Development Of Myxococcus Xanthus Research Articles

Short-range C-signaling restricts cheating behavior during Myxococcus xanthus development.

Bacteria communicate using both long- and short-range signals. Signaling affects community composition, structure, and function. Adherent communities called biofilms impact medicine, agriculture, industry, and the environment. To facilitate the manipulation of biofilms for societal benefits, a better understanding of short-range signaling is necessary. We investigated the susceptibility of short-range C-signaling to cheating during Myxococcus xanthus biofilm development. A mutant deficient in C-signaling fails to form mounds containing spores (i.e., fruiting bodies) but cheats on C-signaling by wild type in starved cell mixtures and forms spores disproportionately. We found that cheating requires sufficient wild-type cells in the initial mix and can occur both before mound formation and later during the sporulation stage of development. By restricting cheating behavior, short-range C-signaling may have been favored evolutionarily rather than long-range diffusible signaling. Cheating restrictions imposed by short-range signaling may have likewise driven the evolution of multicellularity broadly.

Unravelling a diversity of cellular structures and aggregation dynamics during the early development of Myxococcus xanthus.

Aggregation underlies the collective dynamics of a diversity of organisms, enabling the formation of complex structures and emergent behaviours on interaction with the environment. Cellular aggregation constitutes one of the routes to collective motility and multicellular development. Myxococcus xanthus, a social bacterium, is a valuable model for studying the aggregative path to multicellularity, a major transition in the evolutionary history of life. While the collective developmental behaviour of M. xanthus has been largely studied in high cellular densities, there is a lack of understanding at low-density conditions that can be ecologically relevant. In this work, we study the early stages of emergent collective behaviour of M. xanthus under nutrient-poor and low-density conditions, uncovering the formation of diverse cellular structures with different shapes and sizes, ranging from individual cells to networks comprising thousands of cells. We study their motility patterns and their prevalence along development and discuss their cross-scale role on the population's exploratory dynamics. This work contributes to understanding key, yet largely understudied, aspects in the early stages of multicellular development in myxobacteria, shedding light on the dynamics underlying aggregative processes in this and other taxa and study systems.

Regulation of late-acting operons by three transcription factors and a CRISPR-Cas component during Myxococcus xanthus development.

Upon starvation, rod-shaped Myxococcus xanthus bacteria form mounds and then differentiate into round, stress-resistant spores. Little is known about the regulation of late-acting operons important for spore formation. C-signaling has been proposed to activate FruA, which binds DNA cooperatively with MrpC to stimulate transcription of developmental genes. We report that this model can explain regulation of the fadIJ operon involved in spore metabolism, but not that of the spore coat biogenesis operons exoA-I, exoL-P, and nfsA-H. Rather, a mutation in fruA increased the transcript levels from these operons early in development, suggesting negative regulation by FruA, and a mutation in mrpC affected transcript levels from each operon differently. FruA bound to all four promoter regions invitro, but strikingly each promoter region was unique in terms of whether or not MrpC and/or the DNA-binding domain of Nla6 bound, and in terms of cooperative binding. Furthermore, the DevI component of a CRISPR-Cas system is a negative regulator of all four operons, based on transcript measurements. Our results demonstrate complex regulation of sporulation genes by three transcription factors and a CRISPR-Cas component, which we propose produces spores suited to withstand starvation and environmental insults.

Mutation of self-binding sites in the promoter of the MrpC transcriptional regulator leads to asynchronous Myxococcus xanthus development

Mutation of self-binding sites in the promoter of the MrpC transcriptional regulator leads to asynchronous Myxococcus xanthus development

CRP-Like Transcriptional Regulator MrpC Curbs c-di-GMP and 3',3'-cGAMP Nucleotide Levels during Development in Myxococcus xanthus.

