Regulation of Aerobic Succinate Transporter dctA of E. coli by cAMP-CRP, DcuS-DcuR, and EIIAGlc: Succinate as a Carbon Substrate and Signaling Molecule.

INTRODUCTION: C4-dicarboxylates (C4-DC) have emerged as significant growth substrates and signaling molecules for various Enterobacteriaceae during their colonization of mammalian hosts. Particularly noteworthy is the essential role of fumarate respiration during colonization of pathogenic bacteria. To investigate the regulation of aerobic C4-DC metabolism, the study explored the transcriptional control of the main aerobic C4-DC transporter, dctA, under different carbohydrate conditions. In addition, mutants related to carbon catabolite repression (CCR) and C4-DC regulation (DcuS-DcuR) were examined to better understand the regulatory integration of aerobic C4-DC metabolism into CCR. For initial insight into posttranslational regulation, the interaction between the aerobic C4-DC transporter DctA and EIIAGlc from the glucose-specific phosphotransferase system was investigated. METHODS: The expression of dctA was characterized in the presence of various carbohydrates and regulatory mutants affecting CCR. This was accomplished by fusing the dctA promoter (PdctA) to the lacZ reporter gene. Additionally, the interaction between DctA and EIIAGlc of the glucose-specific phosphotransferase system was examined in vivo using a bacterial two-hybrid system. RESULTS: The dctA promoter region contains a class I cAMP-CRP-binding site at position -81.5 and a DcuR-binding site at position -105.5. DcuR, the response regulator of the C4-DC-activated DcuS-DcuR two-component system, and cAMP-CRP stimulate dctA expression. The expression of dctA is subject to the influence of various carbohydrates via cAMP-CRP, which differently modulate cAMP levels. Here we show that EIIAGlc of the glucose-specific phosphotransferase system strongly interacts with DctA, potentially resulting in the exclusion of C4-DCs when preferred carbon substrates, such as sugars, are present. In contrast to the classical inducer exclusion known for lactose permease LacY, inhibition of C4-DC uptake into the cytoplasm affects only its role as a substrate, but not as an inducer since DcuS detects C4-DCs in the periplasmic space ("substrate exclusion"). The work shows an interplay between cAMP-CRP and the DcuS-DcuR regulatory system for the regulation of dctA at both transcriptional and posttranslational levels. CONCLUSION: The study highlights a hierarchical interplay between global (cAMP-CRP) and specific (DcuS-DcuR) regulation of dctA at the transcriptional and posttranslational levels. The integration of global and specific transcriptional regulation of dctA, along with the influence of EIIAGlc on DctA, fine-tunes C4-DC catabolism in response to the availability of other preferred carbon sources. It attributes DctA a central role in the control of aerobic C4-DC catabolism and suggests a new role to EIIAGlc on transporters (control of substrate uptake by substrate exclusion).

Functional characterization of the phosphotransferase system in Parageobacillus thermoglucosidasius.

Parageobacillus thermoglucosidasius is a thermophilic bacterium characterized by rapid growth, low nutrient requirements, and amenability to genetic manipulation. These characteristics along with its ability to ferment a broad range of carbohydrates make P. thermoglucosidasius a potential workhorse in whole-cell biocatalysis. The phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) catalyzes the transport and phosphorylation of carbohydrates and sugar derivatives in bacteria, making it important for their physiological characterization. In this study, the role of PTS elements on the catabolism of PTS and non-PTS substrates was investigated for P. thermoglucosidasius DSM 2542. Knockout of the common enzyme I, part of all PTSs, showed that arbutin, cellobiose, fructose, glucose, glycerol, mannitol, mannose, N-acetylglucosamine, N-acetylmuramic acid, sorbitol, salicin, sucrose, and trehalose were PTS-dependent on translocation and coupled to phosphorylation. The role of each putative PTS was investigated and six PTS-deletion variants could not grow on arbutin, mannitol, N-acetylglucosamine, sorbitol, and trehalose as the main carbon source, or showed diminished growth on N-acetylmuramic acid. We concluded that PTS is a pivotal factor in the sugar metabolism of P. thermoglucosidasius and established six PTS variants important for the translocation of specific carbohydrates. This study lays the groundwork for engineering efforts with P. thermoglucosidasius towards efficient utilization of diverse carbon substrates for whole-cell biocatalysis.

