Suppressor mutations reveal an NtrC-like response regulator, NmpR, for modulation of Type-IV Pili-dependent motility in Myxococcus xanthus

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Abstract
Two-component signaling systems (TCS) regulate bacterial responses to environmental signals through the process of protein phosphorylation. Specifically, sensor histidine kinases (SK) recognize signals and propagate the response via phosphorylation of a cognate response regulator (RR) that functions to initiate transcription of specific genes. Signaling within a single TCS is remarkably specific and cross-talk between TCS is limited. However, regulation of the flow of information through complex signaling networks that include closely related TCS remains largely unknown. Additionally, many bacteria utilize multi-component signaling networks which provide additional genetic and biochemical interactions that must be regulated for signaling fidelity, input and output specificity, and phosphorylation kinetics. Here we describe the characterization of an NtrC-like RR that participates in regulation of Type-IV pilus-dependent motility of Myxococcus xanthus and is thus named NmpR, NtrC Modulator of Pili Regulator. A complex multi-component signaling system including NmpR was revealed by suppressor mutations that restored motility to cells lacking PilR, an evolutionarily conserved RR required for expression of pilA encoding the major Type-IV pilus monomer found in many bacterial species. The system contains at least four signaling proteins: a SK with a protoglobin sensor domain (NmpU), a hybrid SK (NmpS), a phospho-sink protein (NmpT), and an NtrC-like RR (NmpR). We demonstrate that ΔpilR bypass suppressor mutations affect regulation of the NmpRSTU multi-component system, such that NmpR activation is capable of restoring expression of pilA in the absence of PilR. Our findings indicate that pilus gene expression in M. xanthus is regulated by an extended network of TCS which interact to refine control of pilus function. Sensing and responding to the environment are critical for bacterial survival and adaptation. Many bacteria sense environmental signals through biological machinery referred to as two-component signaling systems. These signaling systems are composed of a sensor histidine kinase and a cognate response regulator and serve to regulate gene expression in response to environmental cues. Due to their importance for survival, many bacterial genomes encode multiple two-component systems, some of which arose from gene duplication events. However, the mechanisms by which two-component systems integrate multiple inputs within larger networks remains poorly defined. In this study, we demonstrate that suppressor mutations within a Myxococcus xanthus signaling system of previously unknown function restore Type-IV pilus-dependent motility to cells lacking PilR, a response regulator typically required for production of the pilin monomer PilA. Suppressor mutations in ΔpilR cells led to activation of a related signaling system, resulting in expression of pilA and the corresponding production of PilA to restore motility. Thus, both PilSR and NmpSR coordinately regulate motility in M. xanthus. Collectively, our results expand our understanding of the activation of response regulators via phosphorylation and the mechanisms controlling multi-component signal transduction systems.
Funding Information
  • National Science Foundation (MCB-1244021)
  • National Institutes of Health (AI108255)

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