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 in vitro, 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.

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.

Conserved Proline Residues of Bacillus subtilis Intramembrane Metalloprotease SpoIVFB Are Important for Substrate Interaction and Cleavage.

Intramembrane metalloproteases (IMMPs) regulate diverse biological processes by cleaving membrane-associated substrates within the membrane or near its surface. SpoIVFB is an intramembrane metalloprotease of Bacillus subtilis that cleaves Pro-σK during endosporulation. Intramembrane metalloproteases have a broadly conserved NPDG motif, which in the structure of an archaeal enzyme is located in a short loop that interrupts a transmembrane segment facing the active site. The aspartate residue of the NPDG motif acts as a ligand of the zinc ion involved in catalysis. The functions of other residues in the short loop are less well understood. We found that the predicted short loop of SpoIVFB contains two highly conserved proline residues, P132 of the NPDG motif and P135. Mutational analysis revealed that both proline residues are important for Pro-σK cleavage in Escherichia coli engineered to synthesize the proteins. Substitutions for either residue also impaired the Pro-σK interaction with SpoIVFB in copurification assays. Disulfide cross-linking experiments showed that the predicted short loop of SpoIVFB is in proximity to the N-terminal pro-sequence region (Proregion) of Pro-σK. Alanine substitutions for N129 and P132 of the SpoIVFB NPDG motif reduced cross-linking between its predicted short loop and the Proregion more than a P135A substitution. Conversely, the SpoIVFB P135A substitution reduced Pro-σK cleavage more than the N129A and P132A substitutions during sporulation of B. subtilis. We conclude that all three conserved residues of SpoIVFB are important for substrate interaction and cleavage, and we propose that P135 is necessary to position D137 to act as a zinc ligand. IMPORTANCE Intramembrane metalloproteases (IMMPs) function in numerous signaling pathways. Bacterial IMMPs govern stress responses, including the sporulation of some species, thus enhancing the virulence and persistence of pathogens. Knowledge of IMMP-substrate interactions could aid therapeutic design, but structures of IMMP·substrate complexes are unknown. We examined the interaction of the IMMP SpoIVFB with its substrate Pro-σK, whose cleavage is required for Bacillus subtilis endosporulation. We found that conserved proline residues in a short loop predicted to interrupt a SpoIVFB transmembrane segment are important for Pro-σK binding and cleavage. The corresponding residues of the Escherichia coli IMMP RseP have also been shown to be important for substrate interaction and cleavage, suggesting that this is a broadly conserved feature of IMMPs, potentially suitable as a therapeutic target.

Channels modestly impact compartment-specific ATP levels during Bacillus subtilis sporulation and a rise in the mother cell ATP level is not necessary for Pro-σK cleavage.

Starvation of Bacillus subtilis initiates endosporulation involving formation of mother cell (MC) and forespore (FS) compartments. During engulfment, the MC membrane migrates around the FS and protein channels connect the two compartments. The channels are necessary for postengulfment FS gene expression, which relieves inhibition of SpoIVFB, an intramembrane protease that cleaves Pro-σK , releasing σK into the MC. SpoIVFB has an ATP-binding domain exposed to the MC cytoplasm, but the role of ATP in regulating Pro-σK cleavage has been unclear, as has the impact of the channels on MC and FS ATP levels. Using luciferase produced separately in each compartment to measure relative ATP concentrations during sporulation, we found that the MC ATP concentration rises about twofold coincident with increasing cleavage of Pro-σK , and the FS ATP concentration does not decline. Mutants lacking a channel protein or defective in channel protein turnover exhibited modest and varied effects on ATP levels, which suggested that low ATP concentration does not explain the lack of postengulfment FS gene expression in channel mutants. Furthermore, a rise in the MC ATP level was not necessary for Pro-σK cleavage by SpoIVFB, based on analysis of mutants that bypass the need for relief of SpoIVFB inhibition.

Systematic analysis of the Myxococcus xanthus developmental gene regulatory network supports posttranslational regulation of FruA by C-signaling.

