Formation of a stable RNase Y-RicT (YaaT) complex requires RicA (YmcA) and RicF (YlbF).

In Bacillus subtilis, the RicT (YaaT), RicA (YmcA), and RicF (YlbF) proteins, which form a stable ternary complex, are needed together with RNase Y (Rny) to cleave and thereby stabilize several key transcripts encoding enzymes of intermediary metabolism. We show here that RicT, but not RicA or RicF, forms a stable complex with Rny and that this association requires the presence of RicA and RicF. We propose that RicT is handed off from the ternary complex to Rny. We show further that the two iron-sulfur clusters carried by the ternary Ric complex are required for the formation of the stable RicT-Rny complex. We demonstrate that proteins of the degradosome-like network of B. subtilis, which also interact with Rny, are dispensable for processing of the gapA operon. Thus, Rny participates in distinct RNA-related processes, determined by its binding partners, and a RicT-Rny complex is likely the functional entity for gapA mRNA maturation. IMPORTANCE The action of nucleases on RNA is universal and essential for all forms of life and includes processing steps that lead to the mature and functional forms of certain transcripts. In Bacillus subtilis, it has been shown that key transcripts for energy-producing steps of glycolysis, for nitrogen assimilation, and for oxidative phosphorylation, all of them crucial processes of intermediary metabolism, are cleaved at specific locations, resulting in mRNA stabilization. The proteins required for these cleavages in B. subtilis [Rny (RNase Y), RicA (YmcA), RicF (YlbF), and RicT (YaaT)] are broadly conserved among the firmicutes, including several important pathogens, hinting that regulatory mechanisms they control may also be conserved. Several aspects of these regulatory events have been explored: phenotypes associated with the absence of these proteins have been described, the impact of these absences on the transcriptome has been documented, and there has been significant exploration of the biochemistry and structural biology of Rny and the Ric proteins. The present study further advances our understanding of the association of Ric proteins and Rny and shows that a complex of Rny with RicT is probably the entity that carries out mRNA maturation.

Formation of a stable RNase Y-RicT (YaaT) complex requires RicA (YmcA) and RicF (YlbF).

In Bacillus subtilis , the RicT (YaaT), RicA (YmcA) and RicF (YlbF) proteins, which form a stable ternary complex, are needed together with RNase Y (Rny), to cleave and thereby stabilize several key transcripts encoding enzymes of intermediary metabolism. We show here that RicT, but not RicA or RicF, forms a stable complex with Rny, and that this association requires the presence of RicA and RicF. We propose that RicT is handed off from the ternary complex to Rny. We show further that the two iron-sulfur clusters carried by the ternary Ric complex are required for the formation of the stable RicT-Rny complex. We demonstrate that proteins of the degradosome-like network of B. subtilis , which also interact with Rny, are dispensable for processing of the gapA operon. Thus, Rny participates in distinct RNA-related processes, determined by its binding partners, and a RicT-Rny complex is likely the functional entity for gapA mRNA maturation. IMPORTANCE: The action of nucleases on RNA is universal and essential for all forms of life and includes processing steps that lead to the mature and functional forms of certain transcripts. In B. subtilis it has been shown that key transcripts for energy producing steps of glycolysis, for nitrogen assimilation and for oxidative phosphorylation, all of them crucial processes of intermediary metabolism, are cleaved at specific locations, resulting in mRNA stabilization. The proteins required for these cleavages in B. subtilis (Rny (RNase Y), RicA (YmcA), RicF (YlbF) and RicT (YaaT)) are broadly conserved among the firmicutes, including in several important pathogens, hinting that regulatory mechanisms they control may also be conserved. Several aspects of these regulatory events have been explored: phenotypes associated with the absence of these proteins have been described, the impact of these absences on the transcriptome has been documented, and there has been significant exploration of the biochemistry and structural biology of Rny and the Ric proteins. The present study further advances our understanding of the association of Ric proteins and Rny and shows that a complex of Rny with RicT is probably the entity that carries out mRNA maturation.

Structure-function studies reveal ComEA contains an oligomerization domain essential for transformation in gram-positive bacteria.