ABSTRACTMyxococcus xanthus has a nutrient-regulated biphasic life cycle forming predatory swarms in the presence of nutrients and spore-filled fruiting bodies in the absence of nutrients. The second messenger 3′-5′, 3′-5 cyclic di-GMP (c-di-GMP) is essential during both stages of the life cycle; however, different enzymes involved in c-di-GMP synthesis and degradation as well as several c-di-GMP receptors are important during distinct life cycle stages. To address this stage specificity, we determined transcript levels using transcriptome sequencing (RNA-seq) and transcription start sites using Cappable sequencing (Cappable-seq) during growth and development genome wide. All 70 genes encoding c-di-GMP-associated proteins were expressed, with 28 upregulated and 10 downregulated during development. Specifically, the three genes encoding enzymatically active proteins with a stage-specific function were expressed stage specifically. By combining operon mapping with published chromatin immunoprecipitation sequencing (ChIP-seq) data for MrpC (M. Robinson, B. Son, D. Kroos, L. Kroos, BMC Genomics 15:1123, 2014, http://dx.doi.org/10.1186/1471-2164-15-1123), the cAMP receptor protein (CRP)-like master regulator of development, we identified nine developmentally regulated genes as regulated by MrpC. In particular, MrpC directly represses the expression of dmxB, which encodes the diguanylate cyclase DmxB that is essential for development and responsible for the c-di-GMP increase during development. Moreover, MrpC directly activates the transcription of pmxA, which encodes a bifunctional phosphodiesterase that degrades c-di-GMP and 3′,3′-cGAMP in vitro and is essential for development. Thereby, MrpC regulates and curbs the cellular pools of c-di-GMP and 3′,3′-cGAMP during development. We conclude that temporal regulation of the synthesis of proteins involved in c-di-GMP metabolism contributes to c-di-GMP signaling specificity. MrpC is important for this regulation, thereby being a key regulator of developmental cyclic di-nucleotide metabolism in M. xanthus.

Cell density, alignment, and orientation correlate with C-signal–dependent gene expression during Myxococcus xanthus development

Starving Myxococcus xanthus bacteria use short-range C-signaling to coordinate their movements and construct multicellular mounds, which mature into fruiting bodies as rods differentiate into spherical spores. Differentiation requires efficient C-signaling to drive the expression of developmental genes, but how the arrangement of cells within nascent fruiting bodies (NFBs) affects C-signaling is not fully understood. Here, we used confocal microscopy and cell segmentation to visualize and quantify the arrangement, morphology, and gene expression of cells near the bottom of NFBs at much higher resolution than previously achieved. We discovered that "transitioning cells" (TCs), intermediate in morphology between rods and spores, comprised 10 to 15% of the total population. Spores appeared midway between the center and the edge of NFBs early in their development and near the center as maturation progressed. The developmental pattern, as well as C-signal-dependent gene expression in TCs and spores, were correlated with cell density, the alignment of neighboring rods, and the tangential orientation of rods early in the development of NFBs. These dynamic radial patterns support a model in which the arrangement of cells within the NFBs affects C-signaling efficiency to regulate precisely the expression of developmental genes and cellular differentiation in space and time. Developmental patterns in other bacterial biofilms may likewise rely on short-range signaling to communicate multiple aspects of cellular arrangement, analogous to juxtacrine and paracrine signaling during animal development.

Three PilZ Domain Proteins, PlpA, PixA, and PixB, Have Distinct Functions in Regulation of Motility and Development in Myxococcus xanthus.

