Orphan Hybrid Histidine Protein Kinase SinK Acts as a Signal Integrator To Fine-Tune Multicellular Behavior in Myxococcus xanthus

Abstract

His-Asp phosphorelay (also known as two-component signal transduction) proteins are the predominant mechanism used in most bacteria to control behavior in response to changing environmental conditions. In addition to systems consisting of a simple two-component system utilizing an isolated histidine kinase/response regulator pair, some bacteria are enriched in histidine kinases that serve as signal integration proteins; these kinases are usually characterized by noncanonical domain architecture, and the responses that they regulate may be difficult to identify. The environmental bacterium Myxococcus xanthus is highly enriched in these noncanonical histidine kinases. M. xanthus is renowned for a starvation-induced multicellular developmental program in which some cells are induced to aggregate into fruiting bodies and then differentiate into environmentally resistant spores. Here, we characterize the M. xanthus orphan hybrid histidine kinase SinK (Mxan_4465), which consists of a histidine kinase transmitter followed by two receiver domains (REC1 and REC2). Nonphosphorylatable sinK mutants were analyzed under two distinct developmental conditions and using a new high-resolution developmental assay. These assays revealed that SinK autophosphorylation and REC1 impact the onset of aggregation and/or the mobility of aggregates, while REC2 impacts sporulation efficiency. SinK activity is controlled by a genus-specific hypothetical protein (SinM; Mxan_4466). We propose that SinK serves to fine-tune fruiting body morphology in response to environmental conditions. IMPORTANCE Biofilms are multicellular communities of microorganisms that play important roles in host disease or environmental biofouling. Design of preventative strategies to block biofilms depends on understanding the molecular mechanisms used by microorganisms to build them. The production of biofilms in bacteria often involves two-component signal transduction systems in which one protein component (a kinase) detects an environmental signal and, through phosphotransfer, activates a second protein component (a response regulator) to change the transcription of genes necessary to produce a biofilm. We show that an atypical kinase, SinK, modulates several distinct stages of specialized biofilm produced by the environmental bacterium Myxococcus xanthus. SinK likely integrates multiple signals to fine-tune biofilm formation in response to distinct environmental conditions.

A negative autoregulation network motif is required for synchronizedMyxococcus xanthusdevelopment

“…Movies of M. xanthus development have demonstrated that prior to the onset of sporulation, aggregates are surprisingly dynamic (26, 28). Initial aggregates often dissolve or coalesce, and even mature aggregates can be mobile prior to transition to stationary spore-filled fruiting bodies (26).…”

“…Movies of M. xanthus development have demonstrated that prior to the onset of sporulation, aggregates are surprisingly dynamic (26, 28). Initial aggregates often dissolve or coalesce, and even mature aggregates can be mobile prior to transition to stationary spore-filled fruiting bodies (26). To examine how MrpC NAR affected these transient behaviors, the wild type, P WT - mrpC , and P MUT - mrpC strains were induced to develop under submerged culture conditions, imaged every 30 min from 0 – 72 h in an automated plate reader, and the images assembled into movies.…”

“…M. xanthus strains were induced to develop under submerged culture using 0.15 mL diluted cells per well in 96-well tissue culture plates. After induction of starvation, plates were incubated in a Tecan Spark10M plate reader preheated to 32°C (26). The center of each well was imaged every 30 min from 0 – 72 h post-starvation and images assembled into movies (6 fps) in ImageJ (40).…”

“…The center of each well was imaged every 30 min from 0 – 72 h post-starvation and images assembled into movies (6 fps) in ImageJ (40). For each movie, onset of aggregation (Agg ONSET ), maximum aggregates (Agg MAX ) and final fruiting bodies (Agg FINAL ) were enumerated as described previously (26). The percent of aggregates that transitioned to stationary fruiting bodies was calculated as [Agg FINAL /Agg MAX ].…”

“…We demonstrate that perturbation of MrpC NAR leads to early and over-production of MrpC associated with premature aggregation onset, reduced fruiting body organization, and, unexpectedly, reduced sporulation efficiency. Using a recently developed submerged culture developmental filming method (26), we observed perturbed MrpC NAR resulted in asynchronous development: after formation of aggregates, some cells suddenly exited aggregation centers as fast-moving swarms, while other cells remained in stationary fruiting bodies. Deep convolution neural network analyses indicated these developmental swarms displayed trajectories and velocities that were distinct from cells in the mobile aggregate phase.…”