have multiple critical functions. In Wzx/Wzy- and ABC transporter-dependent pathways, an outer membrane (OM) polysaccharide export (OPX) type translocon exports the polysaccharide across the OM. The paradigm OPX protein WzaE. coli is an octamer, in which the eight C-terminal domains form an -helical OM pore, and the eight copies of the three N-terminal domains (D1-D3) a periplasmic cavity. In synthase-dependent pathways, the OM translocon is a 16- to 18- stranded {beta}-barrel protein. In Myxococcus xanthus, the secreted polysaccharide EPS is synthesized in a Wzx/Wzy-dependent pathway. Here, using experiments and computational structural biology, we characterize EpsX as an OM 18-stranded {beta}-barrel protein important for EPS synthesis and identify AlgE, a {beta}-barrel translocon of a synthase-dependent pathway, as its closest structural homolog. We also find that EpsY, the OPX protein of the EPS pathway, only consists of the periplasmic D1 and D2 domains and lacks the domain for spanning the OM (henceforth D1D2OPX protein). In vivo, EpsX and EpsY mutually stabilize each other, supporting their direct interaction. Based on these observations, we propose a model whereby EpsY and EpsX make up a novel type of translocon for polysaccharide export across the OM. Specifically, in this composite translocon, EpsX functions as the OM-spanning translocon together with the periplasmic D1D2OPX protein EpsY. Based on computational genomics, similar composite systems are present widespread in Gram-negative bacteria. This model provides a framework for these proteins future experimental characterization.

microbiology↗

bioRxiv2022

A bipartite, low-affinity roadblock domain-containing GAP complex regulates bacterial front-rear polarity

The Ras-like GTPase MglA is a key regulator of front-rear polarity in rod-shaped Myxococcus xanthus cells. MglA-GTP localizes to the leading cell pole and stimulates assembly of the two motility machineries. MglA-GTP localization is spatially constrained by its cognate GEF, the RomR/RomX complex, and GAP, the MglB Roadblock-domain protein. 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 as a co-GAP that stimulates MglB GAP activity. The MglB/RomY interaction is low affinity, restricting complex formation 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 MglB GAP activity is spatially precisely confined to the lagging pole, thereby constraining MglA-GTP to the leading pole establishing front-rear polarity.

microbiology↗

bioRxiv2021

The CRP-like transcriptional regulator MrpC curbs c-di-GMP and 3,3 cGAMP nucleotide levels during development in Myxococcus xanthus

Myxococcus xanthus has a nutrient-regulated biphasic lifecycle forming predatory swarms in the presence of nutrients and spore-filled fruiting bodies in the absence of nutrients. The second messenger c-di-GMP is essential during both stages of the lifecycle; however, different enzymes involved in c-di-GMP synthesis and degradation as well as several c-di-GMP receptors are important during distinct lifecycle stages. To address this stage specificity, we determined transcript levels using RNA-seq and transcription start sites using Cappable-seq during growth and development at a genome-wide scale. All 70 genes encoding c-di-GMP associated proteins were expressed, with 28 up-regulated and 10 down-regulated during development. In particular, the three genes encoding enzymatically active proteins with a stage-specific function were expressed stage-specifically. By combining operon mapping with published ChIP-seq data for MrpC (Robinson et al., 2014), the CRP-like master regulator of development, we identified nine developmentally regulated genes as regulated by MrpC. In particular, MrpC directly represses expression of dmxB, which encodes the diguanylate cyclase DmxB that is essential for development and responsible for the c-di-GMP increase during development. Moreover, MrpC directly activates transcription of pmxA, which encodes a bifunctional phosphodiesterase that degrades c-di-GMP and 3, 3 cGAMP in vitro and is essential for development. Thereby, MrpC regulates and curbs the cellular pools of c-di-GMP and 3, 3 cGAMP during development. We conclude that temporal regulation of the synthesis of proteins involved in c-di-GMP metabolism contributes to c-di-GMP signaling specificity. MrpC is important for this regulation, thereby being a key regulator of developmental cyclic di-nucleotide metabolism in M. xanthus. ImportanceThe second messenger c-di-GMP is important during both stages of the nutrient-regulated biphasic lifecycle 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 lifecycle stages. Here, we show that the three genes encoding enzymatically active proteins with a stage-specific function are expressed stage-specifically. Moreover, we find that the master transcriptional regulator of development MrpC directly regulates expression of dmxB, which encodes the diguanylate cyclase DmxB that is essential for development, and of pmxA, which encodes a bifunctional phosphodiesterase that degrades c-di-GMP and 3, 3 cGAMP in vitro and is essential for development. We conclude that temporal regulation of the synthesis of proteins involved in c-di-GMP metabolism contributes to c-di-GMP signaling specificity, and that MrpC plays an important role in this regulation.

microbiology↗

bioRxiv2021

A repurposed, non-canonical cytochrome c, chaperones calcium binding by PilY1 for type IVa pili formation