An essential step in bacterial transformation is the uptake of DNA into the periplasm, across the thick peptidoglycan cell wall of Gram-positive bacteria, or the outer membrane and thin peptidoglycan layer of Gram-negative bacteria. ComEA, a DNA-binding protein widely conserved in transformable bacteria, is required for this uptake step. Here we determine X-ray crystal structures of ComEA from two Gram-positive species, Bacillus subtilis and Geobacillus stearothermophilus, identifying a domain that is absent in Gram-negative bacteria. X-ray crystallographic, genetic, and analytical ultracentrifugation (AUC) analyses reveal that this domain drives ComEA oligomerization, which we show is required for transformation. We use multi-wavelength AUC (MW-AUC) to characterize the interaction between DNA and the ComEA DNA-binding domain. Finally, we present a model for the interaction of the ComEA DNA-binding domain with DNA, suggesting that ComEA oligomerization may provide a pulling force that drives DNA uptake across the thick cell walls of Gram-positive bacteria.

Mechanisms of Transforming DNA Uptake to the Periplasm of Bacillus subtilis.

We demonstrate here that the acquisition of DNase resistance by transforming DNA, often assumed to indicate transport to the cytoplasm, reflects uptake to the periplasm, requiring a reevaluation of conclusions about the roles of several proteins in transformation. The new evidence suggests that the transformation pilus is needed for DNA binding to the cell surface near the cell poles and for the initiation of uptake. The cellular distribution of the membrane-anchored ComEA of Bacillus subtilis does not dramatically change during DNA uptake as does the unanchored ComEA of Vibrio and Neisseria. Instead, our evidence suggests that ComEA stabilizes the attachment of transforming DNA at localized regions in the periplasm and then mediates uptake, probably by a Brownian ratchet mechanism. Following that, the DNA is transferred to periplasmic portions of the channel protein ComEC, which plays a previously unsuspected role in uptake to the periplasm. We show that the transformation endonuclease NucA also facilitates uptake to the periplasm and that the previously demonstrated role of ComFA in the acquisition of DNase resistance derives from the instability of ComGA when ComFA is deleted. These results prompt a new understanding of the early stages of DNA uptake for transformation. IMPORTANCE Transformation is a widely distributed mechanism of bacterial horizontal gene transfer that plays a role in the spread of antibiotic resistance and virulence genes and more generally in evolution. Although transformation was discovered nearly a century ago and most, if not all the proteins required have been identified in several bacterial species, much remains poorly understood about the molecular mechanism of DNA uptake. This study uses epifluorescence microscopy to investigate the passage of labeled DNA into the compartment between the cell wall and the cell membrane of Bacillus subtilis, a necessary early step in transformation. The roles of individual proteins in this process are identified, and their modes of action are clarified.

ComEB protein is dispensable for the transformation but must be translated for the optimal synthesis of comEC.

We show that the ComEB protein is not required for transformation in Bacillus subtilis, despite its expression from within the comE operon under competence control, nor is it required for the correct polar localization of ComGA. We show further that the synthesis of the putative channel protein ComEC is translationally coupled to the upstream comEB open reading frame, so that the translation of comEB and a suboptimal ribosomal-binding site embedded in its sequence are needed for proper comEC expression. Translational coupling appears to be a common mechanism in three major competence operons for the adjustment of protein amounts independent of transcriptional control, probably ensuring the correct stoichiometries for assembly of the transformation machinery. comEB and comFC, respectively, encode cytidine deaminase and a protein resembling type 1 phosphoribosyl transferases and we speculate that nucleotide scavenging proteins are produced under competence control for efficient reutilization of the products of degradation of the non-transforming strand during DNA uptake.

Structure-Function Studies of the Bacillus subtilis Ric Proteins Identify the Fe-S Cluster-Ligating Residues and Their Roles in Development and RNA Processing.