Upon starvation Myxococcus xanthus undergoes multicellular development. Rod-shaped cells move into mounds in which some cells differentiate into spores. Cells begin committing to sporulation at 24-30 h poststarvation, but the mechanisms governing commitment are unknown. FruA and MrpC are transcription factors that are necessary for commitment. They bind cooperatively to promoter regions and activate developmental gene transcription, including that of the dev operon. Leading up to and during the commitment period, dev mRNA increased in wild type, but not in a mutant defective in C-signaling, a short-range signaling interaction between cells that is also necessary for commitment. The C-signaling mutant exhibited ~20-fold less dev mRNA than wild type at 30 h poststarvation, despite a similar level of MrpC and only 2-fold less FruA. Boosting the FruA level twofold in the C-signaling mutant had little effect on the dev mRNA level, and dev mRNA was not less stable in the C-signaling mutant. Neither did high cooperativity of MrpC and FruA binding upstream of the dev promoter explain the data. Rather, our systematic experimental and computational analyses support a model in which C-signaling activates FruA at least ninefold posttranslationally in order to commit a cell to spore formation.

Highly Signal-Responsive Gene Regulatory Network Governing Myxococcus Development.

The bacterium Myxococcus xanthus undergoes multicellular development when starved. Thousands of cells build mounds in which some differentiate into spores. This remarkable feat and the genetic tractability of Myxococcus provide a unique opportunity to understand the evolution of gene regulatory networks (GRNs). Recent work has revealed a GRN involving interconnected cascades of signal-responsive transcriptional activators. Initially, starvation-induced intracellular signals direct changes in gene expression. Subsequently, self-generated extracellular signals provide morphological cues that regulate certain transcriptional activators. However, signals for many of the activators remain to be discovered. A key insight is that activators often work combinatorially, allowing signal integration. The Myxococcus GRN differs strikingly from those governing sporulation of Bacillus and Streptomyces, suggesting that Myxococcus evolved a highly signal-responsive GRN to enable complex multicellular development.

Interaction of intramembrane metalloprotease SpoIVFB with substrate Pro-σK.

Intramembrane proteases (IPs) cleave membrane-associated substrates in nearly all organisms and regulate diverse processes. A better understanding of how these enzymes interact with their substrates is necessary for rational design of IP modulators. We show that interaction of Bacillus subtilis IP SpoIVFB with its substrate Pro-σK depends on particular residues in the interdomain linker of SpoIVFB. The linker plus either the N-terminal membrane domain or the C-terminal cystathione-ß-synthase (CBS) domain of SpoIVFB was sufficient for the interaction but not for cleavage of Pro-σK Chemical cross-linking and mass spectrometry of purified, inactive SpoIVFB-Pro-σK complex indicated residues of the two proteins in proximity. A structural model of the complex was built via partial homology and by using constraints based on cross-linking data. In the model, the Proregion of Pro-σK loops into the membrane domain of SpoIVFB, and the rest of Pro-σK interacts extensively with the linker and the CBS domain of SpoIVFB. The extensive interaction is proposed to allow coordination between ATP binding by the CBS domain and Pro-σK cleavage by the membrane domain.

Ultrasensitive Response of Developing Myxococcus xanthus to the Addition of Nutrient Medium Correlates with the Level of MrpC.

Upon depletion of nutrients, Myxococcus xanthus forms mounds on a solid surface. The differentiation of rod-shaped cells into stress-resistant spores within mounds creates mature fruiting bodies. The developmental process can be perturbed by the addition of nutrient medium before the critical period of commitment to spore formation. The response was investigated by adding a 2-fold dilution series of nutrient medium to starving cells. An ultrasensitive response was observed, as indicated by a steep increase in the spore number after the addition of 12.5% versus 25% nutrient medium. The level of MrpC, which is a key transcription factor in the gene regulatory network, correlated with the spore number after nutrient medium addition. The MrpC level decreased markedly by 3 h after adding nutrient medium but recovered more after the addition of 12.5% than after 25% nutrient medium addition. The difference in MrpC levels was greatest midway during the period of commitment to sporulation, and mound formation was restored after 12.5% nutrient medium addition but not after adding 25% nutrient medium. Although the number of spores formed after 12.5% nutrient medium addition was almost normal, the transcript levels of "late" genes in the regulatory network failed to rise normally during the commitment period. However, at later times, expression from a reporter gene fused to a late promoter was higher after adding 12.5% than after adding 25% nutrient medium, consistent with the spore numbers. The results suggest that a threshold level of MrpC must be achieved in order for mounds to persist and for cells within to differentiate into spores.IMPORTANCE Many signaling and gene regulatory networks convert graded stimuli into all-or-none switch-like responses. Such ultrasensitivity can produce bistability in cell populations, leading to different cell fates and enhancing survival. We discovered an ultrasensitive response of M. xanthus to nutrient medium addition during development. A small change in nutrient medium concentration caused a profound change in the developmental process. The level of the transcription factor MrpC correlated with multicellular mound formation and differentiation into spores. A threshold level of MrpC is proposed to be necessary to initiate mound formation and create a positive feedback loop that may explain the ultrasensitive response. Understanding how this biological switch operates will provide a paradigm for the broadly important topic of cellular behavior in microbial communities.

