Phosphoenolpyruvate Phosphotransferase Research Articles

Natural polysaccharide hydrogel with bioadhesion characters to synergistically enhance berberine's antibacterial effect by regulating the PTS system of Staphylococcus aureus

The global spread of Staphylococcus aureus (S. aureus) not only causes significant economic losses but also poses a serious threat to public health. Consequently, there is an urgent need for multidimensional strategies to develop antimicrobial dressings to combat bacterial infections. In response, we have developed a plant polysaccharide antibacterial hydrogel formed through the self-assembly of edible Kudzu powder (KP) and the natural star molecule berberine (BBR). Rheological tests show that natural polysaccharide KP-BBR hydrogel (BBR@KP) exhibits excellent injectability and adhesion. And the degradation of the hydrogel exceeded 90 % within 3 days. The synergistic effect of these two ingredients enhances the antibacterial activity of BBR and can increase the MIC of BBR to 0.05 mM. Specifically, KP promotes the affinity of the Phosphoenolpyruvate Phosphotransferase System (PTS) of S. aureus, enabling KP, with its bioadhesive properties, to adhere to the bacterial surface and continuously release BBR. Subsequently, BBR effectively exerts its antibacterial effect by inhibiting the synthesis of histidine and isoleucine. Furthermore, the BBR@KP hydrogel exhibits negligible cytotoxicity and hemolytic toxicity, underscoring its favorable biosafety profile. This synergistic natural antibacterial hydrogel, formulated through a green and straightforward methodology, not only holds promise for broad clinical applications but also provides novel perspectives for the utilization and development of plant polysaccharides in the biomedical field.

AI-2 quorum sensing-induced galactose metabolism activation in Streptococcus suis enhances capsular polysaccharide-associated virulence

Bacteria utilize intercellular communication to orchestrate essential cellular processes, adapt to environmental changes, develop antibiotic tolerance, and enhance virulence. This communication, known as quorum sensing (QS), is mediated by the exchange of small signalling molecules called autoinducers. AI-2 QS, regulated by the metabolic enzyme LuxS (S-ribosylhomocysteine lyase), acts as a universal intercellular communication mechanism across gram-positive and gram-negative bacteria and is crucial for diverse bacterial processes. In this study, we demonstrated that in Streptococcus suis (S. suis), a notable zoonotic pathogen, AI-2 QS enhances galactose utilization, upregulates the Leloir pathway for capsular polysaccharide (CPS) precursor production, and boosts CPS synthesis, leading to increased resistance to macrophage phagocytosis. Additionally, our molecular docking and dynamics simulations suggest that, similar to S. pneumoniae, FruA, a fructose-specific phosphoenolpyruvate phosphotransferase system prevalent in gram-positive pathogens, may also function as an AI-2 membrane surface receptor in S. suis. In conclusion, our study demonstrated the significance of AI-2 in the synthesis of galactose metabolism-dependent CPS in S. suis. Additionally, we conducted a preliminary analysis of the potential role of FruA as a membrane surface receptor for S. suis AI-2.

In-patient evolution of a high-persister Escherichia coli strain with reduced in vivo antibiotic susceptibility.

Gram-negative bacterial bloodstream infections (GNB-BSI) are common and frequently lethal. Despite appropriate antibiotic treatment, relapse of GNB-BSI with the same bacterial strain is common and associated with poor clinical outcomes and high healthcare costs. The role of persister cells, which are sub-populations of bacteria that survive for prolonged periods in the presence of bactericidal antibiotics, in relapse of GNB-BSI is unclear. Using a cohort of patients with relapsed GNB-BSI, we aimed to determine how the pathogen evolves within the patient between the initial and subsequent episodes of GNB-BSI and how these changes impact persistence. Using Escherichia coli clinical bloodstream isolate pairs (initial and relapse isolates) from patients with relapsed GNB-BSI, we found that 4/11 (36%) of the relapse isolates displayed a significant increase in persisters cells relative to the initial bloodstream infection isolate. In the relapsed E. coli strain with the greatest increase in persisters (100-fold relative to initial isolate), we determined that the increase was due to a loss-of-function mutation in the ptsI gene encoding Enzyme I of the phosphoenolpyruvate phosphotransferase system. The ptsI mutant was equally virulent in a murine bacteremia infection model but exhibited 10-fold increased survival to antibiotic treatment. This work addresses the controversy regarding the clinical relevance of persister formation by providing compelling data that not only do high-persister mutations arise during bloodstream infection in humans but also that these mutants display increased survival to antibiotic challenge in vivo.

