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Previous work reported that deletion of the Enzyme IIAB subunits (EIIAB Man and manL ) of the glucose phosphotransferase system (PTS) (glucose-PTS, manLMNO ) in Streptococcus sanguinis impacted carbon catabolite repression and bacterial fitness. Here, a single-nucleotide polymorphism in ManN, ManNA91E, produced the unusual phenotype of increased excretion of organic acids and H 2 O 2 yet elevated PTS activities. To characterize the contributions of each component of the glucose-PTS to bacterial fitness, we performed genetic analyses by deleting from S. sanguinis SK36 the entire operon and each EII Man subunit individually; and genes encoding the catabolite control protein A (Δ ccpA ) and the redox regulator Rex (Δ rex ) for comparison. Deletion of each subunit incurred a growth defect on glucose partly due to elevated excretion of H 2 O 2 ; when supplemented with catalase, this defect was rescued, instead resulting in a significantly higher yield than the parent. All glucose-PTS deletion mutants presented an increased antagonism against the oral pathobiont Streptococcus mutans , a phenotype absent in Δ ccpA despite increased H 2 O 2 output. A shift in the pyruvate node toward mixed acid fermentation and increased arginine deiminase activity enhanced pH homeostasis in glucose-PTS mutants but not Δ ccpA . Despite the purported ability of Rex to regulate central carbon metabolism, deletion of rex had no significant impact on most of the phenotypes discussed here. These findings place glucose-PTS in the pivotal position of controlling central carbon flux in streptococci, with critical outcomes impacting acidogenicity, aciduricity, pH homeostasis, and antagonism, highlighting its potential as a therapeutic target for treating diseases with a dysbiotic microbiome., Importance: Management of carbohydrate metabolism and environmental stress is key to the survival of oral commensal species such as S. sanguinis . Antagonism of oral pathobionts and modulation of the environmental pH and oxidative potential by commensals are crucial to the maintenance of microbial homeostasis and prevention of oral diseases including dental caries. It is therefore vital to understand how these species regulate sugar fermentation, production of acids and ammonia, and stress management in an environment known for a feast-and-famine cycle of carbohydrates and similar fluctuations in pH and oxygen tension. Here, we detail that genetic alterations of the glucose-PTS transporter in S. sanguinis can significantly affect the regulation of factors required for bacterial fitness and homeostatic ability independent of known catabolic regulators. It is then discussed how these changes may impact the survival of streptococcal species and affect caries onset., Competing Interests: The authors declare no conflict of interest.

Enzyme-IIA (EIIA Glc , Crr) of the phosphotransferase system (PTS) connects the uptake of glucose-family sugars to the cAMP-Crp regulatory cascade; phosphorylated EIIA Glc enhances cAMP-Crp activity, which then contributes to the antibiotic-mediated accumulation of reactive oxygen species (ROS) and cell death. Defects in PTS cause antibiotic and disinfectant tolerance. We report that mannitol, a carbon source whose uptake does not use EIIA Glc , reduces antibiotic-mediated killing of Escherichia coli without affecting antibiotic minimal inhibitory concentration. Thus, mannitol promotes antibiotic tolerance. The tolerance pathway was defined by the loss of ciprofloxacin lethality from the deletion of ptsI (first gene in PTS), mtlA (mannitol-specific Enzyme-II), cyaA (cAMP synthase), and crp (cAMP receptor protein) but not crr (EIIA Glc ). A crp* mutant, which encodes a constitutively active Crp that bypasses the need for cAMP activation, also decreased mannitol-mediated antibiotic tolerance, as did exogenous cAMP. Thus, inhibition of antibiotic lethality by mannitol involves both PTS-mediated mannitol uptake and suppression of cAMP-Crp action, independent of EIIA Glc . Mannitol suppressed the downstream antibiotic-mediated transcription of genes involved in NADH production and cellular respiration, expression of a superoxide reporter gene ( soxS ), and accumulation of antibiotic-mediated ROS. Similar phenomena were observed with mannose and sorbitol, demonstrating that non-glucose PTS carbon sources can cause antibiotic tolerance by a novel path that reduces the ROS-promoting activity of cAMP-Crp. The work emphasizes that antibiotic tolerance, which contributes to disease relapse and the need for prolonged antibiotic treatment, can result from commonly consumed carbohydrates. This finding, plus mutations that interfere specifically with antibiotic lethality, makes tolerance a high probability event.IMPORTANCEBacterial tolerance constitutes a significant threat to anti-infective therapy and potentially to the use of disinfectants. Deficiency mutations that reduce glucose uptake, central carbon metabolism, and cellular respiration confer antibiotic/disinfectant tolerance by reducing the accumulation of reactive metabolites, such as reactive oxygen species. We identified novel environmental generators of tolerance by showing that non-glucose carbohydrates, such as mannitol, mannose, and sorbitol, generate tolerance to multiple antibiotic classes. Finding that these sugars inhibit a universal, stress-mediated death pathway emphasizes the potential danger of compounds that block the lethal response to severe stress. Immediate practical importance derives from mannitol being a popular food sweetener, a treatment for glaucoma, and a dehydrating agent for treating cerebral edema, including cases caused by bacterial infection: antibiotic tolerance could contra-indicate the use of mannitol and related carbohydrates during antibiotic treatment. Overall, the work shows that the presence of sugars must be considered during antimicrobial and perhaps disinfectant use., Competing Interests: The authors declare no conflict of interest.

