MrpC, a member of the CRP/Fnr superfamily of transcriptional regulators, plays a key role in coordination of the multicellular developmental program in Myxococcus xanthus. Previous reports suggest MrpC is subject to complex regulation including activation by an unusual LonD-dependent proteolytic processing event that removes its unique N-terminal peptide, producing the isoform MrpC2. MrpC2 is proposed to positively autoregulate and regulate transcription of hundreds of genes necessary for both the aggregation and sporulation phases of the developmental program. We demonstrate here that mrpC expression bifurcates corresponding to different cell populations within the developmental program. During our analysis of regulatory events controlling this process, we demonstrate that MrpC2 is not an active isoform; rather, the N-terminal peptide is instead essential for MrpC function in vivo. We also demonstrate that MrpC is instead a negative autoregulator and represses its own expression by specifically competing with its enhancer binding protein, MrpB. These results provide an additional rare example of CRP / EBP coordinated regulation, and significantly revise the model for control of the central developmental transcriptional activator of the M. xanthus developmental program. This article is protected by copyright. All rights reserved.

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 the Myxococcus xanthus developmental 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, T 21 T 22 S 23 S 24, is necessary for MrpC activity by promoting efficient DNA binding. Mass spectrometry analysis indicated the TTSS motif is not directly phosphorylated by Pkn14 in vitro but 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-type M. xanthus under 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.

Transcription factor autoregulation is a simple network motif (recurring circuit) built into genetic regulatory networks that direct cell behavior. Negative autoregulation (NAR) network motifs are particularly abundant in bacteria and provide specific functions, such as buffering against transcriptional noise. Here, we investigate the phenotypic consequence of perturbing NAR of a major transcription factor, MrpC, that controls the multicellular development program of the bacterium Myxococcus xanthus. Launch of the developmental program directs certain cells in the population to first aggregate into haystack-shaped mounds, and then to differentiate into environmentally resistant spores to form mature fruiting bodies. Perturbation of MrpC NAR causes 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, while the remaining cells transition to spores inside residual fruiting bodies. As predicted, disruption of MrpC NAR led to an increased and broadened population distribution of mrpC expression. Examination of MrpC levels in developmental subpopulations during in situ development demonstrated cells locked in the swarms contained intermediate MrpC levels insufficient to promote sporulation. These results suggest an inherent property of NAR motifs that function in multicellular developmental programs is to facilitate synchronized responses.Significance StatementAll organisms use regulatory networks for cellular homeostasis, mediating appropriate responses to environmental changes, or to direct animal development. Understanding how the basic building blocks (motifs) of regulatory networks contribute to these processes is essential to mitigate the effects of mutations in regulatory networks (i.e. cancers) or to synthesize beneficial organisms. In this study, we demonstrate that a common regulatory motif, a transcription factor that represses its own expression, helps synchronize cells that engage in collective behaviors.

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SummaryThe Crp/Fnr family of transcriptional regulators play central roles in transcriptional control of diverse physiological responses. Activation of individual family members is controlled by a surprising diversity of mechanisms tuned to the particular physiological responses or lifestyles that they regulate. MrpC is a Crp/Fnr homolog that plays an essential role in controlling the Myxococcus xanthus developmental 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, T21 T22 S23 S24, is necessary for MrpC activity by promoting efficient DNA binding. Mass spectrometry analysis indicated the TTSS motif is not directly phosphorylated by Pkn14 in vitro but is necessary for efficient Pkn14-dependent phosphorylation on several residues in the remainder of the protein. Pkn8 and Pkn14 kinase activities do not play obvious roles in controlling MrpC activity in wild type M. xanthus under laboratory conditions, but likely modulate MrpC DNA binding in response to unknown environmental conditions. Interestingly, mutational analysis of the TTSS motif caused non-robust developmental phenotypes, revealing that MrpC plays a role in developmental buffering.