Location of the gene for the low-affinity tryptophan-specific permease of Escherichia coli.
Ontology highlight
ABSTRACT:
SUBMITTER: Edwards RM
PROVIDER: S-EPMC1158393 | biostudies-other | 1982 May
REPOSITORIES: biostudies-other
Similar Datasets
Project description:The mtr gene of Escherichia coli K-12 encodes an L-tryptophan-specific permease. This gene was originally identified through the isolation of mutations in the 69-min region of the chromosome, closely linked to argG. Cells with lesions in mtr display a phenotype of 5-methyltryptophan resistance. The mtr gene was cloned by using the mini-Mu system. The amino acid sequence of Mtr (414 codons), deduced by DNA sequence analysis, was found to be 33% identical to that of another single-component transport protein, the tyrosine-specific permease, TyrP. The hydropathy plots of the two permeases were similar. Possible operator sites for the tyrosine and tryptophan repressors are situated within the region of DNA that is likely to be the mtr promoter.
| S-EPMC207163 | biostudies-other
Project description:The PheP protein is a high-affinity phenylalanine-specific permease of the bacterium Escherichia coli. A topological model based on sequence analysis of the putative protein in which PheP has 12 transmembrane segments with both N and C termini located in the cytoplasm had been proposed (J. Pi, P. J. Wookey, and A. J. Pittard, J. Bacteriol. 173:3622-3629, 1991). This topological model of PheP has been further examined by generating protein fusions with alkaline phosphatase. Twenty-five sandwich fusion proteins have been constructed by inserting the 'phoA gene at specific sites within the pheP gene. In general, the PhoA activities of the fusions support a PheP topology model consisting of 12 transmembrane segments with the N and C termini in the cytoplasm. However, alterations to the model, affecting spans III and VI, were indicated by this analysis and were supported by additional site-directed mutagenesis of some of the residues involved.
| S-EPMC177991 | biostudies-other
Project description:We isolated an Escherichia coli mutant strain that suppresses the glycolate-negative phenotype of a strain deficient in both GlcA and LldP transporters of this compound. This suppressing phenotype was assigned to yjcG, a gene whose function was previously unknown, which was found to encode a membrane protein able to transport glycolate. On the basis of sequence similarity, the yjcG gene product was classified as a member of the sodium:solute symporter family. Northern experiments revealed that yjcG is cotranscribed with its neighbor, acs, encoding acetyl coenzyme A synthetase, which is involved in the scavenging acetate. The fortuitous presence of an IS2 element in acs, which impaired yjcG expression by polarity in our parental strain, allowed us to conclude that the alternative glycolate carrier became active after precise excision of IS2 in the suppressed strain. The finding that yjcG encodes a putative membrane carrier for glycolate and the cotranscription of yjcG with acs suggested that the primary function of the yjcG gene product (proposed gene name, actP) could be acetate transport and allowed us to define an operon involved in acetate metabolism. The time course of [1,2-(14)C]acetate uptake and the results of a concentration kinetics analysis performed with cells expressing ActP or cells deficient in ActP supported the the hypothesis that this carrier is an acetate transporter and suggested that there may be another transport system for this monocarboxylate.
| S-EPMC219408 | biostudies-literature
Project description:The crystal structure of the Escherichia coli lactose permease at 3.5 A with a bound substrate has been reported recently. The structure reveals the sugar-protein contacts, which include hydrophobic stacking between the galactopyranosyl ring of substrate and the indole side chain of Trp-151, as proposed previously. The nature of this interaction is studied here by exploiting the luminescence properties of Trp-151 in a mutant devoid of other tryptophan residues. The following phenomena are observed. (i) The fluorescence emission spectrum of Trp-151 and fluorescence-quenching experiments with water-soluble quenchers demonstrate that Trp-151 is in a hydrophilic environment. (ii) Substrate binding leads to a blue shift in the emission spectrum and reduction in accessibility to polar quenchers, indicating that Trp-151 becomes less exposed to aqueous solvent. (iii) The phosphorescence spectrum of Trp-151 is red-shifted in the presence of substrate, indicating charge separation of the triplet state due to a direct stacking interaction between the galactopyranosyl and indole rings. The spectroscopic data fully complement the x-ray structure and demonstrate the feasibility of fluorescence spectroscopy for studying sugar-protein interactions.
