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 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.

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 pilus-dependent motility and the starvation-induced developmental program that results in 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 effectors. We confirm that the stand-alone PilZ domain protein PlpA is important for regulation of motility independently of the Frz chemosensory system and that Pkn1, which is composed of a Ser/Thr kinase 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 pilus-dependent and gliding motility in a Frz-dependent manner 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 independently of the Frz system, likely by setting up the polarity of the two motility systems. Our results support a model whereby PlpA, PixA, and PixB act in independent pathways and have distinct functions in regulation of motility. IMPORTANCE c-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 in a manner that depends on 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. In contrast, PixA acts in a Frz-independent manner to regulate motility. Taking our results together with previous observations, we conclude that PilZ domain proteins and c-di-GMP act in multiple independent pathways to regulate motility and development in M. xanthus.

MglC, a Paralog of Myxococcus xanthus GTPase-Activating Protein MglB, Plays a Divergent Role in Motility Regulation.

UNLABELLED: In order to optimize interactions with their environment and one another, bacteria regulate their motility. In the case of the rod-shaped cells of Myxococcus xanthus, regulated motility is essential for social behaviors. M. xanthus moves over surfaces using type IV pilus-dependent motility and gliding motility. These two motility systems are coordinated by a protein module that controls cell polarity and consists of three polarly localized proteins, the small G protein MglA, the cognate MglA GTPase-activating protein MglB, and the response regulator RomR. Cellular reversals are induced by the Frz chemosensory system, and the output response regulator of this system, FrzZ, interfaces with the MglA/MglB/RomR module to invert cell polarity. Using a computational approach, we identify a paralog of MglB, MXAN_5770 (MglC). Genetic epistasis experiments demonstrate that MglC functions in the same pathway as MglA, MglB, RomR, and FrzZ and is important for regulating cellular reversals. Like MglB, MglC localizes to the cell poles asymmetrically and with a large cluster at the lagging pole. Correct polar localization of MglC depends on RomR and MglB. Consistently, MglC interacts directly with MglB and the C-terminal output domain of RomR, and we identified a surface of MglC that is necessary for the interaction with MglB and for MglC function. Together, our findings identify an additional member of the M. xanthus polarity module involved in regulating motility and demonstrate how gene duplication followed by functional divergence can add a layer of control to the complex cellular processes of motility and motility regulation. IMPORTANCE: Gene duplication and the subsequent divergence of the duplicated genes are important evolutionary mechanisms for increasing both biological complexity and regulation of biological processes. The bacterium Myxococcus xanthus is a soil bacterium with an unusually large genome that carries out several social processes, including predation of other bacterial species and formation of multicellular, spore-filled fruiting bodies. One feature of the large M. xanthus genome is that it contains many gene duplications. Here, we compare the products of one example of gene duplication and divergence, in which a paralog of the cognate MglA GTPase-activating protein MglB has acquired a different and opposing role in the regulation of cellular polarity and motility, processes critical to the bacterium's social behaviors.

Molecular basis and design principles of switchable front-rear polarity and directional migration in Myxococcus xanthus.

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.

Spatiotemporal regulation of switching front-rear cell polarity.

Bacterial cells are spatiotemporally highly organised with proteins localising dynamically to distinct subcellular regions. Motility in the rod-shaped Myxococcus xanthus cells represents an example of signal-induced spatiotemporal regulation of cell polarity. M. xanthus cells move across surfaces with defined front-rear polarity; occasionally, they invert polarity and, in parallel, reverse direction of movement. The polarity module establishes front-rear polarity between reversals and consists of the Ras-like GTPase MglA and its cognate GEF and GAP, that all localise asymmetrically to the cell poles. The Frz chemosensory system constitutes the polarity inversion module and interfaces with the proteins of the polarity module, thereby triggering their polar repositioning. As a result, the polarity proteins, over time, toggle between the cell poles causing cells to oscillate irregularly. Here, we review recent progress in how front-rear polarity is established by the polarity module and inverted by the Frz system and highlight open questions for future studies.

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 life cycle forming predatory swarms in the presence of nutrients and spore-filled fruiting bodies in the absence of nutrients. The second messenger 3'-5', 3'-5 cyclic di-GMP (c-di-GMP) is essential during both stages of the life cycle; however, different enzymes involved in c-di-GMP synthesis and degradation as well as several c-di-GMP receptors are important during distinct life cycle stages. To address this stage specificity, we determined transcript levels using transcriptome sequencing (RNA-seq) and transcription start sites using Cappable sequencing (Cappable-seq) during growth and development genome wide. All 70 genes encoding c-di-GMP-associated proteins were expressed, with 28 upregulated and 10 downregulated during development. Specifically, the three genes encoding enzymatically active proteins with a stage-specific function were expressed stage specifically. By combining operon mapping with published chromatin immunoprecipitation sequencing (ChIP-seq) data for MrpC (M. Robinson, B. Son, D. Kroos, L. Kroos, BMC Genomics 15:1123, 2014, http://dx.doi.org/10.1186/1471-2164-15-1123), the cAMP receptor protein (CRP)-like master regulator of development, we identified nine developmentally regulated genes as regulated by MrpC. In particular, MrpC directly represses the 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 the 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. IMPORTANCE 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. 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 the 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.

CdbA is a DNA-binding protein and c-di-GMP receptor important for nucleoid organization and segregation in Myxococcus xanthus.

Cyclic di-GMP (c-di-GMP) is a second messenger that modulates multiple responses to environmental and cellular signals in bacteria. Here we identify CdbA, a DNA-binding protein of the ribbon-helix-helix family that binds c-di-GMP in Myxococcus xanthus. CdbA is essential for viability, and its depletion causes defects in chromosome organization and segregation leading to a block in cell division. The protein binds to the M. xanthus genome at multiple sites, with moderate sequence specificity; however, its depletion causes only modest changes in transcription. The interactions of CdbA with c-di-GMP and DNA appear to be mutually exclusive and residue substitutions in CdbA regions important for c-di-GMP binding abolish binding to both c-di-GMP and DNA, rendering these protein variants non-functional in vivo. We propose that CdbA acts as a nucleoid-associated protein that contributes to chromosome organization and is modulated by c-di-GMP, thus revealing a link between c-di-GMP signaling and chromosome biology.

Spatial control of the GTPase MglA by localized RomR-RomX GEF and MglB GAP activities enables Myxococcus xanthus motility.

The rod-shaped Myxococcus xanthus cells move with defined front-rear polarity using polarized motility systems. A polarity module consisting of the small GTPase MglA, its cognate GTPase activating protein (GAP) MglB and RomR establishes this polarity. Agl-Glt gliding motility complexes assemble and disassemble at the leading and lagging pole, respectively. These processes are stimulated by MglA-GTP at the leading and MglB at the lagging pole. Here, we identify RomX as an integral component of the polarity module. RomX and RomR form a complex that has MglA guanine nucleotide exchange factor (GEF) activity and also binds MglA-GTP. In vivo RomR recruits RomX to the leading pole forming the RomR-RomX complex that stimulates MglA-GTP formation and binding, resulting in a high local concentration of MglA-GTP. The spatially separated and opposing activities of the RomR-RomX GEF at the leading and the MglB GAP at the lagging cell pole establish front-rear polarity by allowing the spatially separated assembly and disassembly of Agl-Glt motility complexes. Our findings uncover a regulatory system for bacterial cell polarity that incorporates a nucleotide exchange factor as well as an NTPase activating protein for regulation of a nucleotide-dependent molecular switch and demonstrate a spatial organization that is conserved in eukaryotes.