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.

Bacillus subtilis Intramembrane Protease RasP Activity in Escherichia coli and In Vitro.

RasP is a predicted intramembrane metalloprotease of Bacillus subtilis that has been proposed to cleave the stress response anti-sigma factors RsiW and RsiV, the cell division protein FtsL, and remnant signal peptides within their transmembrane segments. To provide evidence for direct effects of RasP on putative substrates, we developed a heterologous coexpression system. Since expression of catalytically inactive RasP E21A inhibited expression of other membrane proteins in Escherichia coli, we added extra transmembrane segments to RasP E21A, which allowed accumulation of most other membrane proteins. A corresponding active version of RasP appeared to promiscuously cleave coexpressed membrane proteins, except those with a large periplasmic domain. However, stable cleavage products were not observed, even in clpP mutant E. coli Fusions of transmembrane segment-containing parts of FtsL and RsiW to E. coli maltose-binding protein (MBP) also resulted in proteins that appeared to be RasP substrates upon coexpression in E. coli, including FtsL with a full-length C-terminal domain (suggesting that prior cleavage by a site 1 protease is unnecessary) and RsiW designed to mimic the PrsW site 1 cleavage product (suggesting that further trimming by extracytoplasmic protease is unnecessary). Purified RasP cleaved His6-MBP-RsiW(73-118) in vitro within the RsiW transmembrane segment based on mass spectrometry analysis, demonstrating that RasP is an intramembrane protease. Surprisingly, purified RasP failed to cleave His6-MBP-FtsL(23-117). We propose that the lack of α-helix-breaking residues in the FtsL transmembrane segment creates a requirement for the membrane environment and/or an additional protein(s) in order for RasP to cleave FtsL.IMPORTANCE Intramembrane proteases govern important signaling pathways in nearly all organisms. In bacteria, they function in stress responses, cell division, pathogenesis, and other processes. Their membrane-associated substrates are typically inferred from genetic studies in the native bacterium. Evidence for direct effects has come sometimes from coexpression of the enzyme and potential substrate in a heterologous host and rarely from biochemical reconstitution of cleavage in vitro We applied these two approaches to the B. subtilis enzyme RasP and its proposed substrates RsiW and FtsL. We discovered potential pitfalls and solutions in heterologous coexpression experiments in E. coli, providing evidence that both substrates are cleaved by RasP in vivo but, surprisingly, that only RsiW was cleaved in vitro, suggesting that FtsL has an additional requirement.

Complex Formed between Intramembrane Metalloprotease SpoIVFB and Its Substrate, Pro-σK.