In Bacillus subtilis, the RicA (YmcA), RicF (YlbF), and RicT (YaaT) proteins accelerate the phosphorylation of the transcription factor Spo0A, contributing to genetic competence, sporulation, and biofilm formation, and are also essential for the correct maturation of several protein-encoding and riboswitch RNAs. These proteins form a stable complex (RicAFT) that carries two [4Fe-4S]+2 clusters. We show here that the complex is a 1:1:1 heterotrimer, and we present the X-ray crystal structures of a RicAF heterotetramer and of a RicA dimer. We also demonstrate that one of the Fe-S clusters (cluster 1) is ligated by cysteine residues donated exclusively by RicT and can be retained when the RicT monomer is purified by itself. Cluster 2 is ligated by C167 from RicT, by C134 and C146 located near the C terminus of RicF, and by C141 at the C terminus of RicA. These findings imply the following novel arrangement: adjacent RicT residues C166 and 167 ligate clusters 1 and 2, respectively, while cluster 2 is ligated by cysteine residues from RicT, RicA, and RicF. Thus, the two clusters must lie close to one another and at the interface of the RicAFT protomers. We also show that the cluster-ligating cysteine residues, and therefore most likely both Fe-S clusters, are essential for cggR-gapA mRNA maturation, for the regulation of ricF transcript stability, and for several Ric-associated developmental phenotypes, including competence for transformation, biofilm formation, and sporulation. Finally, we present evidence that RicAFT, RicAF, and RicA and the RicT monomer may play distinct regulatory roles in vivoIMPORTANCE The RicA, RicF, and RicT proteins are widely conserved among the firmicute bacteria and play multiple roles in Bacillus subtilis Among the phenotypes associated with the inactivation of these proteins are the inability to be genetically transformed or to form biofilms, a decrease in sporulation frequency, and changes in the stability and maturation of multiple RNA species. Despite their importance, the molecular mechanisms of Ric protein activities have not been elucidated and the roles of the two iron-sulfur clusters on the complex of the three proteins are not understood. To unravel the mechanisms of Ric action, molecular characterization of the complex and of its constituent proteins is essential. This report represents a major step toward understanding the structures of the Ric proteins, the arrangement and roles of the Fe-S clusters, and the phenotypes associated with Ric mutations.

Mechanisms of DNA Uptake by Naturally Competent Bacteria.

Transformation is a widespread mechanism of horizontal gene transfer in bacteria. DNA uptake to the periplasmic compartment requires a DNA-uptake pilus and the DNA-binding protein ComEA. In the gram-negative bacteria, DNA is first pulled toward the outer membrane by retraction of the pilus and then taken up by binding to periplasmic ComEA, acting as a Brownian ratchet to prevent backward diffusion. A similar mechanism probably operates in the gram-positive bacteria as well, but these systems have been less well characterized. Transport, defined as movement of a single strand of transforming DNA to the cytosol, requires the channel protein ComEC. Although less is understood about this process, it may be driven by proton symport. In this review we also describe various phenomena that are coordinated with the expression of competence for transformation, such as fratricide, the kin-discriminatory killing of neighboring cells, and competence-mediated growth arrest.

YfmK is an Nε-lysine acetyltransferase that directly acetylates the histone-like protein HBsu in Bacillus subtilis.

Nε-lysine acetylation is an abundant and dynamic regulatory posttranslational modification that remains poorly characterized in bacteria. In bacteria, hundreds of proteins are known to be acetylated, but the biological significance of the majority of these events remains unclear. Previously, we characterized the Bacillus subtilis acetylome and found that the essential histone-like protein HBsu contains seven previously unknown acetylation sites in vivo. Here, we investigate whether acetylation is a regulatory component of the function of HBsu in nucleoid compaction. Using mutations that mimic the acetylated and unacetylated forms of the protein, we show that the inability to acetylate key HBsu lysine residues results in a more compacted nucleoid. We further investigated the mechanism of HBsu acetylation. We screened deletions of the ∼50 putative GNAT domain-encoding genes in B. subtilis for their effects on DNA compaction, and identified five candidates that may encode acetyltransferases acting on HBsu. Genetic bypass experiments demonstrated that two of these, YfmK and YdgE, can acetylate Hbsu, and their potential sites of action on HBsu were identified. Additionally, purified YfmK was able to directly acetylate HBsu in vitro, suggesting that it is the second identified protein acetyltransferase in B. subtilis We propose that at least one physiological function of the acetylation of HBsu at key lysine residues is to regulate nucleoid compaction, analogous to the role of histone acetylation in eukaryotes.

ClpC and MecA, components of a proteolytic machine, prevent Spo0A-P-dependent transcription without degradation.

