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Journal of Molecular Microbiology and... 2015In 1964, Kundig, Ghosh and Roseman reported the discovery of the phosphoenolpyruvate:sugar phosphotransferase system (PTS), which they subsequently proposed might...
In 1964, Kundig, Ghosh and Roseman reported the discovery of the phosphoenolpyruvate:sugar phosphotransferase system (PTS), which they subsequently proposed might catalyze sugar transport as well as sugar phosphorylation. What we have learned in the 50 years since its discovery is that, in addition to these primary functions, the PTS serves as a complex protein kinase system that regulates a wide variety of transport, metabolic and mutagenic processes as well as the expression of numerous genes. Recent operon- and genome-sequencing projects have revealed novel PTS protein-encoding genes, many of which have yet to be functionally defined. The current picture of the PTS is that of a complex system with ramifications in all aspects of cellular physiology. Moreover, its mosaic evolutionary history is unusual and intriguing. The PTS can be considered to serve many prokaryotes in capacities of communication and coordination, as do the nervous systems of animals.
Topics: Bacteria; Escherichia coli; Gene Expression Regulation, Bacterial; Genes, Bacterial; Multigene Family; Operon; Phosphoenolpyruvate Sugar Phosphotransferase System; Phosphorylation; Phosphotransferases
PubMed: 26159069
DOI: 10.1159/000381215 -
The ISME Journal Jun 2023Carbohydrate utilization is critical to microbial survival. The phosphotransferase system (PTS) is a well-documented microbial system with a prominent role in...
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.
Topics: Bacterial Proteins; Cellulose; Histidine; Phosphoenolpyruvate Sugar Phosphotransferase System; Phosphotransferases; Carbohydrates; Firmicutes; DNA
PubMed: 36899058
DOI: 10.1038/s41396-023-01392-2 -
Journal of Molecular Biology May 2019There are two paralogous Escherichia coli phosphotransferase systems, one for sugar import (PTS) and one for nitrogen regulation (PTS), that utilize proteins enzyme I...
There are two paralogous Escherichia coli phosphotransferase systems, one for sugar import (PTS) and one for nitrogen regulation (PTS), that utilize proteins enzyme I (EI) and HPr, and enzyme I (EI) 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:NPr complex formation by the study of transient encounter complexes. NMR paramagnetic relaxation enhancement experiments demonstrated transient encounter complexes of EI not only with the expected partner, NPr, but also with the unexpected partner, HPr. HPr occupies transient sites on EI 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. 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.
Topics: Escherichia coli; Escherichia coli Proteins; Humans; Models, Molecular; Peptide Fragments; Phosphate-Binding Proteins; Phosphoenolpyruvate Sugar Phosphotransferase System; Phosphotransferases; Protein Domains
PubMed: 31071328
DOI: 10.1016/j.jmb.2019.04.040 -
Journal of Bacteriology Oct 2021Genetic truncations in a gene encoding a putative glucose-phosphotransferase system (PTS) protein (, EIIAB) were identified in subpopulations of two separate laboratory...
Genetic truncations in a gene encoding a putative glucose-phosphotransferase system (PTS) protein (, EIIAB) were identified in subpopulations of two separate laboratory stocks of Streptococcus sanguinis SK36; the mutants had reduced PTS activities on glucose and other monosaccharides. To understand the emergence of these mutants, we engineered deletion mutants of and showed that the ManL-deficient strain had improved bacterial viability in the stationary phase and was better able to inhibit the growth of the dental caries pathogen Streptococcus mutans. Transcriptional analysis and biochemical assays suggested that the mutant underwent reprograming of central carbon metabolism that directed pyruvate away from production of lactate, increasing production of hydrogen peroxide (HO) and excretion of pyruvate. Addition of pyruvate to the medium enhanced the survival of SK36 in overnight cultures. Meanwhile, elevated pyruvate levels were detected in the cultures of a small but significant percentage (∼10%) of clinical isolates of oral commensal bacteria. Furthermore, the mutant showed higher expression of the arginine deiminase system than the wild type, which enhanced the ability of the mutant to raise environmental pH when arginine was present. To our surprise, significant discrepancies in genome sequence were identified between strain SK36 obtained from ATCC and the sequence deposited in GenBank. As the conditions that are likely associated with the emergence of spontaneous mutations, i.e., excess carbohydrates and low pH, are those associated with caries development, we propose that glucose-PTS strongly influences commensal-pathogen interactions by altering the production of ammonia, pyruvate, and HO. A health-associated dental microbiome provides a potent defense against pathogens and diseases. Streptococcus sanguinis is an abundant member of a health-associated oral flora that antagonizes pathogens by producing hydrogen peroxide. There is a need for a better understanding of the mechanisms that allow bacteria to survive carbohydrate-rich and acidic environments associated with the development of dental caries. We report the isolation and characterization of spontaneous mutants of S. sanguinis with impairment in glucose transport. The resultant reprograming of the central metabolism in these mutants reduced the production of lactic acid and increased pyruvate accumulation; the latter enables these bacteria to better cope with hydrogen peroxide and low pH. The implications of these discoveries in the development of dental caries are discussed.
