Genetic and Structural Analyses of RRNPP Intercellular Peptide Signaling of Gram-Positive Bacteria.

Bacteria use diffusible chemical messengers, termed pheromones, to coordinate gene expression and behavior among cells in a community by a process known as quorum sensing. Pheromones of many gram-positive bacteria, such as Bacillus and Streptococcus, are small, linear peptides secreted from cells and subsequently detected by sensory receptors such as those belonging to the large family of RRNPP proteins. These proteins are cytoplasmic pheromone receptors sharing a structurally similar pheromone-binding domain that functions allosterically to regulate receptor activity. X-ray crystal structures of prototypical RRNPP members have provided atomic-level insights into their mechanism and regulation by pheromones. This review provides an overview of RRNPP prototype signaling; describes the structure-function of this protein family, which is spread widely among gram-positive bacteria; and suggests approaches to target RRNPP systems in order to manipulate beneficial and harmful bacterial behaviors.

Structure-function studies of Rgg binding to pheromones and target promoters reveal a model of transcription factor interplay.

Regulator gene of glucosyltransferase (Rgg) family proteins, such as Rgg2 and Rgg3, have emerged as primary quorum-sensing regulated transcription factors in Streptococcus species, controlling virulence, antimicrobial resistance, and biofilm formation. Rgg2 and Rgg3 function is regulated by their interaction with oligopeptide quorum-sensing signals called short hydrophobic peptides (SHPs). The molecular basis of Rgg-SHP and Rgg-target DNA promoter specificity was unknown. To close this gap, we determined the cryoelectron microscopy (cryo-EM) structure of Streptococcus thermophilus Rgg3 bound to its quorum-sensing signal, SHP3, and the X-ray crystal structure of Rgg3 alone. Comparison of these structures with that of an Rgg in complex with cyclosporin A (CsA), an inhibitor of SHP-induced Rgg activity, reveals the molecular basis of CsA function. Furthermore, to determine how Rgg proteins recognize DNA promoters, we determined X-ray crystal structures of both Streptococcus dysgalactiae Rgg2 and S. thermophilus Rgg3 in complex with their target DNA promoters. The physiological importance of observed Rgg-DNA interactions was dissected using in vivo genetic experiments and in vitro biochemical assays. Based on these structure-function studies, we present a revised unifying model of Rgg regulatory interplay. In contrast to existing models, where Rgg2 proteins are transcriptional activators and Rgg3 proteins are transcriptional repressors, we propose that both are capable of transcriptional activation. However, when Rgg proteins with different activation requirements compete for the same DNA promoters, those with more stringent activation requirements function as repressors by blocking promoter access of SHP-bound conformationally active Rgg proteins. While a similar gene expression regulatory scenario has not been previously described, in all likelihood it is not unique to streptococci.

One gene, multiple ecological strategies: A biofilm regulator is a capacitor for sustainable diversity.

Many bacteria cycle between sessile and motile forms in which they must sense and respond to internal and external signals to coordinate appropriate physiology. Maintaining fitness requires genetic networks that have been honed in variable environments to integrate these signals. The identity of the major regulators and how their control mechanisms evolved remain largely unknown in most organisms. During four different evolution experiments with the opportunist betaproteobacterium Burkholderia cenocepacia in a biofilm model, mutations were most frequently selected in the conserved gene rpfR RpfR uniquely integrates two major signaling systems-quorum sensing and the motile-sessile switch mediated by cyclic-di-GMP-by two domains that sense, respond to, and control the synthesis of the autoinducer cis-2-dodecenoic acid (BDSF). The BDSF response in turn regulates the activity of diguanylate cyclase and phosphodiesterase domains acting on cyclic-di-GMP. Parallel adaptive substitutions evolved in each of these domains to produce unique life history strategies by regulating cyclic-di-GMP levels, global transcriptional responses, biofilm production, and polysaccharide composition. These phenotypes translated into distinct ecology and biofilm structures that enabled mutants to coexist and produce more biomass than expected from their constituents grown alone. This study shows that when bacterial populations are selected in environments challenging the limits of their plasticity, the evolved mutations not only alter genes at the nexus of signaling networks but also reveal the scope of their regulatory functions.

