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Journal of Bacteriology Apr 2015CodY is a branched-chain amino acid-responsive transcriptional regulator that controls the expression of several dozen transcription units in Bacillus subtilis. The...
UNLABELLED
CodY is a branched-chain amino acid-responsive transcriptional regulator that controls the expression of several dozen transcription units in Bacillus subtilis. The presence of isoleucine, valine, and leucine in the growth medium is essential for achieving high activity of CodY and for efficient regulation of the target genes. We identified three permeases-BcaP, BraB, and BrnQ-that are responsible for the bulk of isoleucine and valine uptake and are also involved in leucine uptake. At least one more permease is capable of efficient leucine uptake, as well as low-affinity transport of isoleucine and valine. The lack of the first three permeases strongly reduced activity of CodY in an amino acid-containing growth medium. BcaP appears to be the most efficient isoleucine and valine permease responsible for their utilization as nitrogen sources. The previously described strong CodY-mediated repression of BcaP provides a mechanism for fine-tuning CodY activity by reducing the availability of amino acids and for delaying the utilization of isoleucine and valine as nitrogen and carbon sources under conditions of nutrient excess.
IMPORTANCE
Bacillus subtilis CodY is a global transcriptional regulator that is activated by branched-chain amino acids (BCAA). Since the level of BCAA achieved by intracellular synthesis is insufficient to fully activate CodY, transport of BCAA from the environment is critical for CodY activation, but the permeases needed for such activation have not been previously identified. This study identifies three such permeases, reports their amino acid transport specificity, and reveals their impact on CodY activation.
Topics: Amino Acids, Branched-Chain; Bacillus subtilis; Bacterial Proteins; Biological Transport; Gene Expression Regulation, Bacterial; Gene Expression Regulation, Enzymologic; Genotype; Membrane Transport Proteins; Mutation; Operon; Transcription Factors
PubMed: 25645558
DOI: 10.1128/JB.02563-14 -
Applied and Environmental Microbiology Nov 2017The glucose/mannose-phosphotransferase system (PTS) permease EII encoded by in the dental caries pathogen has a dominant influence on sugar-specific, CcpA-independent...
The glucose/mannose-phosphotransferase system (PTS) permease EII encoded by in the dental caries pathogen has a dominant influence on sugar-specific, CcpA-independent catabolite repression (CR). Mutations in affect energy metabolism and virulence-associated traits, including biofilm formation, acid tolerance, and competence. Using promoter::reporter fusions, expression of the and the operons, encoding a transcriptional regulator, a fructose-1-phosphate kinase and a fructose-PTS permease EII, respectively, was monitored in response to carbohydrate source and in mutants lacking CcpA, FruR, and components of EII Expression of genes for EII and EII was directly regulated by CcpA and CR, as evinced by and methods. Unexpectedly, not only was the operon negatively regulated by FruR, but also so was Carbohydrate transport by EII had a negative influence on expression of but not In agreement with the proposed role of FruR in regulating these PTS operons, loss of or substantially altered growth on a number of carbohydrates, including fructose. RNA deep sequencing revealed profound changes in gene regulation caused by deletion of or Collectively, these findings demonstrate intimate interconnection of the regulation of two major PTS permeases in and reveal novel and important contributions of fructose metabolism to global regulation of gene expression. The ability of and other streptococcal pathogens to survive and cause human diseases is directly dependent upon their capacity to metabolize a variety of carbohydrates, including glucose and fructose. Our research reveals that metabolism of fructose has broad influences on the regulation of utilization of glucose and other sugars, and mutants with changes in certain genes involved in fructose metabolism display profoundly different abilities to grow and express virulence-related traits. Mutants lacking the FruR regulator or a particular phosphofructokinase, FruK, display changes in expression of a large number of genes encoding transcriptional regulators, enzymes required for energy metabolism, biofilm development, biosynthetic and degradative processes, and tolerance of a spectrum of environmental stressors. Since fructose is a major component of the modern human diet, the results have substantial significance in the context of oral health and the development of dental caries.
Topics: Bacterial Proteins; Dental Caries; Fructose; Gene Expression Regulation, Bacterial; Humans; Membrane Transport Proteins; Operon; Phosphoenolpyruvate Sugar Phosphotransferase System; Streptococcus mutans
PubMed: 28821551
DOI: 10.1128/AEM.01403-17 -
Biochimica Et Biophysica Acta Aug 2002Protein stability, as measured by irreversible protein aggregation, is one of the central difficulties in the handling of detergent-solubilized membrane proteins. We...