PtsN in Pseudomonas aeruginosa Is Phosphorylated by Redundant Upstream Proteins and Impacts Virulence-Related Genes.

The bacterial nitrogen-related phosphotransfer (PTSNtr; here, Nitro-PTS) system bears homology to well-known PTS systems that facilitate saccharide import and phosphorylation. The Nitro-PTS comprises an enzyme I (EI), PtsP; an intermediate phosphate carrier, PtsO; and a terminal acceptor, PtsN, which is thought to exert regulatory effects that depend on its phosphostate. For instance, biofilm formation by Pseudomonas aeruginosa can be impacted by the Nitro-PTS, as deletion of either ptsP or ptsO suppresses Pel exopolysaccharide production and additional deletion of ptsN elevates Pel production. However, the phosphorylation state of PtsN in the presence and absence of its upstream phosphotransferases has not been directly assessed, and other targets of PtsN have not been well defined in P. aeruginosa. We show that PtsN phosphorylation via PtsP requires the GAF domain of PtsP and that PtsN is phosphorylated on histidine 68, as in Pseudomonas putida. We also find that FruB, the fructose EI, can substitute for PtsP in PtsN phosphorylation but only in the absence of PtsO, implicating PtsO as a specificity factor. Unphosphorylatable PtsN had a minimal effect on biofilm formation, suggesting that it is necessary but not sufficient for the reduction of Pel in a ptsP deletion. Finally, we use transcriptomics to show that the phosphostate and the presence of PtsN do not appear to alter the transcription of biofilm-related genes but do influence genes involved in type III secretion, potassium transport, and pyoverdine biosynthesis. Thus, the Nitro-PTS influences several P. aeruginosa behaviors, including the production of its signature virulence factors. IMPORTANCE The PtsN protein impacts the physiology of a number of bacterial species, and its control over downstream targets can be altered by its phosphorylation state. Neither its upstream phosphotransferases nor its downstream targets are well understood in Pseudomonas aeruginosa. Here, we examine PtsN phosphorylation and find that the immediate upstream phosphotransferase acts as a gatekeeper, allowing phosphorylation by only one of two potential upstream proteins. We use transcriptomics to discover that PtsN regulates the expression of gene families that are implicated in virulence. One emerging pattern is a repression hierarchy by different forms of PtsN: its phosphorylated state is more repressive than its unphosphorylated state, but the expression of its targets is even higher in its complete absence.

Functional and structural diversification of incomplete phosphotransferase system in cellulose-degrading clostridia.

Carbohydrate utilization is critical to microbial survival. The phosphotransferase system (PTS) is a well-documented microbial system with a prominent role in carbohydrate metabolism, which can transport carbohydrates through forming a phosphorylation cascade and regulate metabolism by protein phosphorylation or interactions in model strains. However, those PTS-mediated regulated mechanisms have been underexplored in non-model prokaryotes. Here, we performed massive genome mining for PTS components in nearly 15,000 prokaryotic genomes from 4,293 species and revealed a high prevalence of incomplete PTSs in prokaryotes with no association to microbial phylogeny. Among these incomplete PTS carriers, a group of lignocellulose degrading clostridia was identified to have lost PTS sugar transporters and carry a substitution of the conserved histidine residue in the core PTS component, HPr (histidine-phosphorylatable phosphocarrier). Ruminiclostridium cellulolyticum was then selected as a representative to interrogate the function of incomplete PTS components in carbohydrate metabolism. Inactivation of the HPr homolog reduced rather than increased carbohydrate utilization as previously indicated. In addition to regulating distinct transcriptional profiles, PTS associated CcpA (Catabolite Control Protein A) homologs diverged from previously described CcpA with varied metabolic relevance and distinct DNA binding motifs. Furthermore, the DNA binding of CcpA homologs is independent of HPr homolog, which is determined by structural changes at the interface of CcpA homologs, rather than in HPr homolog. These data concordantly support functional and structural diversification of PTS components in metabolic regulation and bring novel understanding of regulatory mechanisms of incomplete PTSs in cellulose-degrading clostridia.