Parallel quorum-sensing system in Vibrio cholerae prevents signal interference inside the host.

Many bacteria use quorum sensing (QS) to regulate virulence factor production in response to changes in population density. QS is mediated through the production, secretion, and detection of signaling molecules called autoinducers (AIs) to modulate population-wide behavioral changes. Four histidine kinases, LuxPQ, CqsS, CqsR and VpsS, have been identified in Vibrio cholerae as QS receptors to activate virulence gene expression at low cell density. Detection of AIs by these receptors leads to virulence gene repression at high cell density. The redundancy among these receptors is puzzling since any one of the four receptors is sufficient to support colonization of V. cholerae in the host small intestine. It is believed that one of the functions of such circuit architecture is to prevent interference on any single QS receptor. However, it is unclear what natural molecules can interfere V. cholerae QS and in what environment interference is detrimental. We show here mutants expressing only CqsR without the other three QS receptors are defective in colonizing the host large intestine. We identified ethanolamine, a common intestinal metabolite that can function as a chemical source of QS interference. Ethanolamine specifically interacts with the ligand-binding CACHE domain of CqsR and induces a premature QS response in V. cholerae mutants expressing only CqsR without the other three QS receptors. The effect of ethanolamine on QS gene expression and host colonization is abolished by mutations that disrupt CqsR signal sensing. V. cholerae defective in producing ethanolamine is still proficient in QS, therefore, ethanolamine functions only as an external cue for CqsR. Our findings suggest the inhibitory effect of ethanolamine on CqsR could be a possible source of QS interference but is masked by the presence of the other parallel QS pathways, allowing V. cholerae to robustly colonize the host.

Structural basis of DSF recognition by its receptor RpfR and its regulatory interaction with the DSF synthase RpfF.

The diffusible signal factors (DSFs) are a family of quorum-sensing autoinducers (AIs) produced and detected by numerous gram-negative bacteria. The DSF family AIs are fatty acids, differing in their acyl chain length, branching, and substitution but having in common a cis-2 double bond that is required for their activity. In both human and plant pathogens, DSFs regulate diverse phenotypes, including virulence factor expression, antibiotic resistance, and biofilm dispersal. Despite their widespread relevance to both human health and agriculture, the molecular basis of DSF recognition by their cellular receptors remained a mystery. Here, we report the first structure-function studies of the DSF receptor regulation of pathogenicity factor R (RpfR). We present the X-ray crystal structure of the RpfR DSF-binding domain in complex with the Burkholderia DSF (BDSF), which to our knowledge is the first structure of a DSF receptor in complex with its AI. To begin to understand the mechanistic role of the BDSF-RpfR contacts observed in the biologically important complex, we have also determined the X-ray crystal structure of the RpfR DSF-binding domain in complex with the inactive, saturated isomer of BDSF, dodecanoic acid (C12:0). In addition to these ligand-receptor complex structures, we report the discovery of a previously overlooked RpfR domain and show that it binds to and negatively regulates the DSF synthase regulation of pathogenicity factor F (RpfF). We have named this RpfR region the RpfF interaction (FI) domain, and we have determined its X-ray crystal structure alone and in complex with RpfF. These X-ray crystal structures, together with extensive complementary in vivo and in vitro functional studies, reveal the molecular basis of DSF recognition and the importance of the cis-2 double bond to DSF function. Finally, we show that throughout cellular growth, the production of BDSF by RpfF is post-translationally controlled by the RpfR N-terminal FI domain, affecting the cellular concentration of the bacterial second messenger bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP). Thus, in addition to describing the molecular basis for the binding and specificity of a DSF for its receptor, we describe a receptor-synthase interaction regulating bacterial quorum-sensing signaling and second messenger signal transduction.

Direct activation of a phospholipase by cyclic GMP-AMP in El Tor Vibrio cholerae.