Bacterial cellulose (BC) is a renewable biomaterial that has attracted significant attention due to its excellent properties and wide applications. Komagataeibacter xylinus CGMCC 2955 is an important BC-producing strain. It primarily produces BC from glucose while simultaneously generating gluconic acid as a by-product, which acidifies the medium and inhibits BC synthesis. To enhance glucose uptake and BC synthesis, we reconstructed the phosphoenolpyruvate-dependent glucose phosphotransferase system (PTS Glc ) and strengthened glycolysis by introducing heterologous genes, resulting in a recombinant strain (GX08PTS03; Δgcd::ptsHIcrr E. coli ::ptsG E. coli ::pfkA E. coli ). Strain GX08PTS03 efficiently utilized glucose for BC production without accumulating gluconic acid. Subsequently, the fermentation process was systematically optimized. Under optimal conditions, strain GX08PTS03 produced 7.74 g/L of BC after 6 days of static fermentation, with a BC yield of 0.39 g/g glucose, which were 87.41 % and 77.27 % higher than those of the wild-type strain, respectively. The BC produced by strain GX08PTS03 exhibited a longer fiber diameter along with a lower porosity, significantly higher solid content, crystallinity, tensile strength, and Young's modulus. This study is novel in reporting that the engineered PTS Glc -based glucose metabolism could effectively enhance the production and properties of BC, providing a future outlook for the biopolymer industry., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

In E. coli K-12, the absence of unphosphorylated PtsN (unphospho-PtsN) has been proposed to cause an L-leucine-sensitive growth phenotype (Leu S ) by hyperactivated K + uptake mediated impairment of the expression of the ilvBN operon, encoding subunits of the L-valine (Val)-sensitive acetohydroxyacid synthase I (AHAS I) that renders residual AHAS activity susceptible to inhibition by Leu and K + . This leads to AHAS insufficiency and a requirement for L-isoleucine (Ile). Herein, we provide an alternate mechanism for the Leu S of the ∆ptsN mutant. Genetic and physiological studies with suppressors of the Leu S indicate that impaired expression of the ilvBN operon jointly caused by the absence of unphospho-PtsN and the presence of Leu coupled to Leu-mediated repression of expression of AHAS III leads to AHAS insufficiency rendering residual AHAS activity susceptible to chronic Val stress that may be generated by exogenous Leu. Hyperactivated K + uptake and an elevated α-ketobutyrate level mediate elevation of ilvBN expression and alleviate the Leu S . The requirement of unphospho-PtsN as a positive regulator of ilvBN expression may buffer Ile biosynthesis against Leu-mediated AHAS insufficiency and protect AHAS I function from chronic endogenous Val generated by Leu and could be realized in certain environments that impair AHAS function., (© 2024 John Wiley & Sons Ltd.)

Glucose is the primary source of energy for many organisms and is efficiently taken up by bacteria through a dedicated transport system that exhibits high specificity. In Escherichia coli, the glucose-specific transporter IICB Glc serves as the major glucose transporter and functions as a component of the phosphoenolpyruvate-dependent phosphotransferase system. Here, we report cryo-electron microscopy (cryo-EM) structures of the glucose-bound IICB Glc protein. The dimeric transporter embedded in lipid nanodiscs was captured in the occluded, inward- and occluded, outward-facing conformations. Together with biochemical and biophysical analyses, and molecular dynamics (MD) simulations, we provide insights into the molecular basis and dynamics for substrate recognition and binding, including the gates regulating the binding sites and their accessibility. By combination of these findings, we present a mechanism for glucose transport across the plasma membrane. Overall, this work provides molecular insights into the structure, dynamics, and mechanism of the IICB Glc transporter in a native-like lipid environment., (© 2024. The Author(s).)

Streptococcus pyogenes [group A streptococcus (GAS)] is a human pathogen capable of infecting diverse tissues. To successfully infect these sites, GAS must detect available nutrients and adapt accordingly. The phosphoenolpyruvate transferase system (PTS) mediates carbohydrate uptake and metabolic gene regulation to adapt to the nutritional environment. Regulation by the PTS can occur through phosphorylation of transcriptional regulators at conserved PTS-regulatory domains (PRDs). GAS has several PRD-containing stand-alone regulators with regulons encoding both metabolic genes and virulence factors [PRD-containing virulence regulators (PCVRs)]. One is RofA, which regulates the expression of virulence genes in multiple GAS serotypes. It was hypothesized that RofA is phosphorylated by the PTS in response to carbohydrate levels to coordinate virulence gene expression. In this study, the RofA regulon of M1T1 strain 5448 was determined using RNA sequencing. Two operons were consistently differentially expressed across growth in the absence of RofA; the pilus operon was downregulated, and the capsule operon was upregulated. This correlated with increased capsule production and decreased adherence to keratinocytes. Purified RofA-His was phosphorylated in vitro by PTS proteins EI and HPr, and phosphorylated RofA-FLAG was detected in vivo when GAS was grown in low-glucose C medium. Phosphorylated RofA was not observed when C medium was supplemented 10-fold with glucose. Mutations of select histidine residues within the putative PRDs contributed to the in vivo phosphorylation of RofA, although phosphorylation of RofA was still observed, suggesting other phosphorylation sites exist in the protein. Together, these findings support the hypothesis that RofA is a PCVR that may couple sugar metabolism with virulence regulation., Competing Interests: The authors declare no conflict of interest.

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)., (© 2024 The Author(s). Published by S. Karger AG, Basel.)

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., (© 2023. The Author(s), under exclusive licence to International Society for Microbial Ecology.)

The bacterial nitrogen-related phosphotransfer (PTS Ntr ; 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., Competing Interests: The authors declare no conflict of interest.

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., (© 2023. The Author(s).)

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., (© 2022 Applied Microbiology International and John Wiley & Sons Ltd.)

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 (PTS Man ) 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 PTS Man (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., (© 2022. The Author(s).)

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 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 (PTS Ntr ) 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, PtsN 1 is the major functional homolog. The unphosphorylated PtsN 1 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 PTS Ntr -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 PTS Ntr , 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 PTS Ntr , KdpDE, and KdpFABC in bacteria and increasing knowledge on microbiome for various niches, the PTS Ntr -KdpDE-KdpFABC pathway can be globally important in the biosphere.

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-Crp–mediated death process can help better control pathogens and maintain beneficial microbiota during antimicrobial treatment.

The par EF0409 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 par EF0409 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 par EF0409 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.