224. Clinical Relevance of Antibiotic Tolerance in Recurrent Gram-negative Bloodstream Infections

Abstract Background Gram-negative bacterial bloodstream infections (GNB-BSI) are common and frequently lethal. Many patients experience recurrent GNB-BSI for unclear reasons. This study explores how antibiotic tolerance may lead to infection relapse.Figure 1:Flow diagram of relapsed versus recurrent infection. Approximately 63% of recurrent GNB-BSI are due to relapse (almost genetically identical isolate) and 37% due to reinfection (genetically distinct isolate).Figure 2:Relapsed GNB-BSI isolate exhibits increased antibiotic tolerance in murine bacteremia model. BALB/c mice infected with paired initial or relapsed GNB-BSI isolate. 50 mg/kg ertapenem added at 3 hours post infection (hpi). Graph shows recovery of viable bacteria from liver tissue. Methods We used a prospective cohort of patients with GNB-BSI at Duke Hospital to identify patients with >1 episode of GNB-BSI due to the same bacterial species. We used whole genome sequencing (WGS) to distinguish reinfection (recurrent infection with different isolate) from relapse (recurrent infection with near-identical isolate). Time-kill curves with meropenem were used to determine the development of antibiotic tolerance between the initial and relapsed Escherichia coli isolates. The biological relevance of antibiotic tolerance was tested using a murine bacteremia model. Results We determined that 63% (30/48) of recurrent GNB-BSI episodes were due to relapse and 37% (18/48) were due to reinfection. We screened 10 relapsed E. coli pairs (initial and relapsed isolates) using meropenem to identify increases in antibiotic tolerance. One isolate pair showed a 100-fold increase in multidrug antibiotic tolerance. We determined the decreased antibiotic killing was due to a loss-of-function mutation in the ptsI gene encoding Enzyme I of the phosphoenolpyruvate phosphotransferase system. To test if the in-vitro tolerance phenotype translated to decreased antibiotic efficacy in-vivo, we developed a murine model of E. coli bacteremia. In our murine bacteremia model, the ptsI mutant was equally virulent as the wild-type, but exhibited 10-fold less killing during antibiotic treatment. Conclusion Our work provides a unique insight into the molecular changes occurring during GNB-BSI and how the pathogen adapts to the host through acquisition of antibiotic tolerance. We address the controversy regarding the clinical relevance of antibiotic tolerance in-vivo by providing compelling data that not only do these mutations arise during bloodstream infection in humans, but the presence of antibiotic tolerance in-vitro likely leads to decreased antibiotic efficacy in-vivo. Further work is required to determine if early detection of antibiotic tolerance could lead to alterations in medical management and ultimately improve patient outcomes. Disclosures Vance G. Fowler, MD, MHS, Amphliphi Biosciences, Integrated Biotherapeutics; C3J, Armata, Valanbio; Akagera, Aridis, Roche, Astra Zeneca: Advisor/Consultant|Genentech, Regeneron, Deep Blue, Basilea, Janssen;: Grant/Research Support|Infectious Diseases Society of America: Honoraria|MedImmune, Allergan, Pfizer, Advanced Liquid Logics, Theravance, Novartis, Merck; Medical Biosurfaces; Locus; Affinergy; Contrafect; Karius;: Grant/Research Support|Novartis, Debiopharm, Genentech, Achaogen, Affinium, Medicines Co., MedImmune, Bayer, Basilea, Affinergy, Janssen, Contrafect, Regeneron, Destiny,: Advisor/Consultant|Sepsis diagnostic: Patent pending|UpToDate: Royalties|Valanbio and ArcBio: Stock Options Joshua T. Thaden, MD, PhD, Resonantia Diagnostics, Inc: Advisor/Consultant

Multi-integrated approach for unraveling small open reading frames potentially associated with secondary metabolism in Streptomyces.

