Cloning of the histidine transport genes from salmonella typhimurium and characterization of an analogous transport system in escherichia coli

The genes for the well-characterized high-affinity histidine transport system of S typhimurium have been cloned in λgt4. Genetic and physiological analyses of the analogous transport system of E coli were undertaken in order that available λ vectors, recombinant DNA techniques, and a genetic selection for transport function might be used to isolate the Salmonella genes. The presence of the transport genes on a 12.4 Kb cloned DNA fragment has been confirmed (1) genetically, by complementation studies; (2) physiologically, by the rates of histidine uptake by bacteria containing this DNA; and (3) by demonstrating that the cloned DNA codes for the previously identified transport proteins J and P. The isolated fragment carries the entire transport operon, the argT gene and the ubiX locus, but neither the purF gene nor the ack/pta loci.     

Regulation of leucine transport by intracellular pH in Bacillus pasteurii

The kinetics, specificity and mechanism of leucine uptake were studied in the alkaliphilic bacterium Bacillus pasteurii DSM 33 (ATCC 11859). Leucine was accumulated up to 200-fold by a sodium-dependent secondary transport system for branched-chain amino acids. Apparent K_t values of 9.6 μM for leucine, 8.9 μM for isoleucine, 9.3 μM for valine, and 0.71 mM for sodium were determined, and maximum uptake activity was observed at an external pH of 8.5 and at 35°C. The effect of several ionophores indicated that transport was energized by the membrane potential and a sodium gradient; each gradient alone was sufficient to drive the uptake of leucine. The activity of the leucine transport system was regulated by the intracellular pH and was inhibited at an internal pH below 7.0. Received: 26 September 1995 / Accepted: 10 December 1995     

Cloning,expression, and nucleotide sequence of livR,the repressor for high-affinity branched-chain amino acid transport in Escherichia coli

The livR gene encoding the repressor for high-affinity branched-chain amino acid transport in Escherichia coli has been cloned from a library prepared from the episome F106. The inserted DNA fragment from the initial cloned plasmid, pANT1, complemented two independent, spontaneously derived, regulatory mutations. Subcloning as well as the creation of deletions with Bal31 exonuclease revealed that the entire regulatory region is contained within a 1.1-kb RsaI-SalI fragment. Expression of the pANT plasmids in E. coli minicells showed that the regulatory region encodes one detectable protein with an apparent molecular weight of 21,000. DNA sequencing revealed one open reading frame of 501 bp encoding a protein with a calculated MW of 19,155. The potential secondary structure of the regulatory protein has been predicted and it suggests that the carboxy terminus may fold into three consecutive alpha helices. These results suggests that the livR gene encodes a repressor which plays a role in the regulation of expression of the livJ and the livK transport genes.     

L-arabinose transport and the L-arabinose binding protein of escherichia coli

The active accumulation of L-arabinose by arabinose induced cultures of Escherichia coli is mediated by 2 independent transport mechanisms. One, specified by the gene locus araE, is membrane bound and possesses a relatively “low affinity.” The other, specified in part by the genetic locus araF, contains as a functional component the L-arabinose binding protein and functions with a “high affinity” for the substrate. The L-arabinose binding protein has been purified, partially characterized, crystallized, and sequenced.     

Characterization of a periplasmic protein related to sn-glycerol-3-phosphate transport in escherichia coli