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 H 2 O 2 , 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.

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 β-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.

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., (© 2021 John Wiley & Sons Ltd.)

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 (EII Glc ). 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 EII Nag , E. coli Mlc interacts with the EIIB domain of both EII Glc and EII Nag . The present data suggest that EII Nag may be the primordial regulator of Mlc, and EII Glc has evolved to interact with Mlc since an EIIA domain was fused to EII Nag 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., (© 2021 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Background: The phosphotransferase system (PTS) modulates the preferential use of sugars in bacteria. It is formed by a protein cascade in which the first two proteins are general (namely enzyme I, EI, and the histidine phosphocarrier protein, HPr) and the others are sugar-specific permeases; the active site of HPr is His15. The HPr kinase/phosphorylase (HPrK/P), involved in the use of carbon sources in Gram-positive, phopshorylates HPr at a serine. The regulator of sigma D protein (Rsd) also binds to HPr. We are designing specific fragments of HPr, which can be used to interfere with those protein-protein interactions (PPIs), where the intact HPr intervenes., Methods: We obtained a fragment (HPr48) comprising the first forty-eight residues of HPr. HPr48 was disordered as shown by fluorescence, far-ultraviolet (UV) circular dichroism (CD), small angle X-ray scattering (SAXS) and nuclear magnetic resonance (NMR)., Results: Secondary structure propensities, from the assigned backbone nuclei, further support the unfolded nature of the fragment. However, HPr48 was capable of binding to: (i) the N-terminal region of EI, EIN; (ii) the intact Rsd; and, (iii) HPrK/P, as shown by fluorescence, far-UV CD, NMR and biolayer interferometry (BLI). The association constants for each protein, as measured by fluorescence and BLI, were in the order of the low micromolar range, similar to those measured between the intact HPr and each of the other macromolecules., Conclusions: Although HPr48 is forty-eight-residue long, it assisted antibiotics to exert antimicrobial activity., General Significance: HPr48 could be used as a lead compound in the development of new antibiotics, or, alternatively, to improve the efficiency of existing ones., (Copyright © 2021 Elsevier B.V. All rights reserved.)

The nitrogen-related PTS Ntr system, present in many Proteobacteria including Escherichia coli, acts as a phosphorelay cascade composed of the EI Ntr , NPr and EIIA Ntr proteins. Phosphotransfer initiates with phosphoenolpyruvate-dependent EI Ntr autophosphorylation, the phosphoryl group is then transferred to NPr and finally to a conserved histidine residue on EIIA Ntr . The reporter metabolites l-glutamine and 2-oxoglutarate reciprocally regulate EI Ntr autophosphorylation (Lee et al., 2013) and consequently the phosphorylation status of the PTS Ntr components is controlled by the availability of nitrogen and carbon. The final phosphate acceptor, EIIA Ntr , regulates a range of cellular process by acting as the central hub of a complex protein-protein interaction network. Contact between EIIA Ntr and its target proteins is usually regulated by the EIIA Ntr 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 EIIA Ntr and a phosphomimic variant of the protein. The ligand fishing data, along with phenotypic analysis, indicated that EIIA Ntr interacts with proteins related to chemotaxis and thereby regulates cell motility. Important metabolic enzymes were also identified as potential EIIA Ntr binding partners., Competing Interests: Declaration of competing interest Authors have no conflict of interest to declare., (Copyright © 2021 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.)

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 (HPr 9-30 ), 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. HPr 9-30 is mainly disordered. We attempted to improve the affinity of HPr 9-30 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 HPr 9-30 . 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.

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.

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., Competing Interests: The authors declare no competing interest.

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 PTS Fru 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 PTS Fru and its relationship to the V. cholerae life cycle. IMPORTANCE Vibrio 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., (Copyright © 2021 Beck et al.)

Translating ribosomes require elongation factor P (EF-P) to incorporate consecutive prolines (XPPX) into nascent peptide chains. The proteome of Corynebacterium glutamicum ATCC 13032 contains a total of 1,468 XPPX motifs, many of which are found in proteins involved in primary and secondary metabolism. We show here that synthesis of EII Glc , the glucose-specific permease of the phosphoenolpyruvate (PEP): sugar phosphotransferase system (PTS) encoded by ptsG, is strongly dependent on EF-P, as an efp deletion mutant grows poorly on glucose as sole carbon source. The amount of EII Glc is strongly reduced in this mutant, which consequently results in a lower rate of glucose uptake. Strikingly, the XPPX motif is essential for the activity of EII Glc , and substitution of the prolines leads to inactivation of the protein. Finally, translation of GntR2, a transcriptional activator of ptsG, is also dependent on EF-P. However, its reduced amount in the efp mutant can be compensated for by other regulators. These results reveal for the first time a translational bottleneck involving production of the major glucose transporter EII Glc , which has implications for future strain engineering strategies., (© 2020 The Authors. Molecular Microbiology published by John Wiley & Sons Ltd.)

The poles of Escherichia coli cells are emerging as hubs for major sensory systems, but the polar determinants that allocate their components to the pole are largely unknown. Here, we describe the discovery of a previously unannotated protein, TmaR, which localizes to the E. coli cell pole when phosphorylated on a tyrosine residue. TmaR is shown here to control the subcellular localization and activity of the general PTS protein Enzyme I (EI) by binding and polar sequestration of EI, thus regulating sugar uptake and metabolism. Depletion or overexpression of TmaR results in EI release from the pole or enhanced recruitment to the pole, which leads to increasing or decreasing the rate of sugar consumption, respectively. Notably, phosphorylation of TmaR is required to release EI and enable its activity. Like TmaR, the ability of EI to be recruited to the pole depends on phosphorylation of one of its tyrosines. In addition to hyperactivity in sugar consumption, the absence of TmaR also leads to detrimental effects on the ability of cells to survive in mild acidic conditions. Our results suggest that this survival defect, which is sugar- and EI-dependent, reflects the difficulty of cells lacking TmaR to enter stationary phase. Our study identifies TmaR as the first, to our knowledge, E. coli protein reported to localize in a tyrosine-dependent manner and to control the activity of other proteins by their polar sequestration and release., Competing Interests: The authors declare no competing interest.