Small open reading frames (smORFs) are widely distributed in various living organisms. However, their functions remain largely unexplored. In addition, annotation and detection of smORFs are limited using existing methods and hindered by their specific properties. In this study, we systematically investigated smORFs and smORF-encoded peptides (SEPs) in Streptomyces, which are well-known bacterial producers of diverse bioactive secondary metabolites. We established a peptidogenomic workflow based on multi-integrated comprehensive database search and database-independent de novo sequencing to identify smORFs in Streptomyces xinghaiensis NRRL B-24674T (S187). In addition, we described SEPome related to the secondary metabolism, which include 68 novel SEPs and 79 common smORFs with Streptomyces coelicolor A3 (2). Functional analysis of universal smORFs revealed enrichment in biosynthetic processes, stress response, ribosomes, and nucleic acid binding. Meanwhile, 5 Cryptic smORF-encoded Peptides (CSEPs) distributed in non-annotated regions of the genome, and non-coding RNAs could encode for CSEPs. A total of 66 new RNAs, including 32 non-coding RNAs (ncRNAs) were revealed, and 4 ncRNA-encoded peptides were identified. Furthermore, an investigation of carbon metabolism showed that NagE functions in spore formation and secondary metabolism in Streptomyces. Particularly, NagE was observed to function in the biosynthesis of anti-complement agents in S. xinghaiensis, suggesting a novel role of the phosphoenolpyruvate phosphotransferase system in microbial secondary metabolism. We thus provide an effective strategy for analyzing public data sets of model strains to identify smORFs for non-model species. The ncRNAs and SEPs present rich sources for engineering streptomycetes to produce bioactive compounds. IMPORTANCE Due to their small size and special chemical features, small open reading frame (smORF)-encoding peptides (SEPs) are often neglected. However, they may play critical roles in regulating gene expression, enzyme activity, and metabolite production. Studies on bacterial microproteins have mainly focused on pathogenic bacteria, which are importance to systematically investigate SEPs in streptomycetes and are rich sources of bioactive secondary metabolites. Our study is the first to perform a global identification of smORFs in streptomycetes. We established a peptidogenomic workflow for non-model microbial strains and identified multiple novel smORFs that are potentially linked to secondary metabolism in streptomycetes. Our multi-integrated approach in this study is meaningful to improve the quality and quantity of the detected smORFs. Ultimately, the workflow we established could be extended to other organisms and would benefit the genome mining of microproteins with critical functions for regulation and engineering useful microorganisms.

Transcriptome analysis reveals the molecular mechanism of cinnamaldehyde against Bacillus cereus spores in ready-to-eat beef

The purpose of this study was to investigate the antibacterial effect and mechanism of cinnamaldehyde on Bacillus cereus spores in ready-to-eat beef. The colour difference and texture of the ready-to-eat beef supplemented with cinnamaldehyde did not differ greatly from the colour and texture of the blank beef. However, cinnamaldehyde has an effective antibacterial effect on the total number of bacterial colonies and B. cereus spores in ready-to-eat beef. Transmission electron microscopy (TEM) analysis revealed that the cell membrane of B. cereus was disrupted by cinnamaldehyde, leading to leakage of intracellular components. Transcriptome sequencing (RNA-seq) indicated that the B. cereus spore resistance regulation system (sigB, sigW, rsbW, rsbV, yfkM and yflT) and phosphoenolpyruvate phosphotransferase system (PTS) (ptsH, ptsI and ptsG) respond positively to cinnamaldehyde in an adverse environment. Intracellular disorders due to damage to the cell membrane involve some transporters (copA, opuBA and opuD) and some oxidative stress systems (ywrO, scdA and katE) in the regulation of the body. However, downregulation of K+ transport channels (kdpD and kdpB), osmotic pressure regulation (opuE) and some oxidative stress (norR and srrA)-related genes may accelerate spore apoptosis. In addition, cinnamaldehyde also effectively inhibits the spore germination-related genes (smc, mreB and gerE). This study provides new insights into the molecular mechanism of the antibacterial effect of cinnamaldehyde on B. cereus spores in ready-to-eat beef.