Phosphotransferase system sugars immediately induce mutations of Cra in an Escherichia coli ptsH mutant.

Most bacteria use the phosphoenolpyruvate (PEP):sugar phosphotransferase system (PTS) to catalyse coupled transport and phosphorylation of sugars. The PTS consists of several sugar-specific components (enzyme IIs) and two general components: enzyme I, encoded by ptsI, and HPr, encoded by ptsH, which are common to most PTS carbohydrates. Although both enzyme I and HPr are believed to be required to utilize these PTS sugars, an E. coli ptsH mutant has been reported to exhibit a leaky growth phenotype on these sugars. Here, we show that this phenomenon occurs because the ptsH mutant undergoes adaptive mutations in the presence of PTS sugars within a few generation times. The ptsH mutant cells once exposed to a PTS sugar showed a growth rate similar to that of the wild-type strain when transferred to fresh medium supplemented with the same PTS sugar, suggesting the acquisition of additional genetic variations. Genome sequencing revealed that the PTS sugar-adapted variants harboured loss-of-function mutations in cra, which increased expression of the fruBKA operon. Our results suggest that the presence of a PTS sugar can exert a strong selective pressure when a general PTS component is defective.

A mannose-sensing AraC-type transcriptional activator regulates cell-cell aggregation of Vibrio cholerae.

In addition to catalyzing coupled transport and phosphorylation of carbohydrates, the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) regulates various physiological processes in most bacteria. Therefore, the transcription of genes encoding the PTS is precisely regulated by transcriptional regulators depending on substrate availability. As the distribution of the mannose-specific PTS (PTSMan) is limited to animal-associated bacteria, it has been suggested to play an important role in host-bacteria interactions. In Vibrio cholerae, mannose is known to inhibit biofilm formation. During host infection, the transcription level of the V. cholerae gene encoding the putative PTSMan (hereafter referred to as manP) significantly increases, and mutations in this gene increase host survival rate. Herein, we show that an AraC-type transcriptional regulator (hereafter referred to as ManR) acts as a transcriptional activator of the mannose operon and is responsible for V. cholerae growth and biofilm inhibition on a mannose or fructose-supplemented medium. ManR activates mannose operon transcription by facilitating RNA polymerase binding to the promoter in response to mannose 6-phosphate and, to a lesser extent, to fructose 1-phosphate. When manP or manR is impaired, the mannose-induced inhibition of biofilm formation was reversed and intestinal colonization was significantly reduced in a Drosophila melanogaster infection model. Our results show that ManR recognizes mannose and fructose in the environment and facilitates V. cholerae survival in the host.

A broadly applicable, stress-mediated bacterial death pathway regulated by the phosphotransferase system (PTS) and the cAMP-Crp cascade.

Recent work indicates that killing of bacteria by diverse antimicrobial classes can involve reactive oxygen species (ROS), as if a common, self-destructive response to antibiotics occurs. However, the ROS-bacterial death theory has been challenged. To better understand stress-mediated bacterial death, we enriched spontaneous antideath mutants of Escherichia coli that survive treatment by diverse bactericidal agents that include antibiotics, disinfectants, and environmental stressors, without a priori consideration of ROS. The mutants retained bacteriostatic susceptibility, thereby ruling out resistance. Surprisingly, pan-tolerance arose from carbohydrate metabolism deficiencies in ptsI (phosphotransferase) and cyaA (adenyl cyclase); these genes displayed the activity of upstream regulators of a widely shared, stress-mediated death pathway. The antideath effect was reversed by genetic complementation, exogenous cAMP, or a Crp variant that bypasses cAMP binding for activation. Downstream events comprised a metabolic shift from the TCA cycle to glycolysis and to the pentose phosphate pathway, suppression of stress-mediated ATP surges, and reduced accumulation of ROS. These observations reveal how upstream signals from diverse stress-mediated lesions stimulate shared, late-stage, ROS-mediated events. Cultures of these stable, pan-tolerant mutants grew normally and were therefore distinct from tolerance derived from growth defects described previously. Pan-tolerance raises the potential for unrestricted disinfectant use to contribute to antibiotic tolerance and resistance. It also weakens host defenses, because three agents (hypochlorite, hydrogen peroxide, and low pH) affected by pan-tolerance are used by the immune system to fight infections. Understanding and manipulating the PtsI-CyaA-Crp­mediated death process can help better control pathogens and maintain beneficial microbiota during antimicrobial treatment.

