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A combination of unusual transcription factors binds cooperatively to control Myxococcus xanthus developmental gene expression.

ABSTRACT: Myxococcus xanthus is a bacterium that undergoes multicellular development requiring coordinate regulation of multiple signaling pathways. One pathway governs aggregation and sporulation of some cells in a starving population and requires C-signaling, whereas another pathway causes programmed cell death and requires the MazF toxin. In response to starvation, the levels of the bifunctional transcription factor/antitoxin MrpC and its related proteolytic fragment MrpC2 are increased, inhibiting the cell death pathway via direct interaction of MrpC with MazF. Herein, we demonstrate that MrpC2 plays a direct role in the transcriptional response to C-signaling. We show that MrpC2 binds to sequences upstream of the C-signal-dependent fmgA promoter. These sequences are present in other C-signal-dependent promoter regions, indicating a general role for MrpC2 in developmental gene regulation. Association of MrpC and/or MrpC2 with the fmgA promoter region in vivo requires FruA, a protein that is similar to response regulators of 2-component signal transduction systems, but may not be phosphorylated. DNA binding studies showed that this association likely involves an unusual mechanism for a response regulator in which FruA and MrpC2 bind cooperatively to adjacent sites upstream of the fmgA promoter. We propose that this unusual mechanism of combinatorial control allows coordination of morphogenetic C-signaling with starvation signaling and cell death, determining spatiotemporal gene expression and cell fate.

SUBMITTER: Mittal S

PROVIDER: S-EPMC2644147 | biostudies-literature | 2009 Feb

REPOSITORIES: biostudies-literature

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A combination of unusual transcription factors binds cooperatively to control Myxococcus xanthus developmental gene expression.

Mittal Sheenu S Kroos Lee L

Proceedings of the National Academy of Sciences of the United States of America 20090129 6

Myxococcus xanthus is a bacterium that undergoes multicellular development requiring coordinate regulation of multiple signaling pathways. One pathway governs aggregation and sporulation of some cells in a starving population and requires C-signaling, whereas another pathway causes programmed cell death and requires the MazF toxin. In response to starvation, the levels of the bifunctional transcription factor/antitoxin MrpC and its related proteolytic fragment MrpC2 are increased, inhibiting the ...[more]

PMID: 19179401

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Project description:BackgroundMyxococcus xanthus is a bacterium that undergoes multicellular development when starved. Cells move to aggregation centers and form fruiting bodies in which cells differentiate into dormant spores. MrpC appears to directly activate transcription of fruA, which also codes for a transcription factor. Both MrpC and FruA are crucial for aggregation and sporulation. The two proteins bind cooperatively in promoter regions of some developmental genes.ResultsChromatin immunoprecipitation followed by DNA sequencing (ChIP-seq) and bioinformatic analysis of cells that had formed nascent fruiting bodies revealed 1608 putative MrpC binding sites. These sites included several known to bind MrpC and they were preferentially distributed in likely promoter regions, especially those of genes up-regulated during development. The up-regulated genes include 22 coding for protein kinases. Some of these are known to be directly involved in fruiting body formation and several negatively regulate MrpC accumulation. Our results also implicate MrpC as a direct activator or repressor of genes coding for several transcription factors known to be important for development, for a major spore protein and several proteins important for spore formation, for proteins involved in extracellular A- and C-signaling, and intracellular ppGpp-signaling during development, and for proteins that control the fate of other proteins or play a role in motility. We found that the putative MrpC binding sites revealed by ChIP-seq are enriched for DNA sequences that strongly resemble a consensus sequence for MrpC binding proposed previously. MrpC2, an N-terminally truncated form of MrpC, bound to DNA sequences matching the consensus in all 11 cases tested. Using longer DNA segments containing 15 of the putative MrpC binding sites from our ChIP-seq analysis as probes in electrophoretic mobility shift assays, evidence for one or more MrpC2 binding site was observed in all cases and evidence for cooperative binding of MrpC2 and FruA was seen in 13 cases.ConclusionsWe conclude that MrpC and MrpC2 bind to promoter regions of hundreds of developmentally-regulated genes in M. xanthus, in many cases cooperatively with FruA. This binding very likely up-regulates protein kinases, and up- or down-regulates other proteins that profoundly influence the developmental process.