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.

Proteomic Analysis of the Colistin-resistant E. coli Clinical Isolate: Explorations of the Resistome.

Antimicrobial resistance is a worldwide problem after the emergence of colistin resistance since it was the last option left to treat carbapenemase-resistant bacterial infections. The mcr gene and its variants are one of the causes for colistin resistance. Besides mcr genes, some other intrinsic genes are also involved in colistin resistance but still need to be explored. The aim of this study was to investigate differential proteins expression of colistin-resistant E. coli clinical isolate and to understand their interactive partners as future drug targets. In this study, we have employed the whole proteome analysis through LC-MS/MS. The advance proteomics tools were used to find differentially expressed proteins in the colistin-resistant Escherichia coli clinical isolate compared to susceptible isolate. Gene ontology and STRING were used for functional annotation and protein-protein interaction networks, respectively. LC-MS/MS analysis showed overexpression of 47 proteins and underexpression of 74 proteins in colistin-resistant E. coli. These proteins belong to DNA replication, transcription and translational process; defense and stress related proteins; proteins of phosphoenol pyruvate phosphotransferase system (PTS) and sugar metabolism. Functional annotation and protein-protein interaction showed translational and cellular metabolic process, sugar metabolism and metabolite interconversion. We conclude that these protein targets and their pathways might be used to develop novel therapeutics against colistin-resistant infections. These proteins could unveil the mechanism of colistin resistance.

PRD-Containing Virulence Regulators (PCVRs) in Pathogenic Bacteria.

Bacterial pathogens rely on a complex network of regulatory proteins to adapt to hostile and nutrient-limiting host environments. The phosphoenolpyruvate phosphotransferase system (PTS) is a conserved pathway in bacteria that couples transport of sugars with phosphorylation to monitor host carbohydrate availability. A family of structurally homologous PTS-regulatory-domain-containing virulence regulators (PCVRs) has been recognized in divergent bacterial pathogens, including Streptococcus pyogenes Mga and Bacillus anthracis AtxA. These paradigm PCVRs undergo phosphorylation, potentially via the PTS, which impacts their dimerization and their activity. Recent work with predicted PCVRs from Streptococcus pneumoniae (MgaSpn) and Enterococcus faecalis (MafR) suggest they interact with DNA like nucleoid-associating proteins. Yet, Mga binds to promoter sequences as a homo-dimeric transcription factor, suggesting a bi-modal interaction with DNA. High-resolution crystal structures of 3 PCVRs have validated the domain structure, but also raised additional questions such as how ubiquitous are PCVRs, is PTS-mediated histidine phosphorylation via potential PCVRs widespread, do specific sugars signal through PCVRs, and do PCVRs interact with DNA both as transcription factors and nucleoid-associating proteins? Here, we will review known and putative PCVRs based on key domain and functional characteristics and consider their roles as both transcription factors and possibly chromatin-structuring proteins.

Potential Probiotic Pediococcus pentosaceus M41 Modulates Its Proteome Differentially for Tolerances Against Heat, Cold, Acid, and Bile Stresses.

Probiotics containing functional food confer health benefits in addition to their nutritional properties. In this study, we have evaluated the differential proteomic responses of a potential novel probiotic Pediococcus pentosaceus M41 under heat, cold, acid, and bile stress conditions. We identified stress response proteins that could provide tolerances against these stresses and could be used as probiotic markers for evaluating stress tolerance. Pediococcus pentosaceus M41 was exposed for 2 h to each condition: 50°C (heat stress), 4°C (cold stress), pH 3.0 (acid stress) and 0.05% bile (bile stress). Proteomic analysis was carried out using 2D-IEF SDS PAGE and LC-MS/MS. Out of 60 identified proteins, 14 upregulated and 6 downregulated proteins were common among all the stress conditions. These proteins were involved in different biological functions such as translation-related proteins, carbohydrate metabolism (phosphoenolpyruvate phosphotransferase), histidine biosynthesis (imidazole glycerol phosphate synthase) and cell wall synthesis (tyrosine-protein kinase CapB). Proteins such as polysaccharide deacetylase, lactate oxidase, transcription repressor NrdR, dihydroxyacetone kinase were upregulated under three out of the four stress conditions. The differential expression of these proteins might be responsible for tolerance and protection of P. pentosaceus M41 against different stress conditions.