Streptococcus pyogenes (group A Streptococcus [GAS]), a major human-specific pathogen, relies on efficient nutrient acquisition for successful infection within its host. The phosphotransferase system (PTS) couples the import of carbohydrates with their phosphorylation prior to metabolism and has been linked to GAS pathogenesis. In a screen of an insertional mutant library of all 14 annotated PTS permease (EIIC) genes in MGAS5005, the annotated β-glucoside PTS transporter ( bglP ) was found to be crucial for GAS growth and survival in human blood and was validated in another M1T1 GAS strain, 5448. In 5448, bglP was shown to be in an operon with a putative phospho-β-glucosidase ( bglB ) downstream and a predicted antiterminator ( licT ) upstream. Using defined nonpolar mutants of the β-glucoside permease ( bglP ) and β-glucosidase enzyme ( bglB ) in 5448, we showed that bglB , not bglP , was important for growth in blood. Furthermore, transcription of the licT-blgPB operon was found to be repressed by glucose and induced by the β-glucoside salicin as the sole carbon source. Investigation of the individual bglP and bglB mutants determined that they influence in vitro growth in the β-glucoside salicin; however, only bglP was necessary for growth in other non-β-glucoside PTS sugars, such as fructose and mannose. Additionally, loss of BglP and BglB suggests that they are important for the regulation of virulence-related genes that control biofilm formation, streptolysin S (SLS)-mediated hemolysis, and localized ulcerative lesion progression during subcutaneous infections in mice. Thus, our results indicate that the β-glucoside PTS transports salicin and its metabolism can differentially influence GAS pathophysiology during soft tissue infection., (Copyright © 2020 American Society for Microbiology.)

The nitrogen-related phosphotransferase system (PTS Ntr ) of Rhizobium leguminosarum bv. viciae 3841 transfers phosphate from PEP via PtsP and NPr to two output regulators, ManX and PtsN. ManX controls central carbon metabolism via the tricarboxylic acid (TCA) cycle, while PtsN controls nitrogen uptake, exopolysaccharide production, and potassium homeostasis, each of which is critical for cellular adaptation and survival. Cellular nitrogen status modulates phosphorylation when glutamine, an abundant amino acid when nitrogen is available, binds to the GAF sensory domain of PtsP, preventing PtsP phosphorylation and subsequent modification of ManX and PtsN. Under nitrogen-rich, carbon-limiting conditions, unphosphorylated ManX stimulates the TCA cycle and carbon oxidation, while unphosphorylated PtsN stimulates potassium uptake. The effects are reversed with the phosphorylation of ManX and PtsN, occurring under nitrogen-limiting, carbon-rich conditions; phosphorylated PtsN triggers uptake and nitrogen metabolism, the TCA cycle and carbon oxidation are decreased, while carbon-storage polymers such as surface polysaccharide are increased. Deleting the GAF domain from PtsP makes cells "blind" to the cellular nitrogen status. PTS Ntr constitutes a switch through which carbon and nitrogen metabolism are rapidly, and reversibly, regulated by protein:protein interactions. PTS Ntr is widely conserved in proteobacteria, highlighting its global importance., Competing Interests: The authors declare no competing interest.

The biotechnology-based production of xylitol has received widespread attention because it can use cheap and renewable lignocellulose as a raw material, thereby decreasing costs and pollution. The simultaneous use of various sugars in lignocellulose hydrolysates is a primary prerequisite for efficient xylitol production. In this study, a ΔptsG and crp* combinatorial strategy was used to generate Escherichia coli W3110 strain IS5-dI, which completely eliminated glucose repression and simultaneously used glucose and xylose. This strain produced 164 g/L xylitol from detoxified corncob hydrolysates during a fed-batch fermentation in a 15-L bioreactor, which was 14.7% higher than the xylitol produced by the starting strain, IS5-d (143 g/L), and the xylitol productivity was 3.04 g/L/h. These results represent the highest xylitol concentration and productivity reported to date for bacteria and hemicellulosic sugars. Additionally, strain IS5-dG, which differs from IS5-dI at CRP amino acid residue 127 (I127G), was tolerant to the toxins in corncob hydrolysates. In a fed-batch fermentation experiment involving a 15-L bioreactor, IS5-dG produced 137 g/L xylitol from non-detoxified corncob hydrolysates, with a productivity of 1.76 g/L/h. On the basis of these results, we believe that IS5-dI and IS5-dG may be useful host strains for the industrial-scale production of xylitol from detoxified or non-detoxified corncob hydrolysates.

Rhizosphere interactions between microorganisms and plants have great influence on plant health. Bacillus cereus C1L, an induced systemic resistance (ISR)-eliciting rhizobacterium from Lilium formosanum , can protect monocot and dicot plants from disease challenges. To identify the ISR-involved bacterial genes, the systemic protection effect of transposon-tagged mutants of B. cereus C1L against southern corn leaf blight (SCLB) was surveyed, and a mutant of the ptsG gene encoding glucose-specific permease of the phosphotransferase system was severely impaired in the abilities of disease suppression and root colonization. The ptsG mutant lost the preferential utilization of glucose and showed reduction of glucose-assisted growth in minimal medium. A promoter-based reporter assay revealed that ptsG expression could be activated by certain sugar constituents of maize root exudates, among which B. cereus C1L exhibited the highest chemotactic response toward glucose, whereas neither of them could attract the ptsG mutant. Additionally, ptsG deficiency almost completely abolished glucose uptake of B. cereus C1L. Metabolite analysis indicated that the lack of ptsG undermined glucose-induced accumulation of acetoin and 2,3-butanediol in B. cereus C1L, both eliciting maize ISR against SCLB. Pretreatments with B. cereus C1L, ptsG mutant, acetoin, and 2,3-butanediol enhanced defense-related reactive oxygen species accumulation and callose deposition at different levels that were positively correlated to their ISR-eliciting activities. Thus, glucose uptake-mediating ptsG participates in ISR elicitation by endowing B. cereus C1L with the full capacities for root colonization and beneficial glucose metabolite production, providing a clue regarding how ISR-mediating rhizobacteria create a mutually beneficial relationship with various plant species.