ABSTRACTIn bacteria, the nucleotide-based second messenger bis-(3′-5′)-cyclic dimeric GMP (c-di-GMP) binds to effectors to generate outputs in response to changes in the environment. In Myxococcus xanthus, c-di-GMP regulates type IV pilus-dependent motility and the starvation-induced developmental program that results in formation of spore-filled fruiting bodies; however, little is known about the effectors that bind c-di-GMP. Here, we systematically inactivated all 24 genes encoding PilZ domain-containing proteins, which are among the most common c-di-GMP effectors. We confirm that the stand-alone PilZ domain protein PlpA is important for regulation of motility independently of the Frz chemosensory system and that Pkn1, which is composed of a Ser/Thr kinase domain and a PilZ domain, is specifically important for development. Moreover, we identify two PilZ domain proteins that have distinct functions in regulating motility and development. PixB, which is composed of two PilZ domains and an acetyltransferase domain, binds c-di-GMP in vitro and regulates type IV pilus-dependent and gliding motility in a Frz-dependent manner as well as development. The acetyltransferase domain is required and sufficient for function during growth, while all three domains and c-di-GMP binding are essential for PixB function during development. PixA is a response regulator composed of a PilZ domain and a receiver domain, binds c-di-GMP in vitro, and regulates motility independently of the Frz system, likely by setting up the polarity of the two motility systems. Our results support a model whereby PlpA, PixA, and PixB act in independent pathways and have distinct functions in regulation of motility.IMPORTANCE c-di-GMP signaling controls bacterial motility in many bacterial species by binding to downstream effector proteins. Here, we identify two PilZ domain-containing proteins in Myxococcus xanthus that bind c-di-GMP. We show that PixB, which contains two PilZ domains and an acetyltransferase domain, acts in a manner that depends on the Frz chemosensory system to regulate motility via the acetyltransferase domain, while the intact protein and c-di-GMP binding are essential for PixB to support development. In contrast, PixA acts in a Frz-independent manner to regulate motility. Taking our results together with previous observations, we conclude that PilZ domain proteins and c-di-GMP act in multiple independent pathways to regulate motility and development in M. xanthus.

Plastic multicellular development of Myxococcus xanthus: genotype-environment interactions in a physical gradient.

In order to investigate the contribution of the physical environment to variation in multicellular development of Myxococcus xanthus, phenotypes developed by different genotypes in a gradient of substrate stiffness conditions were quantitatively characterized. Statistical analysis showed that plastic phenotypes result from the genotype, the substrate conditions and the interaction between them. Also, phenotypes were expressed in two distinguishable scales, the individual and the population levels, and the interaction with the environment showed scale and trait specificity. Overall, our results highlight the constructive role of the physical context in the development of microbial multicellularity, with both ecological and evolutionary implications.

Data-driven modeling reveals cell behaviors controlling self-organization during Myxococcus xanthus development

Collective cell movement is critical to the emergent properties of many multicellular systems, including microbial self-organization in biofilms, embryogenesis, wound healing, and cancer metastasis. However, even the best-studied systems lack a complete picture of how diverse physical and chemical cues act upon individual cells to ensure coordinated multicellular behavior. Known for its social developmental cycle, the bacterium Myxococcus xanthus uses coordinated movement to generate three-dimensional aggregates called fruiting bodies. Despite extensive progress in identifying genes controlling fruiting body development, cell behaviors and cell-cell communication mechanisms that mediate aggregation are largely unknown. We developed an approach to examine emergent behaviors that couples fluorescent cell tracking with data-driven models. A unique feature of this approach is the ability to identify cell behaviors affecting the observed aggregation dynamics without full knowledge of the underlying biological mechanisms. The fluorescent cell tracking revealed large deviations in the behavior of individual cells. Our modeling method indicated that decreased cell motility inside the aggregates, a biased walk toward aggregate centroids, and alignment among neighboring cells in a radial direction to the nearest aggregate are behaviors that enhance aggregation dynamics. Our modeling method also revealed that aggregation is generally robust to perturbations in these behaviors and identified possible compensatory mechanisms. The resulting approach of directly combining behavior quantification with data-driven simulations can be applied to more complex systems of collective cell movement without prior knowledge of the cellular machinery and behavioral cues.

The dev Operon Regulates the Timing of Sporulation during Myxococcus xanthus Development.