Sensing and responding to environmental changes is essential for bacteria to adapt and thrive, and nucleotide-derived second messengers are central signaling systems in this process. The most recently identified bacterial cyclic dinucleotide second messenger, 3', 3'-cyclic GMP-AMP (cGAMP), was first discovered in the El Tor biotype of Vibrio cholerae The cGAMP synthase, DncV, is encoded on the VSP-1 pathogenicity island, which is found in all El Tor isolates that are responsible for the current seventh pandemic of cholera but not in the classical biotype. We determined that unregulated production of DncV inhibits growth in El Tor V. cholerae but has no effect on the classical biotype. This cGAMP-dependent phenotype can be suppressed by null mutations in vc0178 immediately 5' of dncV in VSP-1. VC0178 [renamed as cGAMP-activated phospholipase in Vibrio (CapV)] is predicted to be a patatin-like phospholipase, and coexpression of capV and dncV is sufficient to induce growth inhibition in classical V. cholerae and Escherichia coli Furthermore, cGAMP binds to CapV and directly activates its hydrolase activity in vitro. CapV activated by cGAMP in vivo degrades phospholipids in the cell membrane, releasing 16:1 and 18:1 free fatty acids. Together, we demonstrate that cGAMP activates CapV phospholipase activity to target the cell membrane and suggest that acquisition of this second messenger signaling pathway may contribute to the emergence of the El Tor biotype as the etiological agent behind the seventh cholera pandemic.

Activating mutations in quorum-sensing regulator Rgg2 and its conformational flexibility in the absence of an intermolecular disulfide bond.

Rap/Rgg/NprR/PlcR/PrgX (RRNPP) quorum-sensing systems use extracellular peptide pheromones that are detected by cytoplasmic receptors to regulate gene expression in firmicute bacteria. Rgg-type receptors are allosterically regulated through direct pheromone binding to control transcriptional activity; however, the receptor activation mechanism remains poorly understood. Previous work has identified a disulfide bond between Cys-45 residues within the homodimer interface of Rgg2 from Streptococcus dysgalactiae (Rgg2Sd). Here, we compared two Rgg2Sd(C45S) X-ray crystal structures with that of wild-type Rgg2Sd and found that in the absence of the intermolecular disulfide, the Rgg2Sd dimer interface is destabilized and Rgg2Sd can adopt multiple conformations. One conformation closely resembled the "disulfide-locked" Rgg2Sd secondary and tertiary structures, but another displayed more extensive rigid-body shifts as well as dramatic secondary structure changes. In parallel experiments, a genetic screen was used to identify mutations in rgg2 of Streptococcus pyogenes (rgg2Sp ) that conferred pheromone-independent transcriptional activation of an Rgg2-stimulated promoter. Eight mutations yielding constitutive Rgg2 activity, designated Rgg2Sp*, were identified, and five of them clustered in or near an Rgg2 region that underwent conformational changes in one of the Rgg2Sd(C45S) crystal structures. The Rgg2Sp* mutations increased Rgg2Sp sensitivity to pheromone and pheromone variants while displaying decreased sensitivity to the Rgg2 antagonist cyclosporine A. We propose that Rgg2Sp* mutations invoke shifts in free-energy bias to favor the active state of the protein. Finally, we present evidence for an electrostatic interaction between an N-terminal Asp of the pheromone and Arg-153 within the proposed pheromone-binding pocket of Rgg2Sp.

Rgg protein structure-function and inhibition by cyclic peptide compounds.