Parallel Evolution of Tobramycin Resistance across Species and Environments.

Different species exposed to a common stress may adapt by mutations in shared pathways or in unique systems, depending on how past environments have molded their genomes. Understanding how diverse bacterial pathogens evolve in response to an antimicrobial treatment is a pressing example of this problem, where discovery of molecular parallelism could lead to clinically useful predictions. Evolution experiments with pathogens in environments containing antibiotics, combined with periodic whole-population genome sequencing, can be used to identify many contending routes to antimicrobial resistance. We separately propagated two clinically relevant Gram-negative pathogens, Pseudomonas aeruginosa and Acinetobacter baumannii, in increasing concentrations of tobramycin in two different environments each: planktonic and biofilm. Independently of the pathogen, the populations adapted to tobramycin selection by parallel evolution of mutations in fusA1, encoding elongation factor G, and ptsP, encoding phosphoenolpyruvate phosphotransferase. As neither gene is a direct target of this aminoglycoside, mutations to either are unexpected and underreported causes of resistance. Additionally, both species acquired antibiotic resistance-associated mutations that were more prevalent in the biofilm lifestyle than in the planktonic lifestyle; these mutations were in electron transport chain components in A. baumannii and lipopolysaccharide biosynthesis enzymes in P. aeruginosa populations. Using existing databases, we discovered site-specific parallelism of fusA1 mutations that extends across bacterial phyla and clinical isolates. This study suggests that strong selective pressures, such as antibiotic treatment, may result in high levels of predictability in molecular targets of evolution, despite differences between organisms' genetic backgrounds and environments.IMPORTANCE The rise of antimicrobial resistance is a leading medical threat, motivating efforts to forecast both its evolutionary dynamics and its genetic causes. Aminoglycosides are a major class of antibiotics that disrupt translation, but resistance may occur by a number of mechanisms. Here, we show the repeated evolution of resistance to the aminoglycoside tobramycin in both P. aeruginosa and A. baumannii via mutations in fusA1, encoding elongation factor G, and ptsP, encoding the nitrogen-specific phosphotransferase system. Laboratory evolution and whole-population genome sequencing were used to identify these targets, but mutations at identical amino acid positions were also found in published genomes of diverse bacterial species and clinical isolates. We also identified other resistance mechanisms associated with growth in biofilms that likely interfere with drug binding or uptake. Characterizing the evolution of multiple species in the presence of antibiotics can identify new, repeatable causes of resistance that may be predicted and counteracted by alternative treatment.

Proteomic analysis reveals potential factors associated with enhanced EPS production in Streptococcus thermophilus ASCC 1275

Streptococcus thermophilus ASCC 1275 has two chain length determining genes - epsC and epsD- in its eps gene cluster, and produces two times more EPS in sucrose medium than that in glucose and lactose. Hence, we investigated the influence of sugars (glucose, sucrose and lactose), at log phase (5 h) and stationary phase (10 h), on the global proteomics of S. thermophilus 1275 to understand the differentially expressed proteins (DEPs) during EPS production using isobaric tags for relative and absolute quantitation (iTRAQ)-based proteomic analysis. Among 98 DEPs in sucrose medium, most of them were mapped into EPS biosynthesis pathway and other related metabolisms. There was an upregulation of several proteins involved in sugar transport (phosphoenolpyruvate (PEP) phosphotransferase system), EPS assembly (epsG1D) and amino acid metabolism (methionine, cysteine/arginine metabolism) in sucrose medium. This study showed that increased EPS production in S. thermophilus 1275 requires a well-co-ordinated regulation of pathway involved in both EPS assembly and amino acid metabolism along with the availability of sugars. Thus, it provided valuable insights into the biosynthesis and regulation of EPS in S. thermophilus 1275, and potential gene targets for understanding high-EPS strains.