In Bacillus subtilis, a proteolytic machine composed of MecA, ClpC and ClpP degrades the transcription factor ComK, controlling its accumulation during growth. MecA also inhibits sporulation and biofilm formation by down-regulating spoIIG and sinI, genes that are dependent for their transcription on the phosphorylated protein Spo0A-P. Additionally, MecA has been shown to interact in vitro with Spo0A. Although the inhibitory effect on transcription requires MecA's binding partner ClpC, inhibition is not accompanied by the degradation of Spo0A, pointing to a previously unsuspected regulatory mechanism involving these proteins. Here, we further investigate the MecA and ClpC effects on Spo0A-P-dependent transcription. We show that MecA inhibits the transcription of several Spo0A-P activated genes, but fails to de-repress several Spo0A-P repressed promoters. This demonstrates that MecA and ClpC do not act by preventing the binding of Spo0A-P to its target promoters. Consistent with this, MecA by itself has no effect in vitro on the transcription from PspoIIG while the addition of both MecA and ClpC has a strong inhibitory effect. A complex of MecA and ClpC likely binds to Spo0A-P on its target promoters, preventing the activation of transcription. Thus, components of a degradative machine have been harnessed to directly repress transcription.

Viscous drag on the flagellum activates Bacillus subtilis entry into the K-state.

Bacillus subtilis flagella are not only required for locomotion but also act as sensors that monitor environmental changes. Although how the signal transmission takes place is poorly understood, it has been shown that flagella play an important role in surface sensing by transmitting a mechanical signal to control the DegS-DegU two-component system. Here we report a role for flagella in the regulation of the K-state, which enables transformability and antibiotic tolerance (persistence). Mutations impairing flagellar synthesis are inferred to increase DegU-P, which inhibits the expression of ComK, the master regulator for the K-state, and reduces transformability. Tellingly, both deletion of the flagellin gene and straight filament (hagA233V ) mutations increased DegU phosphorylation despite the fact that both mutants had wild type numbers of basal bodies and the flagellar motors were functional. We propose that higher viscous loads on flagellar motors result in lower DegU-P levels through an unknown signaling mechanism. This flagellar-load based mechanism ensures that cells in the motile subpopulation have a tenfold enhanced likelihood of entering the K-state and taking up DNA from the environment. Further, our results suggest that the developmental states of motility and competence are related and most commonly occur in the same epigenetic cell type.

The RicAFT (YmcA-YlbF-YaaT) complex carries two [4Fe-4S]2+ clusters and may respond to redox changes.

During times of environmental insult, Bacillus subtilis undergoes developmental changes leading to biofilm formation, sporulation and competence. Each of these states is regulated in part by the phosphorylated form of the master response regulator Spo0A (Spo0A∼P). The phosphorylation state of Spo0A is controlled by a multi-component phosphorelay. RicA, RicF and RicT (previously YmcA, YlbF and YaaT) have been shown to be important regulatory proteins for multiple developmental fates. These proteins directly interact and form a stable complex, which has been proposed to accelerate the phosphorelay. Indeed, this complex is sufficient to stimulate the rate of phosphotransfer amongst the phosphorelay proteins in vitro. In this study, we demonstrate that two [4Fe-4S]2+ clusters can be assembled on the complex. As with other iron-sulfur cluster-binding proteins, the complex was also found to bind FAD, hinting that these cofactors may be involved in sensing the cellular redox state. This work provides the first comprehensive characterization of an iron-sulfur protein complex that regulates Spo0A∼P levels. Phylogenetic and genetic evidence suggests that the complex plays a broader role beyond stimulation of the phosphorelay.

A DegU-P and DegQ-Dependent Regulatory Pathway for the K-state in Bacillus subtilis.