| S-EPMC240682 | biostudies-literature
Project description:Expression of lysP, which encodes the lysine-specific transporter LysP in Escherichia coli, is regulated by the concentration of exogenous available lysine. In this study, the LysR-type transcriptional regulator ArgP was identified as the activator of lysP expression. At lysine concentrations higher than 25 μM, lysP expression was shut off and phenocopied an argP deletion mutant. Purified ArgP-His(6) bound to the lysP promoter/control region at a sequence containing a conserved T-N(11)-A motif. Its affinity increased in the presence of lysine but not in the presence of the other known coeffector, arginine. In vivo data suggest that lysine-loaded ArgP and arginine-loaded ArgP compete at the lysP promoter. We propose that lysine-loaded ArgP prevents lysP transcription at the promoter clearance step, as described for the lysine-dependent regulation of argO (R. S. Laishram and J. Gowrishankar, Genes Dev. 21:1258-1272, 2007). The global regulator Lrp also bound to the lysP promoter/control region. An lrp mutant exhibited reduced lysP expression in the absence of external lysine. These results indicate that ArgP is a major regulator of lysP expression but that Lrp modulates lysP transcription under lysine-limiting conditions.
| S-EPMC3133163 | biostudies-literature
Project description:Glucose transporters (GLUTs), expressed in all types of human cells, are responsible for the uptake of sugars as the primary energy source for the normal functions of good cells and for the abnormal growth of cancer cells. The E. coli xylose permease (XylE), a homologue of human GLUTs, has been investigated more thoroughly than other major facilitator proteins in the current literature. In this paper, we present a molecular dynamics (MD) study of an all-atom model system to elucidate the atomistic details and the free-energy landscape along the path of binding a xylopyranose (XYP) from the extracellular space to the inside of the transporter protein XylE. From the MD simulations, the Gibbs free energy of binding was found to be -4.4kcal/mol in agreement with the experimental value of -4.7kcal/mol. The accuracy of our study is further shown in the computed hydration energy of XYP of -14.6kcal/mol in comparison with the experimental data of -15.0kcal/mol. Along the binding path, the Gibbs free energy of the XYP-XylE complex first rises from zero in the dissociated state to approximately 4 kcal/mol in the transition state (when XylE slightly increases its opening toward the extracellular side to accommodate XYP) before dropping down to -9.0 kcal/mol in the bound state. These quantitative insights indicate the fast equilibration between the bound and the unbound states of XylE and XYP. They also serve as an atomistic-dynamic corroboration of the experimental conclusion that XylE is a high-affinity sugar transporter.
| S-EPMC5670004 | biostudies-literature
Project description:The diffusion coefficient of the transcription factor LacI within living Escherichia coli has been measured directly by in vivo tracking to be D = 0.4??m(2)/s. At this rate, simple models of diffusion lead to the expectation that LacI and other proteins will rapidly homogenize throughout the cell. Here, we test this expectation of spatial homogeneity by single-molecule visualization of LacI molecules non-specifically bound to DNA in fixed cells. Contrary to expectation, we find that the distribution depends on the spatial location of its encoding gene. We demonstrate that the spatial distribution of LacI is also determined by the local state of DNA compaction, and that E. coli can dynamically redistribute proteins by modifying the state of its nucleoid. Finally, we show that LacI inhomogeneity increases the strength with which targets located proximally to the LacI gene are regulated. We propose a model for intranucleoid diffusion that can reconcile these results with previous measurements of LacI diffusion, and we discuss the implications of these findings for gene regulation in bacteria and eukaryotes.