Intramembrane metalloproteases (IMMPs) are conserved from bacteria to humans and control many important signaling pathways, but little is known about how IMMPs interact with their substrates. SpoIVFB is an IMMP that cleaves Pro-σ(K) during Bacillus subtilis endospore formation. When catalytically inactive SpoIVFB was coexpressed with C-terminally truncated Pro-σ(K)(1-126) (which can be cleaved by active SpoIVFB) in Escherichia coli, the substrate dramatically improved solubilization of the enzyme from membranes with mild detergents. Both the Pro(1-20) and σ(K)(21-126) parts contributed to improving SpoIVFB solubilization from membranes, but only the σ(K) part was needed to form a stable complex with SpoIVFB in a pulldown assay. The last 10 residues of SpoIVFB were required for improved solubilization from membranes by Pro-σ(K)(1-126) and for normal interaction with the substrate. The inactive SpoIVFB·Pro-σ(K)(1-126)-His6 complex was stable during affinity purification and gel filtration chromatography. Disulfide cross-linking of the purified complex indicated that it resembled the complex formed in vivo Ion mobility-mass spectrometry analysis resulted in an observed mass consistent with a 4:2 SpoIVFB·Pro-σ(K)(1-126)-His6 complex. Stepwise photobleaching of SpoIVFB fused to a fluorescent protein supported the notion that the enzyme is tetrameric during B. subtilis sporulation. The results provide the first evidence that an IMMP acts as a tetramer, give new insights into how SpoIVFB interacts with its substrate, and lay the foundation for further biochemical analysis of the enzyme·substrate complex and future structural studies.

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.

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

UNLABELLED: 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. IMPORTANCE: 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.

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.

Structure of bacterial transcription factor SpoIIID and evidence for a novel mode of DNA binding.

SpoIIID is evolutionarily conserved in endospore-forming bacteria, and it activates or represses many genes during sporulation of Bacillus subtilis. An SpoIIID monomer binds DNA with high affinity and moderate sequence specificity. In addition to a predicted helix-turn-helix motif, SpoIIID has a C-terminal basic region that contributes to DNA binding. The nuclear magnetic resonance (NMR) solution structure of SpoIIID in complex with DNA revealed that SpoIIID does indeed have a helix-turn-helix domain and that it has a novel C-terminal helical extension. Residues in both of these regions interact with DNA, based on the NMR data and on the effects on DNA binding in vitro of SpoIIID with single-alanine substitutions. These data, as well as sequence conservation in SpoIIID binding sites, were used for information-driven docking to model the SpoIIID-DNA complex. The modeling resulted in a single cluster of models in which the recognition helix of the helix-turn-helix domain interacts with the major groove of DNA, as expected. Interestingly, the C-terminal extension, which includes two helices connected by a kink, interacts with the adjacent minor groove of DNA in the models. This predicted novel mode of binding is proposed to explain how a monomer of SpoIIID achieves high-affinity DNA binding. Since SpoIIID is conserved only in endospore-forming bacteria, which include important pathogenic Bacilli and Clostridia, whose ability to sporulate contributes to their environmental persistence, the interaction of the C-terminal extension of SpoIIID with DNA is a potential target for development of sporulation inhibitors.

Biochemical and structural insights into intramembrane metalloprotease mechanisms.

Intramembrane metalloproteases are nearly ubiquitous in living organisms and they function in diverse processes ranging from cholesterol homeostasis and the unfolded protein response in humans to sporulation, stress responses, and virulence of bacteria. Understanding how these enzymes function in membranes is a challenge of fundamental interest with potential applications if modulators can be devised. Progress is described toward a mechanistic understanding, based primarily on molecular genetic and biochemical studies of human S2P and bacterial SpoIVFB and RseP, and on the structure of the membrane domain of an archaeal enzyme. Conserved features of the enzymes appear to include transmembrane helices and loops around the active site zinc ion, which may be near the membrane surface. Extramembrane domains such as PDZ (PSD-95, DLG, ZO-1) or CBS (cystathionine-ß-synthase) domains govern substrate access to the active site, but several different mechanisms of access and cleavage site selection can be envisioned, which might differ depending on the substrate and the enzyme. More work is needed to distinguish between these mechanisms, both for enzymes that have been relatively well-studied, and for enzymes lacking PDZ and CBS domains, which have not been studied. This article is part of a Special Issue entitled: Intramembrane Proteases.

Transcription factor MrpC binds to promoter regions of hundreds of developmentally-regulated genes in Myxococcus xanthus.

BACKGROUND: Myxococcus 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. RESULTS: Chromatin 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. CONCLUSIONS: We 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.