Preferential Regulation of Transient Protein-Protein Interaction by the Macromolecular Crowders.

The environmental condition is a critical regulation factor for protein behavior in solution. Several studies have shown that macromolecular crowders can modulate protein structures, interactions, and functions. Recent publications described the regulation of specific interaction by macromolecular crowders. However, the other category of protein-protein interaction, namely, the transient interaction, is rarely investigated, especially from the perspective of protein structure to study transient interactions between proteins. Here, we used nuclear magnetic resonance and small-angle X-ray/neutron scattering methods to structurally investigate the ensemble of the protein complex in dilute buffer and crowded environments. Histidine phosphocarrier protein (HPr) and the N-terminal domain of enzyme I (EIN) are the important components of the bacterial phosphotransfer system. Our results show that the addition of Ficoll-70 promotes HPr molecules to form the encounter complex with EIN maintained by long-range electrostatic interaction. However, when macromolecular crowder BSA is used, the soft interaction between BSA and HPr perturbs the active site of HPr, driving HPr to form an encounter complex with EIN at the weakly charged interface. Our results indicate that different macromolecular crowders could influence transient EIN-HPr interaction through different mechanisms and provide new insights into protein-protein interaction regulation in native environments.

The PTSNtr-KdpDE-KdpFABC Pathway Contributes to Low Potassium Stress Adaptation and Competitive Nodulation of Sinorhizobium fredii.

The rhizobium-legume symbiosis is essential for sustainable agriculture by reducing nitrogen fertilizer input, but its efficiency varies under fluctuating soil conditions and resources. The nitrogen-related phosphotransferase system (PTSNtr) consisting of PtsP, PtsO, and PtsN is required for optimal nodulation and nitrogen fixation efficiency of the broad-host-range Sinorhizobium fredii CCBAU45436 associated with diverse legumes, though the underlying mechanisms remain elusive. This work characterizes the PtsN-KdpDE-KdpFABC pathway that contributes to low potassium adaptation and competitive nodulation of CCBAU45436. Among three PtsN, PtsN1 is the major functional homolog. The unphosphorylated PtsN1 binds the sensory kinase KdpD through a non-canonical interaction with the GAF domain of KdpD, while the region covering HisKA-HATPase domains mediates the interaction of KdpD with the response regulator KdpE. KdpE directly activates the kdpFABC operon encoding the conserved high-affinity potassium uptake system. Disruption of this signaling pathway leads to reduced nodule number, nodule occupancy, and low potassium adaptation ability, but without notable effects on rhizoplane colonization. The induction of key nodulation genes NIN and ENOD40 in host roots during early symbiotic interactions is impaired when inoculating the kdpBC mutant that shows delayed nodulation. The nodulation defect of the kdpBC mutant can be rescued by supplying replete potassium. Potassium is actively consumed by both prokaryotes and eukaryotes, and components of the PTSNtr-KdpDE-KdpFABC pathway are widely conserved in bacteria, highlighting the global importance of this pathway in bacteria-host interactions. IMPORTANCE In all ecological niches, potassium is actively consumed by diverse prokaryotes and their interacting eukaryote hosts. It is only just emerging that potassium is a key player in host-pathogen interactions, and the role of potassium in mutualistic interactions remains largely unknown. This work is focused on the mutualistic symbiosis between rhizobia and legumes. We report that the nitrogen-related phosphotransferase system PTSNtr, the two-component system KdpDE, and the high-affinity potassium uptake system KdpFABC constitute a pathway that is important for low potassium adaptation and optimal nodulation of rhizobia. Given the widely conserved PTSNtr, KdpDE, and KdpFABC in bacteria and increasing knowledge on microbiome for various niches, the PTSNtr-KdpDE-KdpFABC pathway can be globally important in the biosphere.