Peptide pheromone cell-cell signaling (quorum sensing) regulates the expression of diverse developmental phenotypes (including virulence) in Firmicutes, which includes common human pathogens, e.g., Streptococcus pyogenes and Streptococcus pneumoniae. Cytoplasmic transcription factors known as "Rgg proteins" are peptide pheromone receptors ubiquitous in Firmicutes. Here we present X-ray crystal structures of a Streptococcus Rgg protein alone and in complex with a tight-binding signaling antagonist, the cyclic undecapeptide cyclosporin A. To our knowledge, these represent the first Rgg protein X-ray crystal structures. Based on the results of extensive structure-function analysis, we reveal the peptide pheromone-binding site and the mechanism by which cyclosporin A inhibits activation of the peptide pheromone receptor. Guided by the Rgg-cyclosporin A complex structure, we predicted that the nonimmunosuppressive cyclosporin A analog valspodar would inhibit Rgg activation. Indeed, we found that, like cyclosporin A, valspodar inhibits peptide pheromone activation of conserved Rgg proteins in medically relevant Streptococcus species. Finally, the crystal structures presented here revealed that the Rgg protein DNA-binding domains are covalently linked across their dimerization interface by a disulfide bond formed by a highly conserved cysteine. The DNA-binding domain dimerization interface observed in our structures is essentially identical to the interfaces previously described for other members of the XRE DNA-binding domain family, but the presence of an intermolecular disulfide bond buried in this interface appears to be unique. We hypothesize that this disulfide bond may, under the right conditions, affect Rgg monomer-dimer equilibrium, stabilize Rgg conformation, or serve as a redox-sensitive switch.

The Psp system of Mycobacterium tuberculosis integrates envelope stress-sensing and envelope-preserving functions.

The bacterial envelope integrates essential stress-sensing and adaptive functions; thus, envelope-preserving functions are important for survival. In Gram-negative bacteria, envelope integrity during stress is maintained by the multi-gene Psp response. Mycobacterium tuberculosis was thought to lack the Psp system since it encodes only pspA and no other psp ortholog. Intriguingly, pspA maps downstream from clgR, which encodes a transcription factor regulated by the MprAB-σ(E) envelope-stress-signaling system. clgR inactivation lowered ATP concentration during stress and protonophore treatment-induced clgR-pspA expression, suggesting that these genes express Psp-like functions. We identified a four-gene set - clgR, pspA (rv2744c), rv2743c, rv2742c - that is regulated by clgR and in turn regulates ClgR activity. Regulatory and protein-protein interactions within the set and a requirement of the four genes for functions associated with envelope integrity and surface-stress tolerance indicate that a Psp-like system has evolved in mycobacteria. Among Actinobacteria, the four-gene module occurred only in tuberculous mycobacteria and was required for intramacrophage growth, suggesting links between its function and mycobacterial virulence. Additionally, the four-gene module was required for MprAB-σ(E) stress-signaling activity. The positive feedback between envelope-stress-sensing and envelope-preserving functions allows sustained responses to multiple, envelope-perturbing signals during chronic infection, making the system uniquely suited to tuberculosis pathogenesis.

FUSE Binding Protein 1 Facilitates Persistent Hepatitis C Virus Replication in Hepatoma Cells by Regulating Tumor Suppressor p53.

UNLABELLED: Hepatitis C virus (HCV) is a leading cause of chronic hepatitis C (CHC), liver cirrhosis, and hepatocellular carcinoma (HCC). Immunohistochemistry of archived HCC tumors showed abundant FBP1 expression in HCC tumors with the CHC background. Oncomine data analysis of normal versus HCC tumors with the CHC background indicated a 4-fold increase in FBP1 expression with a concomitant 2.5-fold decrease in the expression of p53. We found that FBP1 promotes HCV replication by inhibiting p53 and regulating BCCIP and TCTP, which are positive and negative regulators of p53, respectively. The severe inhibition of HCV replication in FBP1-knockdown Huh7.5 cells was restored to a normal level by downregulation of either p53 or BCCIP. Although p53 in Huh7.5 cells is transcriptionally inactive as a result of Y220C mutation, we found that the activation and DNA binding ability of Y220C p53 were strongly suppressed by FBP1 but significantly activated upon knockdown of FBP1. Transient expression of FBP1 in FBP1 knockdown cells fully restored the control phenotype in which the DNA binding ability of p53 was strongly suppressed. Using electrophoretic mobility shift assay (EMSA) and isothermal titration calorimetry (ITC), we found no significant difference in in vitro target DNA binding affinity of recombinant wild-type p53 and its Y220C mutant p53. However, in the presence of recombinant FBP1, the DNA binding ability of p53 is strongly inhibited. We confirmed that FBP1 downregulates BCCIP, p21, and p53 and upregulates TCTP under radiation-induced stress. Since FBP1 is overexpressed in most HCC tumors with an HCV background, it may have a role in promoting persistent virus infection and tumorigenesis. IMPORTANCE: It is our novel finding that FUSE binding protein 1 (FBP1) strongly inhibits the function of tumor suppressor p53 and is an essential host cell factor required for HCV replication. Oncomine data analysis of a large number of samples has revealed that overexpression of FBP1 in most HCC tumors with chronic hepatitis C is significantly linked with the decreased expression level of p53. The most significant finding is that FBP1 not only physically interacts with p53 and interferes with its binding to the target DNA but also functions as a negative regulator of p53 under cellular stress. FBP1 is barely detectable in normal differentiated cells; its overexpression in HCC tumors with the CHC background suggests that FBP1 has an important role in promoting HCV infection and HCC tumors by suppressing p53.