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

Myxococcus xanthus uses short-range C-signaling to coordinate multicellular mound formation with sporulation during fruiting body development. A csgA mutant deficient in C-signaling can cheat on wild type (WT) in mixtures and form spores disproportionately, but our understanding of cheating behavior is incomplete. We subjected mixtures of WT and csgA cells at different ratios to co-development and used confocal microscopy and image analysis to quantify the arrangement and morphology of cells. At a ratio of one WT to four csgA cells (1:4), mounds failed to form. At 1:2, only a few mounds and spores formed. At 1:1, mounds formed with a similar number and arrangement of WT and csgA rods early in development, but later the number of csgA spores near the bottom of these nascent fruiting bodies (NFBs) exceeded that of WT. This cheating after mound formation involved csgA forming spores at a greater rate, while WT disappeared at a greater rate, either lysing or exiting NFBs. At 2:1 and 4:1, csgA rods were more abundant than expected throughout the biofilm both before and during mound formation, and cheating continued after mound formation. We conclude that C-signaling restricts cheating behavior by requiring sufficient WT cells in mixtures. Excess cheaters may interfere with positive feedback loops that depend on the cellular arrangement to enhance C-signaling during mound building. Since long-range signaling could not likewise communicate the cellular arrangement, we propose that C-signaling was favored evolutionarily and that other short-range signaling mechanisms provided selective advantages in bacterial biofilm and multicellular animal development. IMPORTANCE: 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.

Substrate engagement by the intramembrane metalloprotease SpoIVFB.

S2P intramembrane metalloproteases regulate diverse signaling pathways across all three domains of life. However, the mechanism by which S2P metalloproteases engage substrates and catalyze peptide hydrolysis within lipid membranes has remained elusive. Here we determine the cryo-EM structure of the S2P family intramembrane metalloprotease SpoIVFB from Bacillus subtilis bound to its native substrate Pro-σK. The structure and accompanying biochemical data demonstrate that SpoIVFB positions Pro-σK at the enzyme active site through a ß-sheet augmentation mechanism, and reveal key interactions between Pro-σK and the interdomain linker connecting SpoIVFB transmembrane and CBS domains. The cryo-EM structure and molecular dynamics simulation reveal a plausible path for water to access the membrane-buried active site of SpoIVFB, and suggest a possible role of membrane lipids in facilitating substrate capture. These results provide key insight into how S2P intramembrane metalloproteases capture and position substrates for hydrolytic proteolysis within the hydrophobic interior of a lipid membrane.

Residues in conserved loops of intramembrane metalloprotease SpoIVFB interact with residues near the cleavage site in pro-σK.

Intramembrane metalloproteases (IMMPs) control critical biological processes by cleaving membrane-associated proteins within a transmembrane segment or at a site near the membrane surface. Phylogenetic analysis divides IMMPs into four groups. SpoIVFB is a group III IMMP that regulates Bacillus subtilis endospore formation by cleaving Pro-σ(K) and releasing the active sigma factor from a membrane. To elucidate the enzyme-substrate interaction, single-cysteine versions of catalytically inactive SpoIVFB and C-terminally truncated Pro-σ(K)(1-126) (which can be cleaved by active SpoIVFB) were coexpressed in Escherichia coli, and proximity was tested by disulfide cross-linking in vivo. As expected, the results provided evidence that catalytic residue Glu-44 of SpoIVFB is near the cleavage site in the substrate. Also near the cleavage site were two residues of SpoIVFB in predicted conserved loops; Pro-135 in a short loop and Val-70 in a longer loop. Pro-135 corresponds to Pro-399 of RseP, a group I IMMP, and Pro-399 was reported previously to interact with substrate near the cleavage site, suggesting a conserved interaction across IMMP subfamilies. Val-70 follows a newly recognized conserved motif, PXGG (X is a large hydrophobic residue), which is in a hydrophobic region predicted to be a membrane reentrant loop. Following the hydrophobic region is a negatively charged region that is conserved in IMMPs of groups I and III. At least two residues with a negatively charged side chain are required in this region for activity of SpoIVFB. The region exhibits other features in IMMPs of groups II and IV. Its possible roles, as well as that of the short loop, are discussed. New insights into IMMP-substrate interaction build toward understanding how IMMPs function and may facilitate manipulation of their activity.