It is of great economic interest to produce succinate from low-grade carbon sources, e.g., lignocellulosic biomass hydrolysate, which mainly contains glucose and xylose. Inactivation of the glucose uptake system PtsG was evaluated for succinate production from xylose-rich feedstocks. Strains with integration of succinate production modules into the chromosome of Escherichia coli were then constructed. These strains have better succinate production performance from xylose-rich feedstocks than strain FZ560 harboring pHL413KF1. Glucose utilization was enhanced in FZ661T by manipulation of the gal operon to allow efficient use of the high-concentration glucose in woody biomass hydrolysate. Up to 906.7 mM (107.0 g/L) succinate was produced from mixed sugars in fed-batch fermentation and more than 461.7 mM (54.5 g/L) succinate was produced from woody hydrolysate in a batch fermentation. In this study, FZ661T was able to produce succinate from woody hydrolysate in minimal medium efficiently, making it attractive for industrial applications in succinate production.

Enterococci are gram-positive pathogens and lead to cause hospital-acquired infections worldwide. Central carbon metabolism was shown as highly induced in Enterococcus faecalis during infection context. Metabolism of α-polysaccharides was previously described as an important factor for host colonisation and biofilm formation. A better characterisation of the adaptation of this bacterium to carbohydrate availabilities may lead to a better understanding of the link between carbohydrate metabolism and the infection process of E. faecalis. Here we show that MalR, a LacI/GalR transcriptional regulator, is the main factor in the regulation of the two divergent operons involved in maltose metabolism in this bacterium. The malR gene is transcribed from the malP promoter, but also from an internal promoter inside the gene located upstream of malR. In the absence of maltose, MalR acts as a repressor and in the presence of glucose, it exerts efficient CcpA-independent carbon catabolite repression. The central PTS protein P-Ser-HPr interacts directly with MalR and enhances its DNA binding capacity, which allows E. faecalis to adapt its metabolism to environmental conditions., (© 2019 John Wiley & Sons Ltd.)

AtxA, the master virulence gene regulator of Bacillus anthracis, is a PRD-Containing Virulence Regulator (PCVR) as indicated by the crystal structure, post-translational modifications and activity of the protein. PCVRs are transcriptional regulators, named for PTS Regulatory Domains (PRDs) subject to phosphorylation by the phosphoenolpyruvate phosphotransferase system (PEP-PTS) and for their impact on virulence gene expression. Here we present data from experiments employing physiological, genetic and biochemical approaches that support a model in which the PTS proteins HPr and Enzyme I (EI) are required for transcription of the atxA gene, rather than phosphorylation of AtxA. We show that atxA transcription is reduced 2.5-fold in a mutant lacking HPr and EI, and that this change is sufficient to affect anthrax toxin production. Mutants harboring HPr proteins altered for phosphotransfer activity were unable to restore atxA transcription to parent levels, suggesting that phosphotransfer activity of HPr and EI is important for regulation of atxA. In a mouse model for anthrax, a HPr - EI - mutant was attenuated for virulence. Virulence was restored by expressing atxA from an alternative, PTS-independent, promoter. Our data support a model in which HPr transfers a phosphate to an unidentified downstream transcriptional regulator to influence atxA gene transcription., (© 2019 John Wiley & Sons Ltd.)

The bicistronic genBA operon (formerly named celBA) of the opportunistic pathogen Enterococcus faecalis, encodes a 6-phospho-β-glucosidase (GenA) and a phosphotransferase system permease EIIC (GenB). It resembles the cel operon of Streptococcus pyogenes, which is implicated in the metabolism of cellobiose. However, genBA mutants grew normally on cellobiose, but not (genA) or only slowly (genB) on gentiobiose and amygdalin. The two glucosides were also found to be the main inducers of the operon, confirming that the encoded proteins are involved in the utilization of β-1,6- rather than β-1,4-linked oligosaccharides. Expression of the genBA operon is regulated by the transcriptional activator GenR, which is encoded by the gene upstream from genB. Thermal shift analysis showed that it binds gentiobiose-6'-P with a Kd of 0.04 mM and with lower affinity also other phospho-sugars. The GenR/gentiobiose-6'-P complex binds to the promoter region upstream from genB. The genBA promoter region contains a cre box and gel-shift experiments demonstrated that the operon is under negative control of the global carbon catabolite regulator CcpA. We also show that the orphan EIIC (GenB) protein needs the EIIA component of the putative OG1RF_10750-OG1RF_10755 operon situated elsewhere on the chromosome to form a functional PTS transporter., (© 2019 John Wiley & Sons Ltd.)

Biofilm formation protects bacteria from stresses including antibiotics and host immune responses. Carbon sources can modulate biofilm formation and host colonization in Vibrio cholerae, but the underlying mechanisms remain unclear. Here, we show that EIIA Glc , a component of the phosphoenolpyruvate (PEP):carbohydrate phosphotransferase system (PTS), regulates the intracellular concentration of the cyclic dinucleotide c-di-GMP, and thus biofilm formation. The availability of preferred sugars such as glucose affects EIIA Glc phosphorylation state, which in turn modulates the interaction of EIIA Glc with a c-di-GMP phosphodiesterase (hereafter referred to as PdeS). In a Drosophila model of V. cholerae infection, sugars in the host diet regulate gut colonization in a manner dependent on the PdeS-EIIA Glc interaction. Our results shed light into the mechanisms by which some nutrients regulate biofilm formation and host colonization.