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 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 a non-canonical cytochrome c (TfcP) with an unusual His/Cys heme ligation and calcium. We provide evidence that TfcP is unlikely to participate in electron transport and has been repurposed to promote calcium binding by PilY1.1 at low calcium concentrations, thereby stabilising PilY1.1 and enabling T4aP function in a broader range of calcium concentrations. These results identify a novel function of cytochromes c and illustrate how incorporating an accessory factor expands the environmental range under which the T4aP system functions.

microbiology↗

bioRxiv2021

Three PilZ domain proteins, PlpA, PixA and PixB, have distinct functions in regulation of motility and development in Myxococcus xanthus

In bacteria, the nucleotide-based second messenger bis-(3-5)-cyclic dimeric GMP (c-di-GMP) binds to effectors to generate outputs in response to changes in the environment. In Myxococcus xanthus, c-di-GMP regulates type IV pili-dependent motility and the starvation-induced developmental program that results in the formation of spore-filled fruiting bodies; however, little is known about the effectors that bind c-di-GMP. Here, we systematically inactivated all 24 genes encoding PilZ domain-containing proteins, which are among the most common c-di-GMP receptors. We confirm that PlpA, a stand-alone PilZ-domain protein, is specifically important for motility and that Pkn1, which is composed of a Ser/Thr domain and a PilZ domain, is specifically important for development. Moreover, we identify two PilZ-domain proteins that have distinct functions in regulating motility and development. PixB, which is composed of two PilZ domains and an acetyltransferase domain, binds c-di-GMP in vitro and regulates type IV pili-dependent and gliding motility upstream of the Frz chemosensory system as well as development. The acetyltransferase domain is required and sufficient for function during growth while all three domains and c-di-GMP binding are essential for PixB function during development. PixA is a response regulator composed of a PilZ domain and a receiver domain, binds c-di-GMP in vitro, and regulates motility downstream of the Frz chemosensory system by setting up the polarity of the two motility systems. Our results support a model whereby the three proteins PlpA, PixA and PixB act in parallel pathways and have distinct functions to regulation of motility. Importancec-di-GMP signaling controls bacterial motility in many bacterial species by binding to downstream effector proteins. Here, we identify two PilZ domain-containing proteins in Myxococcus xanthus that bind c-di-GMP. We show that PixB, which contains two PilZ domains and an acetyltransferase domain, acts upstream of the Frz chemosensory system to regulate motility via the acetyltransferase domain while the intact protein and c-di-GMP binding are essential for PixB to support development. By contrast, PixA acts downstream of the Frz system to regulate motility. Together with previous observations, we conclude that PilZ-domain proteins and c-di-GMP act in multiple parallel pathways to regulate motility and development in M. xanthus.

microbiology↗

bioRxiv2020

PomX, a ParA/MinD ATPase activating protein, is a triple regulator of cell division in Myxococcus xanthus

Cell division is precisely regulated to generate daughter cells of correct size and shape. In the social bacterium Myxococcus xanthus, the tripartite PomX/Y/Z complex directly stimulates positioning of the cytokinetic FtsZ-ring at midcell to mark the division site. The [~]15 MDa PomX/Y/Z complex associates with the nucleoid in a PomZ-dependent manner, translocates to midcell to stimulate FtsZ-ring formation, and undergoes fission during division. We demonstrate that PomX consists of two functionally distinct domains and has three functions. The N-terminal domain interacts with the ParA/MinD ATPase PomZ and stimulates PomZ ATPase activity. The C-terminal domain mediates PomX self-interaction, interaction to PomY, and serves as a scaffold for PomX/Y/Z complex formation. Moreover, the PomX/PomZ interaction is important for fission. These observations together with previous work support that the architecturally diverse ATPase activating proteins of ParA/MinD ATPases are highly modular and use the same mechanism to activate their cognate ATPase via a short positively charged N-terminal extension.

microbiology↗

bioRxiv2020

SMC and the bactofilin/PadC scaffold have distinct yet redundant functions in chromosome segregation and organization in Myxococcus xanthus

In bacteria, ParABS systems and structural maintenance of chromosome (SMC) condensin-like complexes are important for chromosome segregation and organization. The rod-shaped Myxococcus xanthus cells have a unique chromosome arrangement in which a scaffold composed of three bactofilins (BacNOP) and PadC positions the essential ParB{middle dot}parS segregation complexes and the DNA segregation ATPase ParA in the subpolar regions. Here, we identify the Smc and ScpAB subunits of the SMC complex in M. xanthus and demonstrate that SMC is conditionally essential with mutants containing smc or scpAB deletions being temperature sensitive. Lack of SMC caused defects in chromosome segregation and organization. Lack of the BacNOP/PadC scaffold caused chromosome segregation defects but was not essential. Inactivation of SMC was synthetic lethal with lack of the BacNOP/PadC scaffold. Lack of SMC interfered with formation of the BacNOP/PadC scaffold while lack of this scaffold did not interfere with chromosome association by SMC. Altogether, our data support that three systems cooperate to enable chromosome segregation in M. xanthus, whereby ParABS constitutes the basic machinery and SMC and the BacNOP/PadC scaffold have distinct yet redundant roles in this process with SMC supporting individualization of daughter chromosomes and BacNOP/PadC making the ParABS system operate more robustly

microbiology↗