Regulation of Mannitol Metabolism in Enterococcus faecalis and Association with parEF0409 Toxin-Antitoxin Locus Function.

The parEF0409 type I toxin-antitoxin locus is situated between genes for two paralogous mannitol family phosphoenolpyruvate phosphotransferase systems (PTSs). In order to address the possibility that parEF0409 function was associated with sugar metabolism, genetic and phenotypic analyses were performed on the flanking genes. It was found that the genes were transcribed as two operons: the downstream operon essential for mannitol transport and metabolism and the upstream operon performing a regulatory function. In addition to genes for the PTS components, the upstream operon harbors a gene similar to mtlR, the key regulator of mannitol metabolism in other Gram-positive bacteria. We confirmed that this gene is essential for the regulation of the downstream operon and identified putative phosphorylation sites required for carbon catabolite repression and mannitol-specific regulation. Genomic comparisons revealed that this dual-operon organization of mannitol utilization genes is uncommon in enterococci and that the association with a toxin-antitoxin system is unique to Enterococcus faecalis. Finally, we consider possible links between parEF0409 function and mannitol utilization. IMPORTANCE Enterococcus faecalis is both a common member of the human gut microbiota and an opportunistic pathogen. Its evolutionary success is partially due to its metabolic flexibility, in particular its ability to import and metabolize a wide variety of sugars. While a large number of phosphoenolpyruvate phosphotransferase sugar transport systems have been identified in the E. faecalis genome bioinformatically, the specificity and regulation of most of these systems remain undetermined. Here, we characterize a complex system of two operons flanking a type I toxin-antitoxin system required for the transport and metabolism of the common dietary sugar mannitol. We also determine the phylogenetic distribution of mannitol utilization genes in the enterococcal genus and discuss the significance of the association with toxin-antitoxin systems.

N-acetylglucosamine transporter, Ngt1, undergoes sugar-responsive endosomal trafficking in Candida albicans.

N-acetylglucosamine (GlcNAc), an important amino sugar at the infection sites of the fungal pathogen Candida albicans, triggers multiple cellular processes. GlcNAc import at the cell surface is mediated by GlcNAc transporter, Ngt1 which seems to play a critical role during GlcNAc signaling. We have investigated the Ngt1 dynamics that provide a platform for further studies aimed at understanding the mechanistic insights of regulating process(es) in C. albicans. The expression of this transporter is prolific and highly sensitive to even very low levels (˂2 µM) of GlcNAc. Under these conditions, Ngt1 undergoes phosphorylation-associated ubiquitylation as a code for internalization. This ubiquitylation process involves the triggering proteins like protein kinase Snf1, arrestin-related trafficking adaptors (ART) protein Rod1, and yeast ubiquitin ligase Rsp5. Interestingly, analysis of ∆snf1 and ∆rsp5 mutants revealed that while Rsp5 is promoting the endosomal trafficking of Ngt1-GFPɤ, Snf1 hinders the process. Furthermore, colocalization experiments of Ngt1 with Vps17 (an endosomal marker), Sec7 (a trans-Golgi marker), and a vacuolar marker revealed the fate of Ngt1 during sugar-responsive endosomal trafficking. ∆ras1 and ∆ubi4 mutants showed decreased ubiquitylation and delayed endocytosis of Ngt1. According to our knowledge, this is the first report which illustrates the mechanistic insights that are responsible for endosomal trafficking of a GlcNAc transporter in an eukaryotic organism.

Structural Basis of Pore Formation in the Mannose Phosphotransferase System by Pediocin PA-1.