Conformational change-induced repeat domain expansion regulates Rap phosphatase quorum-sensing signal receptors.

The large family of Gram-positive quorum-sensing receptors known as the RNPP proteins consists of receptors homologous to the Rap, NprR, PlcR, and PrgX proteins that are regulated by imported oligopeptide autoinducers. Rap proteins are phosphatases and transcriptional anti-activators, and NprR, PlcR, and PrgX proteins are DNA binding transcription factors. Despite their obvious importance, the mechanistic basis of oligopeptide receptor regulation is largely unknown. Here, we report the X-ray crystal structure of the Bacillus subtilis quorum-sensing receptor RapJ in complex with the centrally important oligopeptide autoinducer competence and sporulation factor (CSF, also termed PhrC), a member of the Phr family of quorum-sensing signals. Furthermore, we present the crystal structure of RapI. Comparison of the RapJ-PhrC, RapI, RapH-Spo0F, and RapF-ComA(C) crystal structures reveals the mechanistic basis of Phr activity. More specifically, when complexed with target proteins, Rap proteins consist of a C-terminal tetratricopeptide repeat (TPR) domain connected by a flexible helix-containing linker to an N-terminal 3-helix bundle. In the absence of a target protein or regulatory peptide, the Rap protein 3-helix bundle adopts different conformations. However, in the peptide-bound conformation, the Rap protein N-terminal 3-helix bundle and linker undergo a radical conformational change, form TPR-like folds, and merge with the existing C-terminal TPR domain. To our knowledge, this is the first example of conformational change-induced repeat domain expansion. Furthermore, upon Phr binding, the entire Rap protein is compressed along the TPR superhelical axis, generating new intramolecular contacts that lock the Rap protein in an inactive state. The fact that Rap proteins are conformationally flexible is surprising considering that it is accepted dogma that TPR proteins do not undergo large conformational changes. Repeat proteins are widely used as scaffolds for the development of designed affinity reagents, and we propose that Rap proteins could be used as scaffolds for engineering novel ligand-switchable affinity reagents.

Antituberculosis thiophenes define a requirement for Pks13 in mycolic acid biosynthesis.

We report a new class of thiophene (TP) compounds that kill Mycobacterium tuberculosis by the previously uncharacterized mechanism of Pks13 inhibition. An F79S mutation near the catalytic Ser55 site in Pks13 conferred TP resistance in M. tuberculosis. Overexpression of wild-type Pks13 resulted in TP resistance, and overexpression of the Pks13(F79S) mutant conferred high resistance. In vitro, TP inhibited fatty acyl-AMP loading onto Pks13. TP inhibited mycolic acid biosynthesis in wild-type M. tuberculosis, but it did so to a much lesser extent in TP-resistant M. tuberculosis. TP treatment was bactericidal and equivalent to treatment with the first-line drug isoniazid, but it was less likely to permit emergent resistance. Combined isoniazid and TP treatment resulted in sterilizing activity. Computational docking identified a possible TP-binding groove within the Pks13 acyl carrier protein domain. This study confirms that M. tuberculosis Pks13 is required for mycolic acid biosynthesis, validates it as a druggable target and demonstrates the therapeutic potential of simultaneously inhibiting multiple targets in the same biosynthetic pathway.