The multicomponent phosphoenolpyruvate (PEP)-dependent sugar-transporting phosphotransferase system (PTS) in Escherichia coli takes up sugar substrates from the medium and concomitantly phosphorylates them, releasing sugar phosphates into the cytoplasm. We have recently provided evidence that many of the integral membrane PTS permeases interact with the fructose PTS (FruA/FruB) [1]. However, the biochemical and physiological significance of this finding was not known. We have carried out molecular genetic/biochemical/physiological studies that show that interactions of the fructose PTS often enhance, but sometimes inhibit the activities of other PTS transporters many fold, depending on the target PTS system under study. Thus, the glucose (Glc), mannose (Man), mannitol (Mtl) and N-acetylglucosamine (NAG) permeases exhibit enhanced in vivo sugar transport and sometimes in vitro PEP-dependent sugar phosphorylation activities while the galactitol (Gat) and trehalose (Tre) systems show inhibited activities. This is observed when the fructose system is induced to high levels and prevented when the fruA/fruB genes are deleted. Overexpression of the fruA and/or fruB genes in the absence of fructose induction during growth also enhances the rates of uptake of other hexoses. The β-galactosidase activities of man, mtl, and gat-lacZ transcriptional fusions and the sugar-specific transphosphorylation activities of these enzyme transporters were not affected either by frustose induction or by fruAB overexpression, showing that the rates of synthesis of the target PTS permeases were not altered. We thus suggest that specific protein-protein interactions within the cytoplasmic membrane regulate transport in vivo (and sometimes the PEP-dependent phosphorylation activities in vitro) of PTS permeases in a physiologically meaningful way that may help to provide a hierarchy of preferred PTS sugars. These observations appear to be applicable in principle to other types of transport systems as well., Competing Interests: The authors have declared that no competing interests exist.

Nisin is used for food preservation due to its antibacterial activity. However, some bacteria survive under the prevailing conditions owing to the acquisition of resistance. This study aimed to characterize nisin-resistant Enterococcus faecalis isolated from raw buffalo milk and investigate their fitness cost. FE-SEM, biofilm and cytochrome c assay were used for characterization. Growth kinetics, HPLC, qPCR and western blotting were performed to confer their fitness cost. Results revealed that nisin-resistant E. faecalis were morphologically different from sensitive strain and internalize more glucose. However, no significant difference was observed in the growth pattern of the resistant strain compared to that of the sensitive strain. A non-phosphotransferase glucose permease (GlcU) was found to be associated with enhanced glucose uptake. Conversely, Mpt, a major phosphotransferase system responsible for glucose uptake, did not play any role, as confirmed by gene expression studies and western blot analysis of HPr protein. The phosphorylation of His-15 residue of HPr phosphoprotein was reduced, while that of the Ser-46 residue increased with progression in nisin resistance, indicating that it may be involved in the regulation of pathogenicity. In conclusion, resistance imposes a significant fitness cost and GlcU plays a key role in maintaining the fitness cost in nisin-resistant variants., (© FEMS 2019.)

Small RNA (sRNA) regulators promote efficient responses to stress, but the mechanisms for prioritizing target mRNA regulation remain poorly understood. This study examines mechanisms underlying hierarchical regulation by the sRNA SgrS, found in enteric bacteria and produced under conditions of metabolic stress. SgrS posttranscriptionally coordinates a nine-gene regulon to restore growth and homeostasis. An in vivo reporter system quantified SgrS-dependent regulation of target genes and established that SgrS exhibits a clear target preference. Regulation of some targets is efficient even at low SgrS levels, whereas higher SgrS concentrations are required to regulate other targets. In vivo and in vitro analyses revealed that RNA structure and the number and position of base pairing sites relative to the start of translation impact the efficiency of regulation of SgrS targets. The RNA chaperone Hfq uses distinct modes of binding to different SgrS mRNA targets, which differentially influences positive and negative regulation. The RNA degradosome plays a larger role in regulation of some SgrS targets compared to others. Collectively, our results suggest that sRNA selection of target mRNAs and regulatory hierarchy are influenced by several molecular features and that the combination of these features precisely tunes the efficiency of regulation of multi-target sRNA regulons., (© 2019 The Authors. Molecular Microbiology Published by John Wiley & Sons Ltd.)

Carbon catabolite repression is a regulatory mechanism to ensure sequential utilization of carbohydrates and is usually accomplished by repression of genes for the transport and metabolism of less preferred carbon compounds by a more preferred one. Although glucose and mannitol share the general components, enzyme I and HPr, of the phosphoenolpyruvate-dependent phosphotransferase system (PTS) for their transport, glucose represses the transport and metabolism of mannitol in a manner dependent on the mannitol operon repressor MtlR in Escherichia coli. In a recent study, we identified the dephosphorylated form of HPr as a regulator determining the glucose preference over mannitol by interacting with and augmenting the repressor activity of MtlR in E. coli. Here, we determined the X-ray structure of the MtlR-HPr complex at 3.5 Å resolution to understand how phosphorylation of HPr impedes its interaction with MtlR. The phosphorylation site (His15) of HPr is located close to Glu108 and Glu140 of MtlR and phosphorylation at His15 causes electrostatic repulsion between the two proteins. Based on this structural insight and comparative sequence analyses, we suggest that the determination of the glucose preference over mannitol solely by the MtlR-HPr interaction is conserved within  the Enterobacteriaceae family.

There are two paralogous Escherichia coli phosphotransferase systems, one for sugar import (PTS sugar ) and one for nitrogen regulation (PTS Ntr ), that utilize proteins enzyme I sugar (EI sugar ) and HPr, and enzyme I Ntr (EI Ntr ) and NPr, respectively. The enzyme I proteins have similar folds, as do their substrates HPr and NPr, yet they show strict specificity for their cognate partner both in stereospecific protein-protein complex formation and in reversible phosphotransfer. Here, we investigate the mechanism of specific EI Ntr :NPr complex formation by the study of transient encounter complexes. NMR paramagnetic relaxation enhancement experiments demonstrated transient encounter complexes of EI Ntr not only with the expected partner, NPr, but also with the unexpected partner, HPr. HPr occupies transient sites on EI Ntr but is unable to complete stereospecific complex formation. By occupying the non-productive transient sites, HPr promotes NPr transient interaction to productive sites closer to the stereospecific binding site and actually enhances specific complex formation between NPr and EI Ntr . The cellular level of HPr is approximately 150 times higher than that of NPr. Thus, our finding suggests a potential mechanism for cross-regulation of enzyme activity through formation of competitive encounter complexes., (Published by Elsevier Ltd.)