Topics: Bacterial Proteins; DNA, Bacterial; Gene Deletion; Gene Expression Regulation, Bacterial; Glucose; Hydrogen Peroxide; Lactic Acid; Phosphotransferases; Pyruvic Acid; Streptococcus sanguis
PubMed: 34460310
DOI: 10.1128/JB.00375-21 -
Nature Communications Apr 2016Rifampin (RIF) phosphotransferase (RPH) confers antibiotic resistance by conversion of RIF and ATP, to inactive phospho-RIF, AMP and Pi. Here we present the crystal...
Rifampin (RIF) phosphotransferase (RPH) confers antibiotic resistance by conversion of RIF and ATP, to inactive phospho-RIF, AMP and Pi. Here we present the crystal structure of RPH from Listeria monocytogenes (RPH-Lm), which reveals that the enzyme is comprised of three domains: two substrate-binding domains (ATP-grasp and RIF-binding domains); and a smaller phosphate-carrying His swivel domain. Using solution small-angle X-ray scattering and mutagenesis, we reveal a mechanism where the swivel domain transits between the spatially distinct substrate-binding sites during catalysis. RPHs are previously uncharacterized dikinases that are widespread in environmental and pathogenic bacteria. These enzymes are members of a large unexplored group of bacterial enzymes with substrate affinities that have yet to be fully explored. Such an enzymatically complex mechanism of antibiotic resistance augments the spectrum of strategies used by bacteria to evade antimicrobial compounds.
Topics: Adenosine Triphosphate; Amino Acid Sequence; Anti-Bacterial Agents; Bacterial Proteins; Binding Sites; Biotransformation; Crystallography, X-Ray; Drug Resistance, Bacterial; Escherichia coli; Gene Expression; Listeria monocytogenes; Models, Molecular; Molecular Sequence Data; Phosphotransferases; Phylogeny; Protein Binding; Protein Structure, Secondary; Protein Structure, Tertiary; Recombinant Proteins; Rifampin; Sequence Alignment
PubMed: 27103605
DOI: 10.1038/ncomms11343 -
Journal of Molecular Microbiology and... 2007Genome sequencing of two different Lactobacillus casei strains (ATCC334 and BL23) is presently going on and preliminary data revealed that this lactic acid bacterium... (Review)
Review
Genome sequencing of two different Lactobacillus casei strains (ATCC334 and BL23) is presently going on and preliminary data revealed that this lactic acid bacterium possesses numerous carbohydrate transport systems probably reflecting its capacity to proliferate under varying environmental conditions. Many carbohydrate transporters belong to the phosphoenolpyruvate:sugar phosphotransferase system (PTS), but all different kinds of non-PTS transporters are present as well and their substrates are known in a few cases. In L. casei regulation of carbohydrate transport and carbon metabolism is mainly achieved by PTS proteins. Carbon catabolite repression (CCR) is mediated via several mechanisms, including the major P-Ser-HPr/catabolite control protein A (CcpA)-dependent mechanism. Catabolite response elements, the target sites for the P-Ser-HPr/CcpA complex, precede numerous genes and operons. PTS regulation domain-containing antiterminators and transcription activators are also present in both L. casei strains. Their activity is usually controlled by two PTS-mediated phosphorylation reactions exerting antagonistic effects on the transcription regulators: P~EIIB-dependent phosphorylation regulates induction of the corresponding genes and P~His-HPr-mediated phosphorylation plays a role in CCR. Carbohydrate transport of L. casei is also regulated via inducer exclusion and inducer expulsion. The presence of glucose, fructose, etc. leads to inhibition of the transport or metabolism of less favorable carbon sources (inducer exclusion) or to the export of accumulated non-metabolizable carbon sources (inducer expulsion). While P-Ser-HPr is essential for inducer exclusion of maltose, it is not necessary for the expulsion of accumulated thio-methyl-beta-D-galactopyranoside. Surprisingly, recent evidence suggests that the PTS of L. casei also plays a role in cold shock response.