Identification of Regulatory Genes and Metabolic Processes Important for Alginate Biosynthesis in Azotobacter vinelandii by Screening of a Transposon Insertion Mutant Library.

Azotobacter vinelandii produces the biopolymer alginate, which has a wide range of industrial and pharmaceutical applications. A random transposon insertion mutant library was constructed from A. vinelandii ATCC12518Tc in order to identify genes and pathways affecting alginate biosynthesis, and about 4,000 mutant strains were screened for altered alginate production. One mutant, containing a mucA disruption, displayed an elevated alginate production level, and several mutants with decreased or abolished alginate production were identified. The regulatory proteins AlgW and AmrZ seem to be required for alginate production in A. vinelandii, similarly to Pseudomonas aeruginosa. An algB mutation did however not affect alginate yield in A. vinelandii although its P. aeruginosa homolog is needed for full alginate production. Inactivation of the fructose phosphoenolpyruvate phosphotransferase system protein FruA resulted in a mutant that did not produce alginate when cultivated in media containing various carbon sources, indicating that this system could have a role in regulation of alginate biosynthesis. Furthermore, impaired or abolished alginate production was observed for strains with disruptions of genes involved in peptidoglycan biosynthesis/recycling and biosynthesis of purines, isoprenoids, TCA cycle intermediates, and various vitamins, suggesting that sufficient access to some of these compounds is important for alginate production. This hypothesis was verified by showing that addition of thiamine, succinate or a mixture of lysine, methionine and diaminopimelate increases alginate yield in the non-mutagenized strain. These results might be used in development of optimized alginate production media or in genetic engineering of A. vinelandii strains for alginate bioproduction.

Influence of the phosphoenolpyruvate:carbohydrate phosphotransferase system on toxin gene expression and virulence in Bacillus anthracis.

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.

Cell Factory Design and Culture Process Optimization for Dehydroshikimate Biosynthesis in Escherichia coli.

3-Dehydroshikimate (DHS) is a useful starting metabolite for the biosynthesis of muconic acid (MA) and shikimic acid (SA), which are precursors of various valuable polymers and drugs. Although DHS biosynthesis has been previously reported in several bacteria, the engineered strains were far from satisfactory, due to their low DHS titers. Here, we created an engineered Escherichia coli cell factory to produce a high titer of DHS as well as an efficient system for the conversion DHS into MA. First, the genes showing negative effects on DHS accumulation in E. coli, such as tyrR (tyrosine dependent transcriptional regulator), ptsG (glucose specific sugar: phosphoenolpyruvate phosphotransferase), and pykA (pyruvate kinase 2), were disrupted. In addition, the genes involved in DHS biosynthesis, such as aroB (DHQ synthase), aroD (DHQ dehydratase), ppsA (phosphoenolpyruvate synthase), galP (D-galactose transporter), aroG (DAHP synthase), and aroF (DAHP synthase), were overexpressed to increase the glucose uptake and flux of intermediates. The redesigned DHS-overproducing E. coli strain grown in an optimized medium produced ~117 g/L DHS in 7-L fed-batch fermentation, which is the highest level of DHS production demonstrated in E. coli. To accomplish the DHS-to-MA conversion, which is originally absent in E. coli, a codon-optimized heterologous gene cassette containing asbF, aroY, and catA was expressed as a single operon under a strong promoter in a DHS-overproducing E. coli strain. This redesigned E. coli grown in an optimized medium produced about 64.5 g/L MA in 7-L fed-batch fermentation, suggesting that the rational cell factory design of DHS and MA biosynthesis could be a feasible way to complement petrochemical-based chemical processes.