Protein stability, as measured by irreversible protein aggregation, is one of the central difficulties in the handling of detergent-solubilized membrane proteins. We present a quantitative analysis of the stability of the Escherichia coli lactose (lac) permease and a series of lac permease fusion proteins containing an insertion of cytochrome(b562), T4 lysozyme or beta-lactamase in the central hydrophilic loop of the permease. The stability of the proteins was evaluated under a variety of storage conditions by both a qualitative SDS-PAGE assay and by a quantitative hplc assay. Long-chain maltoside detergents were more effective at maintaining purified protein in solution than detergents with smaller head groups and/or shorter alkyl tails. A full factorial experiment established that the proteins were insensitive to sodium chloride concentrations, but greatly stabilized by glycerol, low temperature and the combination of glycerol and low temperature. The accurate quantitation of the protein by absorbance spectroscopy required exclusion of all contact with clarified polypropylene or polyvinyl chloride (PVC) materials. Although some of the fusion proteins were more prone to aggregation than the wild-type permease, the stability of a fusion protein containing a cytochrome(b562) insertion was indistinguishable from that of native lac permease.
Topics: Chromatography, High Pressure Liquid; Cytochrome b Group; Detergents; Drug Stability; Electrophoresis, Polyacrylamide Gel; Escherichia coli; Escherichia coli Proteins; Glycerol; Membrane Transport Proteins; Monosaccharide Transport Proteins; Recombinant Fusion Proteins; Solutions; Spectrophotometry, Ultraviolet; Symporters; Temperature
PubMed: 12100995
DOI: 10.1016/s0005-2736(02)00397-8 -
Microbiology Spectrum Jun 2023The soil-dwelling bacterium Listeria monocytogenes survives a multitude of conditions when residing in the outside environment and as a pathogen within host cells. Key...
The soil-dwelling bacterium Listeria monocytogenes survives a multitude of conditions when residing in the outside environment and as a pathogen within host cells. Key to survival within the infected mammalian host is the expression of bacterial gene products necessary for nutrient acquisition. Similar to many bacteria, L. monocytogenes uses peptide import to acquire amino acids. Peptide transport systems play an important role in nutrient uptake as well as in additional functions that include bacterial quorum sensing and signal transduction, recycling of peptidoglycan fragments, adherence to eukaryotic cells, and alterations in antibiotic susceptibility. It has been previously described that CtaP, encoded by , is a multifunctional protein associated with activities that include cysteine transport, resistance to acid, membrane integrity, and bacterial adherence to host cells. is located next to two genes predicted to encode membrane-bound permeases and , termed CtpP1 and CtpP2, respectively. Here, we show that CtpP1 and CtpP2 are required for bacterial growth in the presence of low concentrations of cysteine and for virulence in mouse infection models. Taken together, the data identify distinct nonoverlapping roles for two related permeases that are important for the growth and survival of L. monocytogenes within host cells. Bacterial peptide transport systems are important for nutrient uptake and may additionally function in a variety of other roles, including bacterial communication, signal transduction, and bacterial adherence to eukaryotic cells. Peptide transport systems often consist of a substrate-binding protein associated with a membrane-spanning permease. The environmental bacterial pathogen Listeria monocytogenes uses the substrate-binding protein CtaP not only for cysteine transport but also for resistance to acid, maintenance of membrane integrity, and bacterial adherence to host cells. In this study, we demonstrate complementary yet distinct functional roles for two membrane permeases, CtpP1 and CtpP2, that are encoded by genes linked to and that contribute to bacterial growth, invasion, and pathogenicity.
Topics: Animals; Mice; Listeria monocytogenes; Cysteine; Virulence; Membrane Transport Proteins; Virulence Factors; Bacterial Proteins; Disease Models, Animal; Gene Expression Regulation, Bacterial; Mammals
PubMed: 37199604
DOI: 10.1128/spectrum.03317-22 -
Physiology (Bethesda, Md.) Apr 2006Uptake of nutrients into cells is essential to life and occurs in all organisms at the expense of energy. Whereas in most prokaryotic and simple eukaryotic cells... (Review)
Review
Uptake of nutrients into cells is essential to life and occurs in all organisms at the expense of energy. Whereas in most prokaryotic and simple eukaryotic cells electrochemical transmembrane proton gradients provide the central driving force for nutrient uptake, in higher eukaryotes it is more frequently coupled to sodium movement along the transmembrane sodium gradient, occurs via uniport mechanisms driven by the substrate gradient only, or is linked to the countertransport of a similar organic solute. With the cloning of a large number of mammalian nutrient transport proteins, it became obvious that a few "archaic'' transporters that utilize a transmembrane proton gradient for nutrient transport into cells can still be found in mammals. The present review focuses on the electrogenic peptide transporters as the best studied examples of proton-dependent nutrient transporters in mammals and summarizes the most recent findings on their physiological importance. Taking peptide transport as a general phenomenon found in nature, we also include peptide transport mechanisms in bacteria, yeast, invertebrates, and lower vertebrates, which are not that often addressed in physiology journals.