Myxococcus xanthus undergoes multicellular development when starved. Thousands of rod-shaped cells coordinate their movements and aggregate into mounds in which cells differentiate into spores. Mutations in the dev operon impair development. The dev operon encompasses a clustered regularly interspaced short palindromic repeat-associated (CRISPR-Cas) system. Null mutations in devI, a small gene at the beginning of the dev operon, suppress the developmental defects caused by null mutations in the downstream devR and devS genes but failed to suppress defects caused by a small in-frame deletion in devT We provide evidence that the original mutant has a second-site mutation. We show that devT null mutants exhibit developmental defects indistinguishable from devR and devS null mutants, and a null mutation in devI suppresses the defects of a devT null mutation. The similarity of DevTRS proteins to components of the CRISPR-associated complex for antiviral defense (Cascade), together with our molecular characterization of dev mutants, support a model in which DevTRS form a Cascade-like subcomplex that negatively autoregulates dev transcript accumulation and prevents DevI overproduction that would strongly inhibit sporulation. Our results also suggest that DevI transiently inhibits sporulation when regulated normally. The mechanism of transient inhibition may involve MrpC, a key transcription factor, whose translation appears to be weakly inhibited by DevI. Finally, our characterization of a devI devS mutant indicates that very little exo transcript is required for sporulation, which is surprising since Exo proteins help form the polysaccharide spore coat.IMPORTANCE CRISPR-Cas systems typically function as adaptive immune systems in bacteria. The dev CRISPR-Cas system of M. xanthus has been proposed to prevent bacteriophage infection during development, but how dev controls sporulation has been elusive. Recent evidence supported a model in which DevR and DevS prevent overproduction of DevI, a predicted 40-residue inhibitor of sporulation. We provide genetic evidence that DevT functions together with DevR and DevS to prevent DevI overproduction. We also show that spores form about 6 h earlier in mutants lacking devI than in the wild type. Only a minority of natural isolates appear to have a functional dev promoter and devI, suggesting that a functional dev CRISPR-Cas system evolved recently in niches where delayed sporulation and/or protection from bacteriophage infection proved advantageous.

A gene encoding a potential adenosine 5'-phosphosulphate kinase is necessary for timely development of Myxococcus xanthus.

Microbiology Society journals contain high-quality research papers and topical review articles. We are a not-for-profit publisher and we support and invest in the microbiology community, to the benefit of everyone. This supports our principal goal to develop, expand and strengthen the networks available to our members so that they can generate new knowledge about microbes and ensure that it is shared with other communities.

A Minimal Threshold of c-di-GMP Is Essential for Fruiting Body Formation and Sporulation in Myxococcus xanthus.

Generally, the second messenger bis-(3’-5’)-cyclic dimeric GMP (c-di-GMP) regulates the switch between motile and sessile lifestyles in bacteria. Here, we show that c-di-GMP is an essential regulator of multicellular development in the social bacterium Myxococcus xanthus. In response to starvation, M. xanthus initiates a developmental program that culminates in formation of spore-filled fruiting bodies. We show that c-di-GMP accumulates at elevated levels during development and that this increase is essential for completion of development whereas excess c-di-GMP does not interfere with development. MXAN3735 (renamed DmxB) is identified as a diguanylate cyclase that only functions during development and is responsible for this increased c-di-GMP accumulation. DmxB synthesis is induced in response to starvation, thereby restricting DmxB activity to development. DmxB is essential for development and functions downstream of the Dif chemosensory system to stimulate exopolysaccharide accumulation by inducing transcription of a subset of the genes encoding proteins involved in exopolysaccharide synthesis. The developmental defects in the dmxB mutant are non-cell autonomous and rescued by co-development with a strain proficient in exopolysaccharide synthesis, suggesting reduced exopolysaccharide accumulation as the causative defect in this mutant. The NtrC-like transcriptional regulator EpsI/Nla24, which is required for exopolysaccharide accumulation, is identified as a c-di-GMP receptor, and thus a putative target for DmxB generated c-di-GMP. Because DmxB can be—at least partially—functionally replaced by a heterologous diguanylate cyclase, these results altogether suggest a model in which a minimum threshold level of c-di-GMP is essential for the successful completion of multicellular development in M. xanthus.