HprKXcc is a serine kinase that regulates virulence in the Gram‐negative phytopathogen Xanthomonas campestris

SummaryThe HprK serine kinase is a component of the phosphoenolpyruvate phosphotransferase system (PTS) of bacteria that generally regulates catabolite repression through phosphorylation/dephosphorylation of the PTS protein PtsH at a conserved serine residue. However, many bacteria do not encode a complete PTS or even have an HprK homologue. Xanthomonas campestris pv. campestris (Xcc) is a pathogen that cause black rot disease in crucifer plants and one of the few Gram‐negative bacteria that encodes a homologue of HprK protein (herein HprKXcc). To gain insight into the role of HprKXcc and other PTS‐related components in Xcc we individually mutated and phenotypically assessed the resulting strains. Deletion of hprK Xcc demonstrated its requirement for virulence and other diverse cellular processes associated including extracellular enzyme activity, extracellular‐polysaccharide production and cell motility. Global transcriptome analyses revealed the HprKXcc had a broad regulatory role in Xcc. Additionally, through overexpression, double gene deletion and transcriptome analysis we demonstrated that hprK Xcc shares an epistatic relationship with ptsH. Furthermore, we demonstrate that HprKXcc is a functional serine kinase, which has the ability to phosphorylate PtsH. Taken together, the data illustrates the previously unappreciated global regulatory role of HprKXcc and previously uncharacterized PTS components that control virulence in this pathogen.

Removal of a Membrane Anchor Reveals the Opposing Regulatory Functions of Vibrio cholerae Glucose-Specific Enzyme IIA in Biofilms and the Mammalian Intestine.

The Vibrio cholerae phosphoenolpyruvate phosphotransferase system (PTS) is a well-conserved, multicomponent phosphotransfer cascade that coordinates the bacterial response to carbohydrate availability through direct interactions of its components with protein targets. One such component, glucose-specific enzyme IIA (EIIAGlc), is a master regulator that coordinates bacterial metabolism, nutrient uptake, and behavior by direct interactions with cytoplasmic and membrane-associated protein partners. Here, we show that an amphipathic helix (AH) at the N terminus of V.cholerae EIIAGlc serves as a membrane association domain that is dispensable for interactions with cytoplasmic partners but essential for regulation of integral membrane protein partners. By deleting this AH, we reveal previously unappreciated opposing regulatory functions for EIIAGlc at the membrane and in the cytoplasm and show that these opposing functions are active in the laboratory biofilm and the mammalian intestine. Phosphotransfer through the PTS proceeds in the absence of the EIIAGlc AH, while PTS-dependent sugar transport is blocked. This demonstrates that the AH couples phosphotransfer to sugar transport and refutes the paradigm of EIIAGlc as a simple phosphotransfer component in PTS-dependent transport. Our findings show that Vibrio cholerae EIIAGlc, a central regulator of pathogen metabolism, contributes to optimization of bacterial physiology by integrating metabolic cues arising from the cytoplasm with nutritional cues arising from the environment. Because pathogen carbon metabolism alters the intestinal environment, we propose that it may be manipulated to minimize the metabolic cost of intestinal infection.IMPORTANCE The V.cholerae phosphoenolpyruvate phosphotransferase system (PTS) is a well-conserved, multicomponent phosphotransfer cascade that regulates cellular physiology and virulence in response to nutritional signals. Glucose-specific enzyme IIA (EIIAGlc), a component of the PTS, is a master regulator that coordinates bacterial metabolism, nutrient uptake, and behavior by direct interactions with protein partners. We show that an amphipathic helix (AH) at the N terminus of V.cholerae EIIAGlc serves as a membrane association domain that is dispensable for interactions with cytoplasmic partners but essential for regulation of integral membrane protein partners. By removing this amphipathic helix, hidden, opposing roles for cytoplasmic partners of EIIAGlc in both biofilm formation and metabolism within the mammalian intestine are revealed. This study defines a novel paradigm for AH function in integrating opposing regulatory functions in the cytoplasm and at the bacterial cell membrane and highlights the PTS as a target for metabolic modulation of the intestinal environment.

Transcriptomic Insights Into the Growth Phase- and Sugar-Associated Changes in the Exopolysaccharide Production of a High EPS-Producing Streptococcus thermophilus ASCC 1275.

