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MrpC, a member of the CRP/Fnr transcription factor superfamily, is necessary to induce and control the multicellular developmental program of the bacterium,Myxococcus xanthus. During development, certain cells in the population first swarm into haystack-shaped aggregates and then differentiate into environmentally resistant spores to form mature fruiting bodies (a specialized biofilm).mrpCtranscriptional regulation is controlled by negative autoregulation (NAR).

Wild type and mutantmrpCpromoter regions were fused to a fluorescent reporter to examine effects onmrpCexpression in the population and in single cellsin situ. Phenotypic consequences of the mutantmrpCpromoter were assayed by deep convolution neural network analysis of developmental movies, sporulation efficiency assays, and anti-MrpC immunoblot. In situ analysis of single cell MrpC levels in distinct populations were assayed with an MrpC-mNeonGreen reporter.

Disruption of MrpC binding sites within themrpCpromoter region led to increased and broadened distribution ofmrpCexpression levels between individual cells in the population. Expression ofmrpCfrom the mutant promoter led to a striking phenotype in which cells lose synchronized transition from aggregation to sporulation. Instead, some cells abruptly exit aggregation centers and remain locked in a cohesive swarming state we termed developmental swarms, while the remaining cells transition to spores inside residual fruiting bodies.In situexamination of a fluorescent reporter for MrpC levels in developmental subpopulations demonstrated cells locked in the developmental swarms contained MrpC levels that do not reach the levels observed in fruiting bodies.

Increased cell-to-cell variation inmrpCexpression upon disruption of MrpC binding sites within its promoter is consistent with NAR motifs functioning to reducing noise. Noise reduction may be key to synchronized transition of cells in the aggregation state to the sporulation state. We hypothesize a novel subpopulation of cells trapped as developmental swarms arise from intermediate levels of MrpC that are sufficient to promote aggregation but insufficient to trigger sporulation. Failure to transition to higher levels of MrpC necessary to induce sporulation may indicate cells in developmental swarms lack an additional positive feedback signal required to boost MrpC levels.

 

The Crp/Fnr family of transcriptional regulators play central roles in transcriptional control of diverse physiological responses, and are activated by a surprising diversity of mechanisms. MrpC is a Crp/Fnr homolog that controls theMyxococcus xanthusdevelopmental program. A long‐standing model proposed that MrpC activity is controlled by the Pkn8/Pkn14 serine/threonine kinase cascade, which phosphorylates MrpC on threonine residue(s) located in its extreme amino‐terminus. In this study, we demonstrate that a stretch of consecutive threonine and serine residues, T₂₁T₂₂S₂₃S_24,is necessary for MrpC activity by promoting efficient DNA binding. Mass spectrometry analysis indicated the TTSS motif is not directly phosphorylated by Pkn14in vitrobut is necessary for efficient Pkn14‐dependent phosphorylation on several residues in the remainder of the protein. In an important correction to a long‐standing model, we show Pkn8 and Pkn14 kinase activities do not play obvious roles in controlling MrpC activity in wild‐typeM. xanthusunder laboratory conditions. Instead, we propose Pkn14 modulates MrpC DNA binding in response to unknown environmental conditions. Interestingly, substitutions in the TTSS motif caused developmental defects that varied between biological replicates, revealing that MrpC plays a role in promoting a robust developmental phenotype.

 

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 (REC 1 and REC 2 ). 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 REC 1 impact the onset of aggregation and/or the mobility of aggregates, while REC 2 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.