| S-EPMC3472691 | biostudies-literature
Project description:Synechocystis sp. strain PCC 6803 has five genes for putative Na(+)/H(+) antiporters (designated nhaS1, nhaS2, nhaS3, nhaS4, and nhaS5). The deduced amino acid sequences of NhaS1 and NhaS2 are similar to that of NhaP, the Na(+)/H(+) antiporter of Pseudomonas aeruginosa, whereas those of NhaS3, NhaS4, and NhaS5 resemble that of NapA, the Na(+)/H(+) antiporter of Enterococcus hirae. We successfully induced the expression of nhaS1, nhaS3, and nhaS4 under control of an Na(+)-dependent promoter in Escherichia coli TO114, a strain that is deficient in Na(+)/H(+) antiport activity. Inverted membrane vesicles prepared from TO114 nhaS1 and TO114 nhaS3 cells exhibited Na(+)(Li(+))/H(+) antiport activity. Kinetic analysis of this activity revealed that nhaS1 encodes a low-affinity Na(+)/H(+) antiporter with a K(m) of 7.7 mM for Na(+) ions and a K(m) of 2.5 mM for Li(+) ions, while nhaS3 encodes a high-affinity Na(+)/H(+) antiporter with a K(m) of 0.7 mM for Na(+) ions and a K(m) of 0.01 mM for Li(+) ions. Transformation of E. coli TO114 with the nhaS1 and nhaS3 genes increased cellular tolerance to high concentrations of Na(+) and Li(+) ions, as well as to depletion of K(+) ions during cell growth. To our knowledge, this is the first functional characterization of Na(+)/H(+) antiporters from a cyanobacterium. Inverted membrane vesicles prepared from TO114 nhaS4 cells did not have Na(+)/H(+) antiport activity, and the cells themselves were as sensitive to Na(+) and Li(+) ions as the original TO114 cells. However, the TO114 nhaS4 cells were tolerant to depletion of K(+) ions. Taking into account these results and the growth characteristics of Synechocystis mutants in which nhaS genes had been inactivated by targeted disruption, we discuss possible roles of NhaS1, NhaS3, and NhaS4 in Synechocystis.
| S-EPMC95012 | biostudies-literature
Project description:Building a three-dimensional model of the sucrose permease of Escherichia coli (CscB) with the X-ray crystal structure lactose permease (LacY) as template reveals a similar overall fold for CscB. Moreover, despite only 28% sequence identity and a marked difference in substrate specificity, the structural organization of the residues involved in sugar-binding and H(+) translocation is conserved in CscB. Functional analyses of mutants in the homologous key residues provide strong evidence that they play a similar critical role in the mechanisms of CscB and LacY.
| S-EPMC2786776 | biostudies-literature
Project description:To improve the thermostability of tryptophan synthase, the molecular modification of tryptophan synthase was carried out by rational molecular engineering. First, B-FITTER software was used to analyze the temperature factor (B-factor) of each amino acid residue in the crystal structure of tryptophan synthase. A key amino acid residue, G395, which adversely affected the thermal stability of the enzyme, was identified, and then, a mutant library was constructed by site-specific saturation mutation. A mutant (G395S) enzyme with significantly improved thermal stability was screened from the saturated mutant library. Error-prone PCR was used to conduct a directed evolution of the mutant enzyme (G395S). Compared with the parent, the mutant enzyme (G395S /A191T) had a Km of 0.21 mM and a catalytic efficiency kcat/Km of 5.38 mM-1∙s-1, which was 4.8 times higher than that of the wild-type strain. The conditions for L-tryptophan synthesis by the mutated enzyme were a L-serine concentration of 50 mmol/L, a reaction temperature of 40 °C, pH of 8, a reaction time of 12 h, and an L-tryptophan yield of 81%. The thermal stability of the enzyme can be improved by using an appropriate rational design strategy to modify the correct site. The catalytic activity of tryptophan synthase was increased by directed evolution.
| S-EPMC7232222 | biostudies-literature