Topics: Amino Acid Sequence; Animals; Bacteria; Biological Transport; Eukaryotic Cells; Humans; Invertebrates; Membrane Transport Proteins; Molecular Sequence Data; Peptide Transporter 1; Phylogeny; Prokaryotic Cells; Protons; Substrate Specificity; Symporters; Vertebrates; Yeasts
PubMed: 16565475
DOI: 10.1152/physiol.00054.2005 -
Biochemistry May 1993In this study, wild-type lac permease and lac permease mutated at each of the eight cysteinyl residues in the molecule were solubilized from the membrane, purified, and... (Comparative Study)
Comparative Study
In this study, wild-type lac permease and lac permease mutated at each of the eight cysteinyl residues in the molecule were solubilized from the membrane, purified, and reconstituted into proteoliposomes. Lactose equilibrium exchange and efflux activities of mutants with Ser in place of Cys117, Cys176, Cys234, Cys333, Cys353, or Cys355 are essentially the same as wild-type permease. In contrast, mutants in Cys148 and Cys154 exhibit diminished exchange and efflux activities. These mutants in Cys148 and Cys154, except for the C148S mutant, have previously been shown to slow down active transport as well [Van Iwaarden, P.R., Driessen, A. J. M., Menick, D. R., Kaback, H.R., & Konings, W. N. (1991) J. Biol. Chem. 266, 15688-15692]. C148S permease shows monophasic kinetics with a high apparent KM with respect to external lactose in the exchange reaction under nonequilibrium conditions, whereas wild-type permease exhibits biphasic kinetics with both a high and low KM component. Moreover, the absence of the low Km pathway in the C148S permease is correlated with the absence of a high-affinity binding site for p-nitrophenyl alpha-D-galactopyranoside (NPG). Interestingly, the affinity of the permease for NPG appears to increase with the hydrophobicity of the side chain at position 154 (Ser < Cys < Gly < Val). Finally, the presence of a high-affinity binding site for NPG in C154V is consistent with the biphasic exchange kinetics exhibited by this mutant. The results are discussed in the context of a model in which lac permease has two substrate binding sites, a catalytic site and a regulatory site.
Topics: Amino Acid Sequence; Binding Sites; Cysteine; Escherichia coli; Escherichia coli Proteins; Hydrogen-Ion Concentration; Kinetics; Lactose; Liposomes; Membrane Transport Proteins; Molecular Sequence Data; Monosaccharide Transport Proteins; Mutagenesis; Nitrophenylgalactosides; Structure-Activity Relationship; Symporters
PubMed: 8499445
DOI: 10.1021/bi00071a017 -
Biophysical Journal Mar 2022The lac operon of Escherichia coli is repressed several 100-fold in the presence of glucose. This repression has been attributed to cAMP receptor protein-mediated...
The lac operon of Escherichia coli is repressed several 100-fold in the presence of glucose. This repression has been attributed to cAMP receptor protein-mediated inhibition of lac transcription and EIIA-mediated inhibition of lactose transport (inducer exclusion). The growing evidence against the first mechanism has led to the postulate that the repression is driven by inducer exclusion. Although inducer exclusion reduces the permease activity only 2-fold in fully induced cells, it could be more potent in partially induced cells. Here, we show that even in partially induced cells, inducer exclusion reduces the permease activity no more than 6-fold. Moreover, the repression is so small because these experiments are performed in the presence of chloramphenicol. Indeed, when glucose is added to a culture growing on glycerol and TMG, but no chloramphenicol, lac expression is repressed 900-fold. This repression is primarily due to reversal of the positive feedback loop, i.e., the decline of the intracellular TMG level leads to a lower permease level, which reduces the intracellular TMG level even further. The repression in the absence of chloramphenicol is therefore primarily due to positive feedback, which does not exist during measurements of inducer exclusion.
Topics: Chloramphenicol; Escherichia coli; Glucose; Lac Operon; Lactose; Membrane Transport Proteins
PubMed: 35065916
DOI: 10.1016/j.bpj.2022.01.016 -
The Journal of Biological Chemistry Apr 1994Yeast uracil permease follows the secretory pathway to the plasma membrane and is phosphorylated on serine residues in a post-Golgi compartment. The protein was found to...