The K-state in the model bacterium Bacillus subtilis is associated with transformability (competence) as well as with growth arrest and tolerance for antibiotics. Entry into the K-state is determined by the stochastic activation of the transcription factor ComK and occurs in about ∼15% of the population in domesticated strains. Although the upstream mechanisms that regulate the K-state have been intensively studied and are well understood, it has remained unexplained why undomesticated isolates of B. subtilis are poorly transformable compared to their domesticated counterparts. We show here that this is because fewer cells enter the K-state, suggesting that a regulatory pathway limiting entry to the K-state is missing in domesticated strains. We find that loss of this limitation is largely due to an inactivating point mutation in the promoter of degQ. The resulting low level of DegQ decreases the concentration of phosphorylated DegU, which leads to the de-repression of the srfA operon and ultimately to the stabilization of ComK. As a result, more cells reach the threshold concentration of ComK needed to activate the auto-regulatory loop at the comK promoter. In addition, we demonstrate that the activation of srfA transcription in undomesticated strains is transient, turning off abruptly as cells enter the stationary phase. Thus, the K-state and transformability are more transient and less frequently expressed in the undomesticated strains. This limitation is more extreme than appreciated from studies of domesticated strains. Selection has apparently limited both the frequency and the duration of the bistably expressed K-state in wild strains, likely because of the high cost of growth arrest associated with the K-state. Future modeling of K-state regulation and of the fitness advantages and costs of the K-state must take these features into account.

A protein complex supports the production of Spo0A-P and plays additional roles for biofilms and the K-state in Bacillus subtilis.

Bacillus subtilis can enter three developmental pathways to form spores, biofilms or K-state cells. The K-state confers competence for transformation and antibiotic tolerance. Transition into each of these states requires a stable protein complex formed by YlbF, YmcA and YaaT. We have reported that this complex acts in sporulation by accelerating the phosphorylation of the response regulator Spo0A. Phosphorelay acceleration was also predicted to explain their involvement in biofilm formation and the K-state. This view has been challenged in the case of biofilms, by the suggestion that the three proteins act in association with the mRNA degradation protein RNaseY (Rny) to destabilize the sinR transcript. Here, we reaffirm the roles of the three proteins in supporting the phosphorylation of Spo0A for all three developmental pathways and show that in their absence sinR mRNA is not stabilized. We demonstrate that the three proteins also play unknown Spo0A-P-independent roles in the expression of biofilm matrix and in the production of ComK, the master transcription factor for competence. Finally, we show that domesticated strains of B. subtilis carry a mutation in sigH, which influences the expression kinetics of the early spore gene spoIIG, thereby increasing the penetrance of the ylbF, ymcA and yaaT sporulation phenotypes.

Temporal Regulation of the Bacillus subtilis Acetylome and Evidence for a Role of MreB Acetylation in Cell Wall Growth.

Nε-Lysine acetylation has been recognized as a ubiquitous regulatory posttranslational modification that influences a variety of important biological processes in eukaryotic cells. Recently, it has been realized that acetylation is also prevalent in bacteria. Bacteria contain hundreds of acetylated proteins, with functions affecting diverse cellular pathways. Still, little is known about the regulation or biological relevance of nearly all of these modifications. Here we characterize the cellular growth-associated regulation of the Bacillus subtilis acetylome. Using acetylation enrichment and quantitative mass spectrometry, we investigate the logarithmic and stationary growth phases, identifying over 2,300 unique acetylation sites on proteins that function in essential cellular pathways. We determine an acetylation motif, EK(ac)(D/Y/E), which resembles the eukaryotic mitochondrial acetylation signature, and a distinct stationary-phase-enriched motif. By comparing the changes in acetylation with protein abundances, we discover a subset of critical acetylation events that are temporally regulated during cell growth. We functionally characterize the stationary-phase-enriched acetylation on the essential shape-determining protein MreB. Using bioinformatics, mutational analysis, and fluorescence microscopy, we define a potential role for the temporal acetylation of MreB in restricting cell wall growth and cell diameter. IMPORTANCE: The past decade highlighted Nε-lysine acetylation as a prevalent posttranslational modification in bacteria. However, knowledge regarding the physiological importance and temporal regulation of acetylation has remained limited. To uncover potential regulatory roles for acetylation, we analyzed how acetylation patterns and abundances change between growth phases in B. subtilis. To demonstrate that the identification of cell growth-dependent modifications can point to critical regulatory acetylation events, we further characterized MreB, the cell shape-determining protein. Our findings led us to propose a role for MreB acetylation in controlling cell width by restricting cell wall growth.

ComGA-RelA interaction and persistence in the Bacillus subtilis†K-state.