Bacteriocins are ribosomally synthesized bacterial antimicrobial peptides that have a narrow spectrum of antibacterial activity against species closely related to the producers. Pediocin-like (or class IIa) bacteriocins (PLBs) exhibit antibacterial activity against several Gram-positive bacterial strains by forming pores in the cytoplasmic membrane of target cells with a specific receptor, the mannose phosphotransferase system (man-PTS). In this study, we report the cryo-electron microscopy structures of man-PTS from Listeria monocytogenes alone and its complex with pediocin PA-1, the first and most extensively studied representative PLB, at resolutions of 3.12 and 2.45 Å, respectively. The structures revealed that the binding of pediocin PA-1 opens the Core domain of man-PTS away from its Vmotif domain, creating a pore through the cytoplasmic membranes of target cells. During this process, the N-terminal ß-sheet region of pediocin PA-1 can specifically attach to the extracellular surface of the man-PTS Core domain, whereas the C-terminal half penetrates the membrane and cracks the man-PTS like a wedge. Thus, our findings shed light on a design of novel PLBs that can kill the target pathogenic bacteria. IMPORTANCE Listeria monocytogenes is a ubiquitous microorganism responsible for listeriosis, a rare but severe disease in humans, who become infected by ingesting contaminated food products (i.e., dairy, meat, fish, and vegetables): the disease has a fatality rate of 33%. Pediocin PA-1 is an important commercial additive used in food production to inhibit Listeria species. The mannose phosphotransferase system (man-PTS) is responsible for the sensitivity of Listeria monocytogenes to pediocin PA-1. In this study, we report the cryo-EM structures of man-PTS from Listeria monocytogenes alone and its complex with pediocin PA-1 at resolutions of 3.12 and 2.45 Å, respectively. Our results facilitate the understanding of the mode of action of class IIa bacteriocins as an alternative to antibiotics.

Evidence for reciprocal evolution of the global repressor Mlc and its cognate phosphotransferase system sugar transporter.

Because the bacterial phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) is involved in the regulation of various physiological processes in addition to carbohydrate transport, its expression is precisely regulated in response to the availability of PTS sugars. The PTS consists of enzyme I and histidine phosphocarrier protein, and several sugar-specific enzymes II. In Escherichia coli, genes for enzymes II specific for glucose and related sugars are co-regulated by the global repressor Mlc, and glucose induction of the Mlc regulon genes is achieved by its interaction with glucose-specific enzyme II (EIIGlc ). In this study, we revealed that, in Vibrio species, which are phylogenetically older than Enterobacteriaceae, the membrane sequestration of Mlc and thereby the induction of its regulon genes is mediated by N-acetylglucosamine (NAG)-specific EII. While Vibrio Mlc interacts only with the EIIB domain of EIINag , E. coli Mlc interacts with the EIIB domain of both EIIGlc and EIINag . The present data suggest that EIINag may be the primordial regulator of Mlc, and EIIGlc has evolved to interact with Mlc since an EIIA domain was fused to EIINag in Enterobacteriaceae. Our findings provide insight into the coevolutionary dynamics between a transcription factor and its cognate regulator according to long-term resource availability in the bacterial natural habitat.

HprSR Is a Reactive Chlorine Species-Sensing, Two-Component System in Escherichia coli.

Two-component systems (TCS) are signaling pathways that allow bacterial cells to sense, respond to, and adapt to fluctuating environments. Among the classical TCS of Escherichia coli, HprSR has recently been shown to be involved in the regulation of msrPQ, which encodes the periplasmic methionine sulfoxide reductase system. In this study, we demonstrated that hypochlorous acid (HOCl) induces the expression of msrPQ in an HprSR-dependent manner, whereas H2O2, NO, and paraquat (a superoxide generator) do not. Therefore, HprS appears to be an HOCl-sensing histidine kinase. Using a directed mutagenesis approach, we showed that Met residues located in the periplasmic loop of HprS are important for its activity: we provide evidence that as HOCl preferentially oxidizes Met residues, HprS could be activated via the reversible oxidation of its methionine residues, meaning that MsrPQ plays a role in switching HprSR off. We propose that the activation of HprS by HOCl could occur through a Met redox switch. HprSR appears to be the first characterized TCS able to detect reactive chlorine species (RCS) in E. coli. This study represents an important step toward understanding the mechanisms of RCS resistance in prokaryotes. IMPORTANCE Understanding how bacteria respond to oxidative stress at the molecular level is crucial in the fight against pathogens. HOCl is one of the most potent industrial and physiological microbicidal oxidants. Therefore, bacteria have developed counterstrategies to survive HOCl-induced stress. Over the last decade, important insights into these bacterial protection factors have been obtained. Our work establishes HprSR as a reactive chlorine species-sensing, two-component system in Escherichia coli MG1655, which regulates the expression of msrPQ, two genes encoding, a repair system for HOCl-oxidized proteins. Moreover, we provide evidence suggesting that HOCl could activate HprS through a methionine redox switch.