Yeast uracil permease follows the secretory pathway to the plasma membrane and is phosphorylated on serine residues in a post-Golgi compartment. The protein was found to be rather stable in growing cells, but its turnover rate (half-life of about 7 h) was much faster than that of most yeast proteins. Several adverse conditions triggered the rapid degradation of uracil permease, and so a loss of uracil uptake. Turnover was rapid when yeast cells were starved of either nitrogen, phosphate, or carbon, and as they approached the stationary growth phase. Rapid permease degradation was also promoted by the inhibition of protein synthesis. The degradation of uracil permease in response to several stresses was strikingly slower in the two mutants, end3 and end4, that are deficient in the internalization step of receptor-mediated endocytosis. Thus, internalization is the first step in the permease degradative pathway. Uracil permease is degraded in the vacuole, since pep4 mutant cells lacking vacuolar protease activities accumulated large amounts of uracil permease, which was located within the vacuole by immunofluorescence. We have yet to determine whether adverse conditions enhance permease endocytosis and subsequent degradation or divert internalized uracil permease from a recycling to a degradative pathway.
Topics: Amino Acid Sequence; Biological Transport; Cell Membrane; Cycloheximide; Endocytosis; Genes, Fungal; Kinetics; Membrane Transport Proteins; Molecular Sequence Data; Mutagenesis; Nitrogen; Nucleotide Transport Proteins; Phosphorylation; Protein Processing, Post-Translational; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Time Factors; Uracil
PubMed: 8144575
DOI: No ID Found -
The Journal of Biological Chemistry Dec 2013The evolutionarily broad family nucleobase-cation symporter-2 (NCS2) encompasses transporters that are conserved in binding site architecture but diverse in substrate...
The evolutionarily broad family nucleobase-cation symporter-2 (NCS2) encompasses transporters that are conserved in binding site architecture but diverse in substrate selectivity. Putative purine transporters of this family fall into one of two homology clusters: COG2233, represented by well studied xanthine and/or uric acid permeases, and COG2252, consisting of transporters for adenine, guanine, and/or hypoxanthine that remain unknown with respect to structure-function relationships. We analyzed the COG2252 genes of Escherichia coli K-12 with homology modeling, functional overexpression, and mutagenesis and showed that they encode high affinity permeases for the uptake of adenine (PurP and YicO) or guanine and hypoxanthine (YjcD and YgfQ). The two pairs of paralogs differ clearly in their substrate and ligand preferences. Of 25 putative inhibitors tested, PurP and YicO recognize with low micromolar affinity N(6)-benzoyladenine, 2,6-diaminopurine, and purine, whereas YjcD and YgfQ recognize 1-methylguanine, 8-azaguanine, 6-thioguanine, and 6-mercaptopurine and do not recognize any of the PurP ligands. Furthermore, the permeases PurP and YjcD were subjected to site-directed mutagenesis at highly conserved sites of transmembrane segments 1, 3, 8, 9, and 10, which have been studied also in COG2233 homologs. Residues irreplaceable for uptake activity or crucial for substrate selectivity were found at positions occupied by similar role amino acids in the Escherichia coli xanthine- and uric acid-transporting homologs (XanQ and UacT, respectively) and predicted to be at or around the binding site. Our results support the contention that the distantly related transporters of COG2233 and COG2252 use topologically similar side chain determinants to dictate their function and the distinct purine selectivity profiles.
Topics: Escherichia coli K12; Escherichia coli Proteins; Ligands; Membrane Transport Proteins; Models, Molecular; Mutagenesis, Site-Directed; Nucleoside Transport Proteins; Protein Structure, Tertiary; Structural Homology, Protein; Structure-Activity Relationship
PubMed: 24214977
DOI: 10.1074/jbc.M113.523340 -
The Journal of Biological Chemistry Oct 1998This report describes an extensive mutational analysis of the most carboxyl-terminal membrane-spanning sequence of Escherichia coli lac permease (TM12). In addition to...
This report describes an extensive mutational analysis of the most carboxyl-terminal membrane-spanning sequence of Escherichia coli lac permease (TM12). In addition to identifying residues important for lactose transport function, the analysis revealed that numerous mutations made lac permease highly toxic to cells. In the most extreme cases, production of such proteins at very low steady-state levels reduced cell viability greater than 10(4)-fold. Both frameshift and missense mutations led to toxicity, with the frameshift mutations having the strongest effects observed. The toxic missense mutations corresponded to changes in TM12 expected to interfere with membrane insertion or folding, such as the introduction of charged residues or prolines in the putative helix. The results suggest that cellular toxicity may be a relatively common consequence of mutations altering integral membrane protein folding. An analogous toxicity might contribute to the pathogenesis of several degenerative diseases caused by mutant membrane proteins, such as retinitis pigmentosa, Charcot-Marie-Tooth syndrome, and Alzheimer's disease.
Topics: Amino Acid Sequence; Biological Transport; DNA Mutational Analysis; Escherichia coli; Escherichia coli Proteins; Frameshift Mutation; Lactose; Membrane Transport Proteins; Molecular Sequence Data; Monosaccharide Transport Proteins; Mutagenesis, Insertional; Mutation, Missense; Protein Structure, Secondary; Symporters
PubMed: 9774425
DOI: 10.1074/jbc.273.43.28078