Topics: Biological Transport; Carbohydrate Metabolism; Carbon; Cold Temperature; Lacticaseibacillus casei; Phosphorylation; Phosphotransferases
PubMed: 17183208
DOI: 10.1159/000096456 -
BMC Microbiology Mar 2016Streptococcus mutans is the primary etiological agent of human dental caries. It can metabolize a wide variety of carbohydrates and produce large amounts of organic...
BACKGROUND
Streptococcus mutans is the primary etiological agent of human dental caries. It can metabolize a wide variety of carbohydrates and produce large amounts of organic acids that cause enamel demineralization. Phosphoenolpyruvate-dependent sugar phosphotransferase system (PTS) plays an important role in carbohydrates uptake of S. mutans. The ptxA and ptxB genes in S. mutans encode putative enzyme IIA and enzyme IIB of the L-ascorbate-specific PTS. The aim of this study was to analyze the function of these proteins and understand the transcriptional regulatory mechanism.
RESULTS
ptxA (-), ptxB (-), as well as ptxA (-) , ptxB (-) double-deletion mutants all had more extended lag phase and lower growth yield than wild-type strain UA159 when grown in the medium using L-ascorbate as the sole carbon source. Acid production and acid killing assays showed that the absence of the ptxA and ptxB genes resulted in a reduction in the capacity for acidogenesis, and all three mutant strains did not survive an acid shock. According to biofilm and extracellular polysaccharides (EPS) formation analysis, all the mutant strains formed much less prolific biofilms with small amounts of EPS than wild-type UA159 when using L-ascorbate as the sole carbon source. Moreover, PCR analysis and quantitative real-time PCR revealed that sgaT, ptxA, ptxB, SMU.273, SMU.274 and SMU.275 appear to be parts of the same operon. The transcription levels of these genes were all elevated in the presence of L-ascorbate, and the expression of ptxA gene decreased significantly once ptxB gene was knockout.
CONCLUSIONS
The ptxA and ptxB genes are involved in the growth, aciduricity, acidogenesis, and formation of biofilms and EPS of S. mutans when L-ascorbate is the sole carbon source. In addition, the expression of ptxA is regulated by ptxB. ptxA, ptxB, and the upstream gene sgaT, the downstream genes SMU.273, SMU.274 and SMU.275 appear to be parts of the same operon, and L-ascorbate is a potential inducer of the operon.
Topics: Ascorbic Acid; Bacterial Proteins; Biofilms; Dental Caries; Humans; Operon; Phosphotransferases; Streptococcus mutans
PubMed: 27001419
DOI: 10.1186/s12866-016-0668-9 -
Molecular Microbiology Apr 2017The nitrogen-related phosphotransferase system (PTS ) is composed of the EI , NPr and EIIA proteins that form a phosphorylation cascade from phosphoenolpyruvate. PTS is...