Residual Helicity at the Active Site of the Histidine Phosphocarrier, HPr, Modulates Binding Affinity to Its Natural Partners.

The phosphoenolpyruvate-dependent phosphotransferase system (PTS) modulates the preferential use of sugars in bacteria. The first proteins in the cascade are common to all organisms (EI and HPr). The active site of HPr involves a histidine (His15) located immediately before the beginning of the first α-helix. The regulator of sigma D (Rsd) protein also binds to HPr. The region of HPr comprising residues Gly9-Ala30 (HPr9-30), involving the first α-helix (Ala16-Thr27) and the preceding active site loop, binds to both the N-terminal region of EI and intact Rsd. HPr9-30 is mainly disordered. We attempted to improve the affinity of HPr9-30 to both proteins by mutating its sequence to increase its helicity. We designed peptides that led to a marginally larger population in solution of the helical structure of HPr9-30. Molecular simulations also suggested a modest increment in the helical population of mutants, when compared to the wild-type. The mutants, however, were bound with a less favorable affinity than the wild-type to both the N-terminal of EI (EIN) or Rsd, as tested by isothermal titration calorimetry and fluorescence. Furthermore, mutants showed lower antibacterial properties against Staphylococcus aureus than the wild-type peptide. Therefore, we concluded that in HPr, a compromise between binding to its partners and residual structure at the active site must exist to carry out its function.

The protein-protein interaction network of the Escherichia coli EIIANtr regulatory protein reveals a role in cell motility and metabolic control.

The nitrogen-related PTSNtr system, present in many Proteobacteria including Escherichia coli, acts as a phosphorelay cascade composed of the EINtr, NPr and EIIANtr proteins. Phosphotransfer initiates with phosphoenolpyruvate-dependent EINtr autophosphorylation, the phosphoryl group is then transferred to NPr and finally to a conserved histidine residue on EIIANtr. The reporter metabolites l-glutamine and 2-oxoglutarate reciprocally regulate EINtr autophosphorylation (Lee et al., 2013) and consequently the phosphorylation status of the PTSNtr components is controlled by the availability of nitrogen and carbon. The final phosphate acceptor, EIIANtr, regulates a range of cellular process by acting as the central hub of a complex protein-protein interaction network. Contact between EIIANtr and its target proteins is usually regulated by the EIIANtr phosphorylation status. In this study we performed ligand fishing assays coupled to label-free quantitative proteomics to examine the protein-protein interaction network of E. coli EIIANtr and a phosphomimic variant of the protein. The ligand fishing data, along with phenotypic analysis, indicated that EIIANtr interacts with proteins related to chemotaxis and thereby regulates cell motility. Important metabolic enzymes were also identified as potential EIIANtr binding partners.

The Antimicrobial Activity of the Glycocin Sublancin Is Dependent on an Active Phosphoenolpyruvate-Sugar Phosphotransferase System.