The bistably expressed K-state of Bacillus subtilis is characterized by two distinct features; transformability and arrested growth when K-state cells are exposed to fresh medium. The arrest is manifested by a failure to assemble replisomes and by decreased rates of cell growth and rRNA synthesis. These phenotypes are all partially explained by the presence of the AAA(+) protein ComGA, which is also required for the binding of transforming DNA to the cell surface and for the assembly of the transformation pilus that mediates DNA transport. We have discovered that ComGA interacts with RelA and that the ComGA-dependent inhibition of rRNA synthesis is largely bypassed in strains that cannot synthesize the alarmone (p)ppGpp. We propose that the interaction of ComGA with RelA prevents the hydrolysis of (p)ppGpp in K-state cells, which are thus trapped in a non-growing state until ComGA is degraded. We show that some K-state cells exhibit tolerance to antibiotics, a form of type 1 persistence, and we propose that the bistable expression of both transformability and the growth arrest are bet-hedging adaptations that improve fitness in the face of varying environments, such as those presumably encountered by B. subtilis in the soil.

Regulation by the modulation of gene expression variability.

Classically, transcription is regulated so that the average expression per cell changes, often with a distribution that extends across the population. Roggiani and Goulian (M. Roggiani and M. Goulian, J. Bacteriol. 197:1976-1987, 2015, doi:http://dx.doi.org/10.1128/JB.00074-15) have shown that this is what happens when the torCAD operon of Escherichia coli is induced anaerobically by the addition of trimethylamine-N-oxide (TMAO). However, when the same inducer is added to aerobically growing cells, only a subset of the cells respond, although the mean expression per cell is similar to that obtained anaerobically. Thus, in the presence of oxygen, the variance but not the expression mean is altered. The regulation of gene expression variance appears to be due to noise in the phosphorelay that governs torCAD transcription.

Complex formation and processing of the minor transformation pilins of Bacillus subtilis.

Transformation in most bacteria is dependent on orthologues of Type 2 secretion and Type 4 pilus system proteins. In each system, pilin proteins (major and minor) are required to make the pilus structure and are essential to the process, although the precise roles of the minor pilins remain unclear. We have explored protein-protein interactions among the competence minor pilins of Bacillus subtilis through in vitro binding studies, immunopurification and mass spectrometry. We demonstrate that the minor pilins directly interact, and the minor pilin ComGG interacts with most of the known proteins required for transformation. We find that ComGG requires other ComG proteins for its stabilization and for processing by the pre-pilin peptidase. These observations, C-terminal mutations in ComGG that prevent processing and the inaccessibility of pre-ComGG to externally added protease suggest a model in which pre-ComGG must be associated with other minor pilins for processing to take place. We propose that ComGG does not become a transmembrane protein until after processing. These behaviours contrast with that of pre-ComGC, the major pilin, which is accessible to externally added protease and requires only the peptidase to be processed. The roles of the pilins and of the pilus in transformation are discussed.

Genome Sequence of the Bacillus subtilis Biofilm-Forming Transformable Strain PS216.

Bacillus subtilis PS216, a strain isolated in Slovenia, has been sequenced. PS216 is transformable and forms robust biofilms, making it useful for the study of competence regulation in an undomesticated bacterium.

A complex of YlbF, YmcA and YaaT regulates sporulation, competence and biofilm formation by accelerating the phosphorylation of Spo0A.

Bacillus subtilis has adopted a bet-hedging strategy to ensure survival in changing environments. From a clonal population, numerous sub-populations can emerge, expressing different sets of genes that govern the developmental processes of sporulation, competence and biofilm formation. The master transcriptional regulator Spo0A controls the entry into all three fates and the production of the phosphorylated active form of Spo0A is precisely regulated via a phosphorelay, involving at least four proteins. Two proteins, YmcA and YlbF were previously shown to play an unidentified role in the regulation of biofilm formation, and in addition, YlbF was shown to regulate competence and sporulation. Using an unbiased proteomics screen, we demonstrate that YmcA and YlbF interact with a third protein, YaaT to form a tripartite complex. We show that all three proteins are required for proper establishment of the three above-mentioned developmental states. We show that the complex regulates the activity of Spo0A in vivo and, using in vitro reconstitution experiments, determine that they stimulate the phosphorelay, probably by interacting with Spo0F and Spo0B. We propose that the YmcA-YlbF-YaaT ternary complex is required to increase Spo0A~P levels above the thresholds needed to induce development.