Cyclic di-GMP inhibits Vibrio cholerae motility by repressing induction of transcription and inducing extracellular polysaccharide production.

Cyclic di-GMP (c-di-GMP) controls the transition between sessility and motility in many bacterial species. This regulation is achieved by a variety of mechanisms including alteration of transcription initiation and inhibition of flagellar function. How c-di-GMP inhibits the motility of Vibrio cholerae has not been determined. FlrA, a homologue of the c-di-GMP binding Pseudomonas aeruginosa motility regulator FleQ, is the master regulator of the V. cholerae flagellar biosynthesis regulon. Here we show that binding of c-di-GMP to FlrA abrogates binding of FlrA to the promoter of the flrBC operon, deactivating expression of the flagellar biosynthesis regulon. FlrA does not regulate expression of extracellular Vibrio polysaccharide (VPS) synthesis genes. Mutation of the FlrA amino acids R135 and R176 to histidine abrogates binding of c-di-GMP to FlrA, rendering FlrA active in the presence of high levels of c-di-GMP. Surprisingly, c-di-GMP still inhibited the motility of V. cholerae only expressing the c-di-GMP blind FlrA(R176H) mutant. We determined that this flagellar transcription-independent inhibition is due to activation of VPS production by c-di-GMP. Therefore, c-di-GMP prevents motility of V. cholerae by two distinct but functionally redundant mechanisms.

Structural basis of response regulator inhibition by a bacterial anti-activator protein.

The complex interplay between the response regulator ComA, the anti-activator RapF, and the signaling peptide PhrF controls competence development in Bacillus subtilis. More specifically, ComA drives the expression of genetic competence genes, while RapF inhibits the interaction of ComA with its target promoters. The signaling peptide PhrF accumulates at high cell density and upregulates genetic competence by antagonizing the interaction of RapF and ComA. How RapF functions mechanistically to inhibit ComA activity and how PhrF in turn antagonizes the RapF-ComA interaction were unknown. Here we present the X-ray crystal structure of RapF in complex with the ComA DNA binding domain. Along with biochemical and genetic studies, the X-ray crystal structure reveals how RapF mechanistically regulates ComA function. Interestingly, we found that a RapF surface mimics DNA to block ComA binding to its target promoters. Furthermore, RapF is a monomer either alone or in complex with PhrF, and it undergoes a conformational change upon binding to PhrF, which likely causes the dissociation of ComA from the RapF-ComA complex. Finally, we compare the structure of RapF complexed with the ComA DNA binding domain and the structure of RapH complexed with Spo0F. This comparison reveals that RapF and RapH have strikingly similar overall structures, and that they have evolved different, non-overlapping surfaces to interact with diverse cellular targets. To our knowledge, the data presented here reveal the first atomic level insight into the inhibition of response regulator DNA binding by an anti-activator. Compounds that affect the interaction of Rap and Rap-like proteins with their target domains could serve to regulate medically and commercially important phenotypes in numerous Bacillus species, such as sporulation in B. anthracis and sporulation and the production of Cry protein endotoxin in B. thuringiensis.

Structural basis of response regulator dephosphorylation by Rap phosphatases.