A gene encoding a potential adenosine 5'-phosphosulphate kinase is necessary for timely development of Myxococcus xanthus.

A Myxococcus xanthus gene, MXAN3487, was identified by transposon mutagenesis to be required for the expression of mcuABC, an operon coding for part of the chaperone-usher (CU) system in this bacterium. The MXAN3487 protein displays sequence and structural homology to adenosine 5'-phosphosulphate (APS) kinase family members and contains putative motifs for ATP and APS binding. Although the MXAN3487 locus is not linked to other sulphate assimilation genes, its protein product may have APS kinase activity in vivo and the importance of the ATP-binding site for activity was demonstrated. Expression of MXAN3487 was not affected by sulphate availability, suggesting that MXAN3487 may not function in a reductive sulphate assimilation pathway. Deletion of MXAN3487 significantly delayed fruiting body formation and the production of McuA, a spore coat protein secreted by the M. xanthus Mcu CU system. Based on these observations and data from our previous studies, we propose that MXAN3487 may phosphorylate molecules structurally related to APS, generating metabolites necessary for M. xanthus development, and that MXAN3487 exerts a positive effect on the mcuABC operon whose expression is morphogenesis dependent.

An iso-15 : 0 O-alkylglycerol moiety is the key structure of the E-signal in Myxococcus xanthus.

The E-signal is one of five intercellular signals (named A- to E-signal) guiding fruiting body development in Myxococcus xanthus, and it has been shown to be a combination of the branched-chain fatty acid (FA) iso-15 : 0 and the diacylmonoalkyl ether lipid TG1. Developmental mutants HB015 (Δbkd MXAN_4265::kan) and elbD (MXAN_1528::kan) are blocked at different stages of fruiting body and spore formation as they cannot form the required iso-FA or the actual ether lipid, respectively. In order to define the structural basis of the E-signal, different mono- and triglycerides containing ether or ester bonds were synthesized and used for complementation of these mutants. Here, the monoalkylglyceride dl-1-O-(13-methyltetradecyl)glycerol exhibited comparably high levels of complementation in both mutants, restoring fruiting body and spore formation, identifying iso-15 : 0 O-alkylglycerol, part of the natural lipid TG1, as the 'signalophore' of E-signalling.

Myxococcus CsgA, Drosophila Sniffer, and human HSD10 are cardiolipin phospholipases.

Myxococcus xanthus development requires CsgA, a member of the short-chain alcohol dehydrogenase (SCAD) family of proteins. We show that CsgA and SocA, a protein that can replace CsgA function in vivo, oxidize the 2'-OH glycerol moiety on cardiolipin and phosphatidylglycerol to produce diacylglycerol (DAG), dihydroxyacetone, and orthophosphate. A lipid extract enriched in DAGs from wild-type cells initiates development and lipid body production in a csgA mutant to bypass the mutational block. This novel phospholipase C-like reaction is widespread. SCADs that prevent neurodegenerative disorders, such as Drosophila Sniffer and human HSD10, oxidize cardiolipin with similar kinetic parameters. HSD10 exhibits a strong preference for cardiolipin with oxidized fatty acids. This activity is inhibited in the presence of the amyloid β peptide. Three HSD10 variants associated with neurodegenerative disorders are inactive with cardiolipin. We suggest that HSD10 protects humans from reactive oxygen species by removing damaged cardiolipin before it induces apoptosis.

DevI is an evolutionarily young negative regulator of Myxococcus xanthus development.