Antimicrobial resistance is a global challenge that is compounded by the limited number of available targets. Glycocins are antimicrobial glycopeptides that are believed to have novel targets. Previous studies have shown that the mechanism of action of the glycocin sublancin 168 involves the glucose uptake system. The phosphoenolpyruvate:sugar phosphotransferase system (PTS) phosphorylates the C6 hydroxyl group on glucose during import. Since sublancin carries a glucose on a Cys on an exposed loop, we investigated whether phosphorylation of this glucose might be involved in its mechanism of action by replacement with xylose. Surprisingly, the xylose analog was more active than wild-type sublancin and still required the glucose PTS for activity. Overexpression of the individual components of the PTS rendered cells more sensitive to sublancin, and their resistance frequency was considerably decreased. These observations suggest that sublancin is activated in some form by the glucose PTS or that sublancin imparts a deleterious gain-of-function on the PTS. Superresolution microscopy studies with fluorescent sublancin and fluorescently labeled PTS proteins revealed localization of both at the poles of cells. Resistant mutants raised under conditions that would minimize mutation of the PTS revealed mutations in FliQ, a protein involved in the flagellar protein export process. Overexpression of FliQ lead to decreased sensitivity of cells to sublancin. Collectively, these findings enforce a model in which the PTS is required for sublancin activity, either by inducing a deleterious gain-of-function or by activating or transporting sublancin.

Structure elucidation of the elusive Enzyme I monomer reveals the molecular mechanisms linking oligomerization and enzymatic activity.

Enzyme I (EI) is a phosphotransferase enzyme responsible for converting phosphoenolpyruvate (PEP) into pyruvate. This reaction initiates a five-step phosphorylation cascade in the bacterial phosphotransferase (PTS) transduction pathway. Under physiological conditions, EI exists in an equilibrium between a functional dimer and an inactive monomer. The monomer-dimer equilibrium is a crucial factor regulating EI activity and the phosphorylation state of the overall PTS. Experimental studies of EI's monomeric state have yet been hampered by the dimer's high thermodynamic stability, which prevents its characterization by standard structural techniques. In this study, we modified the dimerization domain of EI (EIC) by mutating three amino acids involved in the formation of intersubunit salt bridges. The engineered variant forms an active dimer in solution that can bind and hydrolyze PEP. Using hydrostatic pressure as an additional perturbation, we were then able to study the complete dissociation of the variant from 1 bar to 2.5 kbar in the absence and the presence of EI natural ligands. Backbone residual dipolar couplings collected under high-pressure conditions allowed us to determine the conformational ensemble of the isolated EIC monomeric state in solution. Our calculations reveal that three catalytic loops near the dimerization interface become unstructured upon monomerization, preventing the monomeric enzyme from binding its natural substrate. This study provides an atomic-level characterization of EI's monomeric state and highlights the role of the catalytic loops as allosteric connectors controlling both the activity and oligomerization of the enzyme.

Cra and cAMP Receptor Protein Have Opposing Roles in the Regulation of fruB in Vibrio cholerae.

The Gram-negative bacterium Vibrio cholerae adapts to changes in the environment by selectively producing the necessary machinery to take up and metabolize available carbohydrates. The import of fructose by the fructose-specific phosphoenolpyruvate (PEP) phosphotransferase system (PTS) is of particular interest because of its putative connection to cholera pathogenesis and persistence. Here, we describe the expression and regulation of fruB, which encodes an EIIA-FPr fusion protein as part of the fructose-specific PTS in V. cholerae Using a series of transcriptional reporter fusions and additional biochemical and genetic assays, we identified Cra (catabolite repressor/activator) and cAMP receptor protein (CRP) as regulators of fruB expression and determined that this regulation is dependent upon the presence or absence of PTS sugars. Cra functions as a repressor, downregulating fruB expression in the absence of fructose when components of PTSFru are not needed. CRP functions as an activator of fruB expression. We also report that Cra and CRP can affect fruB expression independently; however, CRP can modulate cra expression in the presence of fructose and glucose. Evidence from this work provides the foundation for continued investigations into PTSFru and its relationship to the V. cholerae life cycle.IMPORTANCEVibrio cholerae is the causative agent of cholera disease. While current treatments of care are accessible, we still lack an understanding of the molecular mechanisms that allow V. cholerae to survive in both aquatic reservoirs and the human small intestine, where pathogenesis occurs. Central to V. cholerae's survival is its ability to use available carbon sources. Here, we investigate the regulation of fruB, which encodes a protein central to the import and metabolism of fructose. We show that fruB expression is controlled by the transcriptional regulators Cra and CRP. This work contributes toward a clearer understanding of how carbon source availability impacts the physiology and, potentially, the persistence of the pathogen.