The nitrogen-related phosphotransferase system (PTS ) is composed of the EI , NPr and EIIA proteins that form a phosphorylation cascade from phosphoenolpyruvate. PTS is a global regulatory system present in most Gram-negative bacteria that controls some pivotal processes such as potassium and phosphate homeostasis, virulence, nitrogen fixation and ABC transport activation. In the soil bacterium Azotobacter vinelandii, unphosphorylated EIIA negatively regulates the expression of genes related to the synthesis of the bioplastic polyester poly-β-hydroxybutyrate (PHB) and cyst-specific lipids alkylresorcinols (ARs). The mechanism by which EIIA controls gene expression in A. vinelandii is not known. Here, we show that, in presence of unphosphorylated EIIA , the stability of the stationary phase sigma factor RpoS, which is necessary for transcriptional activation of PHB and ARs synthesis related genes, is reduced, and that the inactivation of genes coding for ClpAP protease complex in strains that carry unphosphorylated EIIA , restored the levels and in vivo stability of RpoS, as well as the synthesis of PHB and ARs. Taken together, our results reveal a novel mechanism, by which EIIA globally controls gene expression in A. vinelandii, where the unphosphorylated EIIA induces the degradation of RpoS by the proteolytic complex ClpAP.
Topics: Azotobacter vinelandii; Bacterial Proteins; Escherichia coli Proteins; Gene Expression Regulation, Bacterial; Hydroxybutyrates; Nitrogen Fixation; Phosphoenolpyruvate; Phosphoenolpyruvate Sugar Phosphotransferase System; Phosphorylation; Phosphotransferases; Polyesters; Potassium; Sigma Factor; Transcriptional Activation
PubMed: 28097724
DOI: 10.1111/mmi.13621 -
Journal of Bacteriology May 2023The bacterial nitrogen-related phosphotransfer (PTS; here, Nitro-PTS) system bears homology to well-known PTS systems that facilitate saccharide import and...
The bacterial nitrogen-related phosphotransfer (PTS; 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 or suppresses Pel exopolysaccharide production and additional deletion of 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 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. 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.
Topics: Bacterial Proteins; Pseudomonas aeruginosa; Virulence; Phosphorylation; Phosphotransferases; Phosphoenolpyruvate Sugar Phosphotransferase System; Gene Expression Regulation, Bacterial
PubMed: 37074168
DOI: 10.1128/jb.00453-22 -
PloS One 2019Certain bacterial species target the polysaccharide glycosaminoglycans (GAGs) of animal extracellular matrices for colonization and/or infection. GAGs such as hyaluronan...
Certain bacterial species target the polysaccharide glycosaminoglycans (GAGs) of animal extracellular matrices for colonization and/or infection. GAGs such as hyaluronan and chondroitin sulfate consist of repeating disaccharide units of uronate and amino sugar residues, and are depolymerized to unsaturated disaccharides by bacterial extracellular or cell-surface polysaccharide lyase. The disaccharides are degraded and metabolized by cytoplasmic enzymes such as unsaturated glucuronyl hydrolase, isomerase, and reductase. The genes encoding these enzymes are assembled to form a GAG genetic cluster. Here, we demonstrate the Streptococcus agalactiae phosphotransferase system (PTS) for import of unsaturated hyaluronan disaccharide. S. agalactiae NEM316 was found to depolymerize and assimilate hyaluronan, whereas its mutant with a disruption in the PTS genes included in the GAG cluster was unable to grow on hyaluronan, while retaining the ability to depolymerize hyaluronan. Using toluene-treated wild-type cells, the PTS activity for import of unsaturated hyaluronan disaccharide was significantly higher than that observed in the absence of the substrate. In contrast, the PTS mutant was unable to import unsaturated hyaluronan disaccharide, indicating that the corresponding PTS is the only importer of fragmented hyaluronan, which is suitable for PTS to phosphorylate the substrate at the C-6 position. This is distinct from Streptobacillus moniliformis ATP-binding cassette transporter for import of sulfated and non-sulfated fragmented GAGs without substrate modification. The three-dimensional structure of streptococcal EIIA, one of the PTS components, was found to contain a Rossman-fold motif by X-ray crystallization. Docking of EIIA with another component EIIB by modeling provided structural insights into the phosphate transfer mechanism. This study is the first to identify the substrate (unsaturated hyaluronan disaccharide) recognized and imported by the streptococcal PTS. The PTS and ABC transporter for import of GAGs shed light on bacterial clever colonization/infection system targeting various animal polysaccharides.
Topics: Amino Acid Sequence; Disaccharides; Escherichia coli; Extracellular Matrix; Hyaluronic Acid; Models, Biological; Models, Molecular; Phosphotransferases; Streptococcus
PubMed: 31697725
DOI: 10.1371/journal.pone.0224753