Bacteriocins are bacterial antimicrobial peptides that, unlike classical peptide antibiotics, are products of ribosomal synthesis and usually have a narrow spectrum of antibacterial activity against species closely related to the producers. Pediocin-like bacteriocins (PLBs) belong to the class IIa of the bacteriocins of Gram-positive bacteria. PLBs possess high activity against pathogenic bacteria from Listeria and Enterococcus genera. Molecular target for PLBs is a membrane protein complex - bacterial mannose-phosphotransferase. PLBs can be synthesized by components of symbiotic microflora and participate in the maintenance of homeostasis in various compartments of the digestive tract and on the surface of epithelial tissues contacting the external environment. PLBs could give a rise to a new group of antibiotics of narrow spectrum of activity.

Many bacteria take up carbohydrates by membrane-integral sugar specific phosphoenolpyruvate-dependent carbohydrate:phosphotransferase systems (PTS). Although the PTS is centrally involved in regulation of carbon metabolism in different bacteria, little is known about localization and putative oligomerization of the permease subunits (EII). Here, we analyzed localization of the fructose specific PtsF and the glucose specific PtsG transporters, as well as the general components EI and HPr from Corynebacterium glutamicum using widefield and single molecule localization microscopy. PtsF and PtsG form membrane embedded clusters that localize in a punctate pattern. Size, number and fluorescence of the membrane clusters change upon presence or absence of the transported substrate, and a direct influence of EI and HPr was not observed. In presence of the transport substrate, EII clusters significantly increased in size. Photo-activated localization microscopy data revealed that, in presence of different carbon sources, the number of EII proteins per cluster remains the same, however, the density of these clusters reduces. Our work reveals a simple mechanism for efficient membrane occupancy regulation. Clusters of PTS EII transporters are densely packed in absence of a suitable substrate. In presence of a transported substrate, the EII proteins in individual clusters occupy larger membrane areas., (© 2019 The Authors. Molecular Microbiology Published by John Wiley & Sons Ltd.)

Signal transduction systems dictate various cellular behaviors in response to environmental changes. To operate cellular programs appropriately, organisms have sophisticated regulatory factors to optimize the signal response. The PhoP/PhoQ master virulence regulatory system of the intracellular pathogen Salmonella enterica is activated inside acidic macrophage phagosomes. Here we report that Salmonella delays the activation of this system inside macrophages using an inhibitory protein, EIIA Ntr (a component of the nitrogen-metabolic phosphotransferase system). We establish that EIIA Ntr directly restrains PhoP binding to its target promoter, thereby negatively controlling the expression of PhoP-activated genes. PhoP furthers its activation by promoting Lon-mediated degradation of EIIA Ntr at acidic pH. These results suggest that Salmonella ensures robust activation of its virulence system by suspending the activation of PhoP until a sufficient level of active PhoP is present to overcome the inhibitory effect of EIIA Ntr Our findings reveal how a pathogen precisely and efficiently operates its virulence program during infection. IMPORTANCE To accomplish successful infection, pathogens must operate their virulence programs in a precise, time-sensitive, and coordinated manner. A major question is how pathogens control the timing of virulence gene expression during infection. Here we report that the intracellular pathogen Salmonella controls the timing and level of virulence gene expression by using an inhibitory protein, EIIA Ntr A DNA binding master virulence regulator, PhoP, controls various virulence genes inside acidic phagosomes. Salmonella decreases EIIA Ntr amounts at acidic pH in a Lon- and PhoP-dependent manner. This, in turn, promotes expression of the PhoP-activated virulence program because EIIA Ntr hampers activation of PhoP-regulated genes by interfering with PhoP binding to DNA. EIIA Ntr enables Salmonella to impede the activation of PhoP-regulated gene expression inside macrophages. Our findings suggest that Salmonella achieves programmed delay of virulence gene activation by adjusting levels of an inhibitory factor., (Copyright © 2019 Choi et al.)

Background: Many bacteria transport cellobiose via a phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS). In Listeria monocytogenes, two pairs of soluble PTS components (EIIACel1/EIIBCel1 and EIIACel2/EIIBCel2) and the permease EIICCel1 were suggested to contribute to cellobiose uptake. Interestingly, utilization of several carbohydrates, including cellobiose, strongly represses virulence gene expression by inhibiting PrfA, the virulence gene activator., Results: The LevR-like transcription regulator CelR activates expression of the cellobiose-induced PTS operons celB1-celC1-celA1, celB2-celA2, and the EIIC-encoding monocistronic celC2. Phosphorylation by P∼His-HPr at His550 activates CelR, whereas phosphorylation by P∼EIIBCel1 or P∼EIIBCel2 at His823 inhibits it. Replacement of His823 with Ala or deletion of both celA or celB genes caused constitutive CelR regulon expression. Mutants lacking EIICCel1, CelR or both EIIACel exhibitedslow cellobiose consumption. Deletion of celC1 or celR prevented virulence gene repression by the disaccharide, but not by glucose and fructose. Surprisingly, deletion of both celA genes caused virulence gene repression even during growth on non-repressing carbohydrates. No cellobiose-related phenotype was found for the celC2 mutant., Conclusion: The two EIIA/BCel pairs are similarly efficient as phosphoryl donors in EIICCel1-catalyzed cellobiose transport and CelR regulation. The permanent virulence gene repression in the celA double mutant further supports a role of PTSCel components in PrfA regulation., (© 2019 S. Karger AG, Basel.)