In a previous study, incorporation of high exopolysaccharide (EPS) producing dairy starter bacterium Streptococcus thermophilus ASCC 1275 was found to improve functionality of low fat mozzarella cheese and yogurt. This bacterium in its eps gene cluster has a unique pair of chain length determining genes, epsC- epsD, when compared to other sequenced S. thermophilus strains. Hence, the aim of this study was to understand the regulatory mechanism of EPS production in this bacterium using transcriptomic analysis to provide opportunities to improve the yield of EPS. As sugars are considered as one of the major determinants of EPS production, after preliminary screening, we selected three sugars, glucose, sucrose and lactose to identify the EPS producing mechanism of this bacterium in M17 medium. Complete RNA-seq analysis was performed using Illumina HiSeq 2000 sequencing system on S. thermophilus 1275 grown in three different sugars at two-time points, 5 h (log phase) and 10 h (stationary phase) to recognize the genes involved in sugar uptake, UDP-sugar formation, EPS assembly and export of EPS outside the bacterial cell. S. thermophilus 1275 was found to produce high amount of EPS (∼430 mg/L) in sucrose (1%) supplemented M17 medium when compared to other two sugars. Differential gene expression analysis revealed the involvement of phosphoenolpyruvate phosphotransferase system (PEP-PTS) for glucose and sucrose uptake, and lacS gene for lactose uptake. The pathways for the formation of UDP-glucose and UDP-galactose were highly upregulated in all the three sugars. In the presence of sucrose, eps1C1D2C2D were found to be highly expressed which refers to high EPS production. Protein homology study suggested the presence of Wzx/Wzy-dependent EPS synthesis and transport pathway in this bacterium. KEGG pathway and COG functional enrichment analysis were also performed to support the result. This is the first report providing the transcriptomic insights into the EPS production mechanism of a common dairy bacterium, S. thermophilus.

Carbon catabolite regulation in Streptomyces: new insights and lessons learned.

One of the most significant control mechanisms of the physiological processes in the genus Streptomyces is carbon catabolite repression (CCR). This mechanism controls the expression of genes involved in the uptake and utilization of alternative carbon sources in Streptomyces and is mostly independent of the phosphoenolpyruvate phosphotransferase system (PTS). CCR also affects morphological differentiation and the synthesis of secondary metabolites, although not all secondary metabolite genes are equally sensitive to the control by the carbon source. Even when the outcome effect of CCR in bacteria is the same, their essential mechanisms can be rather different. Although usually, glucose elicits this phenomenon, other rapidly metabolized carbon sources can also cause CCR. Multiple efforts have been put through to the understanding of the mechanism of CCR in this genus. However, a reasonable mechanism to explain the nature of this process in Streptomyces does not yet exist. Several examples of primary and secondary metabolites subject to CCR will be examined in this review. Additionally, recent advances in the metabolites and protein factors involved in the Streptomyces CCR, as well as their mechanisms will be described and discussed in this review.

Mode of Interaction of the Signal-Transducing Protein EIIA(Glc) with the Maltose ABC Transporter in the Process of Inducer Exclusion.

Enzyme IIA(Glc) (EIIA(Glc)) of the phosphoenolpyruvate phosphotransferase system for the uptake of glucose in Escherichia coli and Salmonella inhibits the maltose ATP-binding cassette transporter (MalE-FGK2) by interaction with the nucleotide-binding and -hydrolyzing subunit MalK, a process termed inducer exclusion. We have investigated binding of EIIA(Glc) to the MalK dimer by cysteine cross-linking in proteoliposomes. The results prove that the binding site I of EIIA(Glc) is contacting the N-terminal subdomain of MalK while the binding site II is relatively close to the C-terminal (regulatory) subdomain, in agreement with a crystal structure [ Chen , S. , Oldham , M. L. , Davidson , A. L. , and Chen , J. ( 2013 ) Nature 499 , 364 - 368 ]. Moreover, EIIA(Glc) was found to bind to the MalK dimer regardless of its conformational state. Deletion of the amphipathic N-terminal peptide of EIIA(Glc), which is required for inhibition, reduced formation of cross-linked products. Using a spin-labeled transporter variant and EPR spectroscopy, we demonstrate that EIIA(Glc) arrests the transport cycle by inhibiting the ATP-dependent closure of the MalK dimer.