During starvation-induced development of Myxococcus xanthus, thousands of rod-shaped cells form mounds in which they differentiate into spores. The dev locus includes eight genes followed by clustered regularly interspaced short palindromic repeats (CRISPRs), comprising a CRISPR-Cas system (Cas stands for CRISPR associated) typically involved in RNA interference. Mutations in devS or devR of a lab reference strain permit mound formation but impair sporulation. We report that natural isolates of M. xanthus capable of normal development are highly polymorphic in the promoter region of the dev operon. We show that the dev promoter is predicted to be nonfunctional in most natural isolates and is dispensable for development of a laboratory reference strain. Moreover, deletion of the dev promoter or the small gene immediately downstream of it, here designated devI (development inhibitor), suppressed the sporulation defect of devS or devR mutants in the lab strain. Complementation experiments and the result of introducing a premature stop codon in devI support a model in which DevRS proteins negatively autoregulate expression of devI, whose 40-residue protein product DevI inhibits sporulation if overexpressed. DevI appears to act in a cell-autonomous manner since experiments with conditioned medium and with cell mixtures gave no indication of extracellular effects. Strikingly, we report that devI is entirely absent from most M. xanthus natural isolates and was only recently integrated into the developmental programs of some lineages. These results provide important new insights into both the evolutionary history of the dev operon and its mechanistic role in M. xanthus sporulation. Certain mutations in the dev CRISPR-Cas (clustered regularly interspaced short palindromic repeat-associated) system of Myxococcus xanthus impair sporulation. The link between development and a CRISPR-Cas system has been a mystery. Surprisingly, DNA sequencing of natural isolates revealed that many appear to lack a functional dev promoter, yet these strains sporulate normally. Deletion of the dev promoter or the small gene downstream of it suppressed the sporulation defect of a lab strain with mutations in dev genes encoding Cas proteins. The results support a model in which the Cas proteins DevRS prevent overexpression of the small gene devI, which codes for an inhibitor of sporulation. Phylogenetic analysis of natural isolates suggests that devI and the dev promoter were only recently acquired in some lineages.

Identification of a putative flavin adenine dinucleotide-binding monooxygenase as a regulator for Myxococcus xanthus development.

Gene clusters coding for the chaperone/usher (CU) pathway are widely distributed in many important environmental and pathogenic microbes; however, information about the regulatory machineries controlling CU gene expression during multicellular morphogenesis is missing. The Myxococcus xanthus Mcu system, encoded by the mcuABCD gene cluster, represents a prototype of the archaic CU family that functions in spore coat formation. Using genome-wide transposon mutagenesis, we identified MXAN2872 to be a potential regulator of the mcuABC operon and demonstrated the necessity of MXAN2872 for mcuABC expression and fruiting body morphogenesis in early development. In silico, biochemical, and genetic analyses suggest that MXAN2872 encodes a Baeyer-Villiger monooxygenase (BVMO) of flavoproteins, and the potential cofactor-binding site as well as the BVMO fingerprint sequence is important for the regulatory role of the MXAN2872 protein. The expression profile of mcuA in strains with an MXAN2872 deletion and point mutation agrees well with the timing of cell aggregation of these mutants. Furthermore, McuA could not be detected either in a fruA-null mutant, where starvation-induced aggregation was completely blocked, or in the glycerol-induced spores, where sporulation was uncoupled from cell aggregation. In sum, the present work uncovers a positive role for MXAN2872, a metabolic enzyme-encoding gene, in controlling M. xanthus development. MXAN2872 functions by affecting the onset of cell aggregation, thereby leading to a secondary effect on the timing of mcuABC expression of this model organism. Identification of the players that drive Myxococcus xanthus fruiting body formation is necessary for studying the mechanism of multicellular morphogenesis in this model organism. This study identifies MXAN2872, a gene encoding a putative flavin adenine dinucleotide-binding monooxygenase, to be a new interesting regulator regulating the timing of developmental aggregation. In addition, MXAN2872 seems to affect the expression of the chaperone/usher gene cluster mcu in a manner that is aggregation dependent. Thus, in organisms characterized by a developmental cycle, expression of the chaperone/usher pathway can be controlled by morphological checkpoints, demonstrating another layer of complexity in the regulation of this conserved protein secretion pathway.