The Bacterial Phosphoenolpyruvate (PEP) : Sugar Phosphotransferase System (PTS) mediates the uptake and phosphorylation of carbohydrates, and controls the carbon- and nitrogen metabolism in response to the availability of sugars. PTS occur in eubacteria and in a few archaebacteria but not in animals and plants. All PTS comprise two cytoplasmic phosphotransferase proteins (EI and HPr) and a species-dependent, variable number of sugar-specific enzyme II complexes (IIA, IIB, IIC, IID). EI and HPr transfer phosphorylgroups from PEP to the IIA units. Cytoplasmic IIA and IIB units sequentially transfer phosphates to the sugar, which is transported by the IIC and IICIID integral membrane protein complexes. Phosphorylation by IIB and translocation by IIC(IID) are tightly coupled. The IIC(IID) sugar transporters of the PTS are in the focus of this review. There are four structurally different PTS transporter superfamilies (glucose, glucitol, ascorbate, mannose) . Crystal structures are available for transporters of two superfamilies: bcIIC mal (MalT, 5IWS, 6BVG) and bcIIC chb (ChbC, 3QNQ) of B. subtilis from the glucose family, and IIC asc (UlaA, 4RP9, 5ZOV) of E. coli from the ascorbate superfamily . They are homodimers and each protomer has an independent transport pathway which functions by an elevator-type alternating-access mechanism. bcIIC mal and bcIIC chb have the same fold, IIC asc has a completely different fold. Biochemical and biophysical data accumulated in the past with the transporters for mannitol (IICBA mtl ) and glucose (IICB glc ) are reviewed and discussed in the context of the bcIIC mal crystal structures. The transporters of the mannose superfamily are dimers of protomers consisting of a IIC and a IID protein chain. The crystal structure is not known and the topology difficult to predict. Biochemical data indicate that the IICIID complex employs a different transport mechanism . Species specific IICIID serve as a gateway for the penetration of bacteriophage lambda DNA across, and insertion of class IIa bacteriocins into the inner membrane. PTS transporters are inserted into the membrane by SecYEG translocon and have specific lipid requirements. Immunoelectron- and fluorescence microscopy indicate a non-random distribution and supramolecular complexes of PTS proteins.

The presence of some sugars (e.g. glucose) downregulates the activity of the Pu promoter of plasmid pWW0 of Pseudomonas putida mt-2, which drives the upper TOL operon for biodegradation of m-xylene. Genetic evidence produced 20 years ago documented an effect of the EIIA Ntr (PtsN) protein of the nitrogen-related phosphoenolpyruvate-dependent phosphotransferase system (PTS Ntr ) in such a C-source control of Pu activity. In this study, we have exploited the wealth of recent information on the PTS of P. putida as well as transcriptomic data available in the last few years on this bacterium to revisit this question - and the role of EIIA Ntr as such. To this end, we examined Pu output under physiological conditions known to either phosphorylate PTS proteins to saturation or to deplete them altogether from high-energy phosphate. The results showed that Pu activity is checked by EIIA Ntr regardless of its phosphorylation state. However, such inhibition is intensified during growth on glucose (which correlates with more phosphate-free EIIA Ntr ) and partially relieved in fructose, which triggers phosphorylation of PTS proteins. These data explain former inconsistencies on the Pu-PTS Ntr interplay and provides a better understanding of the metabolic and regulatory retroactivity between the TOL plasmid and its host metabolism., (© 2018 Society for Applied Microbiology and John Wiley & Sons Ltd.)

The phosphoenolpyruvate-dependent phosphotransferase system (PTS) is the main carbohydrate uptake system in Bacillus subtilis A typical PTS consists of two general proteins, enzyme I (EI) and a histidine-containing protein (HPr), as well as a specific carbohydrate transporter (or enzyme II [EII]), all of which transfer the phosphoryl group from phosphoenolpyruvate to the transported carbohydrate. The specific PTS transporters are formed by multidomain proteins or single-domain subunits. These domains are domain C (EIIC), the transmembrane channel for the carbohydrate transport; domain B (EIIB), the membrane-bound domain responsible for phosphorylation of the carbohydrate; and domain A (EIIA), the mediator between HPr(H15∼P) and EIIB. There are 16 PTS transporters in B. subtilis , 6 of which, i.e., NagP, MalP, MurP, TreP, SacP, and SacX, contain no EIIA domain. Deletion of the single-EIIA-containing transporters showed that there is cross talk between the noncognate EIIA and EIIB domains in PTS. By deletion of all EIIA-containing proteins, strain KM455 (ΔEIIA) was constructed, and the EIIA-containing proteins were individually introduced into the strain. In this way, the PTS transporters of the glucose family, namely, PtsG, GamP, and PtsA (also known as YpqE), enabled growth with maltose, N -acetylglucosamine, sucrose, or trehalose as the sole carbon source. Construction of TkmA-EIIA fusion proteins confirmed the probable interaction between the EIIAs of the glucose family of PTS transporters and the EIIA-deficient PTS transporters. Likewise, we have shown that SacX is mainly phosphorylated by PtsA and GamP. PtsG and GmuA were also able to phosphorylate SacX, albeit less well than GamP and PtsA. IMPORTANCE The phosphoenolpyruvate-dependent phosphotransferase system (PTS) not only is a carbohydrate uptake system in B. subtilis but also plays an important role in sensing the nutrient fluctuation in the medium. This sensing system enables the cells to respond to these fluctuations properly. The PTS transporters have a pivotal role in this sensing system since they are carbohydrate specific. In this study, we tried to understand the interactions among these transporters which revealed the cross talk among PTSs. Three PTS proteins, namely, PtsG (the specific transporter of glucose), GamP (the specific transporter of glucosamine), and PtsA (a cytoplasmic single-domain EIIA protein) were shown to play the major role in the interaction among the PTSs., (Copyright © 2018 Morabbi Heravi and Altenbuchner.)