Bacterial Rap family proteins have been most extensively studied in Bacillus subtilis, where they regulate activities including sporulation, genetic competence, antibiotic expression, and the movement of the ICEBs1 transposon. One subset of Rap proteins consists of phosphatases that control B. subtilis and B. anthracis sporulation by dephosphorylating the response regulator Spo0F. The mechanistic basis of Rap phosphatase activity was unknown. Here we present the RapH-Spo0F X-ray crystal structure, which shows that Rap proteins consist of a 3-helix bundle and a tetratricopeptide repeat domain. Extensive biochemical and genetic functional studies reveal the importance of the observed RapH-Spo0F interactions, including the catalytic role of a glutamine in the RapH 3-helix bundle that inserts into the Spo0F active site. We show that in addition to dephosphorylating Spo0F, RapH can antagonize sporulation by sterically blocking phosphoryl transfer to and from Spo0F. Our structure-function analysis of the RapH-Spo0F interaction identified Rap protein residues critical for Spo0F phosphatase activity. This information enabled us to assign Spo0F phosphatase activity to a Rap protein based on sequence alone, which was not previously possible. Finally, as the ultimate test of our newfound understanding of the structural requirements for Rap phosphatase function, a non-phosphatase Rap protein that inhibits the binding of the response regulator ComA to DNA was rationally engineered to dephosphorylate Spo0F. In addition to revealing the mechanistic basis of response regulator dephosphorylation by Rap proteins, our studies support the previously proposed T-loop-Y allostery model of receiver domain regulation that restricts the aromatic "switch" residue to an internal position when the ß4-α4 loop adopts an active-site proximal conformation.

A plasmid-encoded phosphatase regulates Bacillus subtilis biofilm architecture, sporulation, and genetic competence.

Bacillus subtilis biofilm formation is tightly regulated by elaborate signaling pathways. In contrast to domesticated lab strains of B. subtilis which form smooth, essentially featureless colonies, undomesticated strains such as NCIB 3610 form architecturally complex biofilms. NCIB 3610 also contains an 80-kb plasmid absent from laboratory strains, and mutations in a plasmid-encoded homolog of a Rap protein, RapP, caused a hyperrugose biofilm phenotype. Here we explored the role of rapP phrP in biofilm formation. We found that RapP is a phosphatase that dephosphorylates the intermediate response regulator Spo0F. RapP appears to employ a catalytic glutamate to dephosphorylate the Spo0F aspartyl phosphate, and the implications of the RapP catalytic glutamate are discussed. In addition to regulating B. subtilis biofilm formation, we found that RapP regulates sporulation and genetic competence as a result of its ability to dephosphorylate Spo0F. Interestingly, while rap phr gene cassettes routinely form regulatory pairs; i.e., the mature phr gene product inhibits the activity of the rap gene product, the phrP gene product did not inhibit RapP activity in our assays. RapP activity was, however, inhibited by PhrH in vivo but not in vitro. Additional genetic analysis suggests that RapP is directly inhibited by peptide binding. We speculate that PhrH could be subject to posttranslational modification in vivo and directly inhibit RapP activity or, more likely, PhrH upregulates the expression of a peptide that, in turn, directly binds to RapP and inhibits its Spo0F phosphatase activity.

Erratum: 498 Structural Determination of the CqsR CACHE Domain and its Autoinducer - CORRIGENDUM.

[This corrects the article DOI: 10.1017/cts.2023.503.].

Identification of small molecules that antagonize diguanylate cyclase enzymes to inhibit biofilm formation.

Bacterial biofilm formation is responsible for numerous chronic infections, causing a severe health burden. Many of these infections cannot be resolved, as bacteria in biofilms are resistant to the host's immune defenses and antibiotic therapy. New strategies to treat biofilm-based infections are critically needed. Cyclic di-GMP (c-di-GMP) is a widely conserved second-messenger signal essential for biofilm formation. As this signaling system is found only in bacteria, it is an attractive target for the development of new antibiofilm interventions. Here, we describe the results of a high-throughput screen to identify small-molecule inhibitors of diguanylate cyclase (DGC) enzymes that synthesize c-di-GMP. We report seven small molecules that antagonize these enzymes and inhibit biofilm formation by Vibrio cholerae. Moreover, two of these compounds significantly reduce the total concentration of c-di-GMP in V. cholerae, one of which also inhibits biofilm formation by Pseudomonas aeruginosa in a continuous-flow system. These molecules represent the first compounds described that are able to inhibit DGC activity to prevent biofilm formation.

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.