Identification of a mutant locus that bypasses the BsgA protease requirement for social development in Myxococcus xanthus.

The BsgA protease is required for the earliest morphological changes observed in Myxococcus xanthus development. We hypothesize that the BsgA protease is required to cleave an inhibitor of the developmental program, and isolation of genetic bypass suppressors of a bsgA mutant was used to identify signaling components controlling development downstream of the BsgA protease. Strain M955 was created by transposon mutagenesis of a bsgA mutant followed by screening for strains that could develop despite the absence of the BsgA protease. Strain M955 was able to aggregate, form fruiting bodies, and partially restored the production of viable spores in comparison to the parental bsgA mutant. The bsgA Tn5Ω955 strain partially restored developmental expression to a subset of genes normally induced during development, and expressed one developmentally induced fusion at higher amounts during vegetative growth in comparison to wild-type cells. The transposon in strain M955 was localized to a Ribonuclease D homolog that appears to exist in an operon with a downstream aminopeptidase-encoding gene. The identification of a third distinct bypass suppressor of the BsgA protease suggests that the BsgA protease may regulate a potentially complex pathway during the initiation of the M. xanthus developmental program.

Combinatorial regulation of the dev operon by MrpC2 and FruA during Myxococcus xanthus development.

Proper expression of the dev operon is important for normal development of Myxococcus xanthus. When starved, these bacteria coordinate their gliding movements to build mounds that become fruiting bodies as some cells differentiate into spores. Mutations in the devTRS genes impair sporulation. Expression of the operon occurs within nascent fruiting bodies and depends in part on C signaling. Here, we report that expression of the dev operon, like that of several other C-signal-dependent genes, is subject to combinatorial control by the transcription factors MrpC2 and FruA. A DNA fragment upstream of the dev promoter was bound by a protein in an extract containing MrpC2, protecting the region spanning positions -77 to -54. Mutations in this region impaired binding of purified MrpC2 and abolished developmental expression of reporter fusions. The association of MrpC2 and/or its longer form, MrpC, with the dev promoter region depended on FruA in vivo, based on chromatin immunoprecipitation analysis, and purified FruA appeared to bind cooperatively with MrpC2 to DNA just upstream of the dev promoter in vitro. We conclude that cooperative binding of the two proteins to this promoter-proximal site is crucial for dev expression. 5' deletion analysis implied a second upstream positive regulatory site, which corresponded to a site of weak cooperative binding of MrpC2 and FruA and boosted dev expression 24 h into development. This site is unique among the C-signal-dependent genes studied so far. Deletion of this site in the M. xanthus chromosome did not impair sporulation under laboratory conditions.

Nutrient-regulated proteolysis of MrpC halts expression of genes important for commitment to sporulation during Myxococcus xanthus development.

Starved Myxococcus xanthus cells glide to aggregation centers and form fruiting bodies in which rod-shaped cells differentiate into ovoid spores. Commitment to development was investigated by adding nutrients at specific times after starvation and determining whether development halted or proceeded. At 24 h poststarvation, some rod-shaped cells were committed to subsequent shape change and to becoming sonication-resistant spores, but nutrients caused partial disaggregation of fruiting bodies. By 30 h poststarvation, 10-fold more cells were committed to becoming sonication-resistant spores, and compact fruiting bodies persisted after nutrient addition. During the critical period of commitment around 24 to 30 h poststarvation, the transcription factors MrpC and FruA cooperatively regulate genes important for sporulation. FruA responds to short-range C-signaling, which increases as cells form fruiting bodies. MrpC was found to be highly sensitive to nutrient-regulated proteolysis both before and during the critical period of commitment to sporulation. The rapid turnover of MrpC upon nutrient addition to developing cells halted expression of the dev operon, which is important for sporulation. Regulated proteolysis of MrpC appeared to involve ATP-independent metalloprotease activity and may provide a mechanism for monitoring whether starvation persists and halting commitment to sporulation if nutrients reappear.