Project description:BackgroundThe Myxococcales are well known for their predatory and developmental social processes, and for the molecular complexity of regulation of these processes. Many species within this order have unusually large genomes compared to other bacteria, and their genomes have many genes that are unique to one specific sequenced species or strain. Here, we describe RNAseq based transcriptome analysis of the FruA regulon of Myxococcus xanthus and a comparative RNAseq analysis of two Myxococcus species, M. xanthus and Myxococcus stipitatus, as they respond to starvation and begin forming fruiting bodies.ResultsWe show that both species have large numbers of genes that are developmentally regulated, with over half the genome showing statistically significant changes in expression during development in each species. We also included a non-fruiting mutant of M. xanthus that is missing the transcriptional regulator FruA to identify the direct and indirect FruA regulon and to identify transcriptional changes that are specific to fruiting and not just the starvation response. We then identified Interpro gene ontologies and COG annotations that are significantly up- or down-regulated during development in each species. Our analyses support previous data for M. xanthus showing developmental upregulation of signal transduction genes, and downregulation of genes related to cell-cycle, translation, metabolism, and in some cases, DNA replication. Gene expression in M. stipitatus follows similar trends. Although not all specific genes show similar regulation patterns in both species, many critical developmental genes in M. xanthus have conserved expression patterns in M. stipitatus, and some groups of otherwise unstudied orthologous genes share expression patterns.ConclusionsBy identifying the FruA regulon and identifying genes that are similarly and uniquely regulated in two different species, this work provides a more complete picture of transcription during Myxococcus development. We also provide an R script to allow other scientists to mine our data for genes whose expression patterns match a user-selected gene of interest.

Project description:Myxococcus xanthus is a gram-negative bacterium which has a complex life cycle. Autochemotaxis, a process whereby cells release a self-generated signaling molecule, may be the principal mechanism facilitating directed motility in both the vegetative swarming and developmental aggregation stages of this life cycle. The process requires the Frz signal transduction system, including FrzZ, a protein which is composed of two domains, both showing homology to the enteric chemotaxis response regulator CheY. The first domain of FrzZ (FrzZ1), when expressed as bait in the yeast two-hybrid system and screened against a library, was shown to potentially interact with the C-terminal portion of a protein encoding an ATP-binding cassette (AbcA). The activation domain-AbcA fusion protein did not interact with the second domain of FrzZ (FrzZ2) or with two other M. xanthus response regulator-containing proteins presented as bait, suggesting that the FrzZ1-AbcA interaction may be specific. Cloning and sequencing of the upstream region of the abcA gene showed the ATP-binding cassette to be linked to a large hydrophobic, potentially membrane-spanning domain. This domain organization is characteristic of a subgroup of ABC transporters which perform export functions. Cloning and sequencing downstream of abcA indicated that the ABC transporter is at the start of an operon containing three open reading frames. An insertion mutation in the abcA gene resulted in cells displaying the frizzy aggregation phenotype, providing additional evidence that FrzZ and AbcA may be part of the same signal transduction pathway. Cells with mutations in genes downstream of abcA showed no developmental defects. Analysis of the proposed exporter role of AbcA in cell mixing experiments showed that the ABC transporter mutant could be rescued by extracellular complementation. We speculate that the AbcA protein may be involved in the export of a molecule required for the autochemotactic process.

Dataset Information

A combination of unusual transcription factors binds cooperatively to control Myxococcus xanthus developmental gene expression.

Publications

A combination of unusual transcription factors binds cooperatively to control Myxococcus xanthus developmental gene expression.

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