The cold osmotic shock procedure releases a protein (GLPT) from the cell envelope of Escherichia coli that is related to the transport of sn-glycerol-3-phosphate in this organism. The evidence for this correlation is as follows: (1) GLPT is under the regulatory control of the glpR gene. (2) Some glpT mutants that were isolated as phosphonomycin resistant clones do not synthesize GLPT. Revertants of these mutants (growth on sn-glycerol 3-phosphate) again synthesize GLPT. (3) Some amber mutations in glpT reduce the amount of GLPT while suppressed strains produce normal amounts. (4) Transfer of a plasmid carrying the glpT genes into a strain lacking GLPT and sn-glycerol-3-phosphate transport restores both functions in the recipient. Transport and GLPT synthesis in the plasmid carrying strain are increased 2- to 3-fold over a fully induced wild-type strain, but appear to be constitutive. GLPT is a soluble protein of molecular weight 160,000 composed of 4 identical subunits. The 160,000 molecular weight complex is stable in 1% sodium dodecylsulfate at room temperature. Upon boiling in 1% sodium dodecylsulfate GLPT dissociates into its subunits. Likewise, 8 M urea at room temperature dissociates GLPT into its subunits. Dialysis of dissociated GLPT against phosphate or Tris-HCl buffer, pH 7.0, allows renaturation to the tetrameric form. The protein is acidic in nature (isoelectric point 4.4). In contrast to the typical transport-related periplasmic-binding proteins, no conditions could be found where pure GLPT exhibited binding activity toward its supposed substrate, sn-glycerol-3-phosphate. In vivo new appearance of transport activity for sn-glycerol-3-phosphate transport occurs only shortly before cell division. However, GLPT synthesis does not fluctuate during the cell cycle. The available evidence indicates a cell-division-dependent processing of GLPT in the cell envelope as a reason for the alteration in transport activity. Transport in whole cells is sensitive to the cold osmotic shock procedure, demonstrating the participation of an essential periplasmic component. However, isolated membrane vesicles that are devoid of periplasmic components, including GLPT, are fully active in sn-glycerol-3-phosphate transport. Therefore, we conclude that GLPT is essential in overcoming a diffusion barrier for sn-glycerol-3-phosphate established by the outer membrane. Attempts to isolate mutants that are transport negative in whole cells due to a defect in GLPT but are active in isolated membrane vesicles have failed so far. All GLPT mutants tested, whether or not they synthesize GLPT, are not active in isolated membrane vesicles. Iodination of whole cells with [¹²⁵I] followed by osmotic shock reveals that several shock-releasable proteins including GLPT become radioactively labeled. This indicates that some portions of GLPT are accessible to the external medium.     

Changes in leucine transport activity in Chironomus riparius larvae after short-term exposure to potassium dichromate and fenitrothion

The effect of sublethal concentrations of potassium dichromate and fenitrothion on sodium-leucine cotransport in brush border membrane vesicles from Chironomus riparius larvae has been investigated. Exposure to potassium dichromate and fenitrothion caused a dose- and time-dependent inhibition of leucine uptake. Transport inhibition is easily detectable at doses 100-fold lower than LD50. Kinetic experiments showed that inhibition was mainly caused by a decrease of the Vmax (680 +/- 53 vs. 382 +/- 23 and 555 +/- 27 nmol/15s/mg protein in control and exposed larvae to K2Cr2O7 and fenitrothion, respectively). Inhibition is possibly related to a variation of sodium ions permeability as evidenced by increased membrane lipid peroxidation. Appropriate control experiments ruled out that the observed differences could be due to changes in general features of membrane preparations. Transport inhibition observed in larvae exposed to potassium dichromate was accompanied by changes in ascorbate peroxidase and dehydroascorbate reductase activities, whereas those exposed to fenitrothion displayed an increase in transaminase activity. The possible value of leucine uptake as biochemical biomarker is briefly discussed. Arch. Insect Biochem. Physiol. 55:90-101, 2004.     

大肠杆菌中外源基因的表达调节

大肠杆菌已经被广泛地应用于表达各种外源基因,但基因的表达受到多种因素的调节,而且不同的外源基因在大肠杆菌中的表达效率也有很大差异。本文从转录水平调节、翻译水平调节、培养条件调节等方面综述了大肠杆菌中外源基因的表达调节,以便认识其规律,有助于使用有效的方法提高外源基因在大肠杆菌中的表达效率。     

大肠杆菌tyrR基因对aroP的表达调控

大肠杆菌的tyrR基因编码芳香氨基酸生物合成和运输途径中的一种全局性调控蛋白质,该蛋白质控制着包括自身编码基因tyrR在内的涉及苯丙氨酸、酪氨酸、色氨酸合成与运输的8个转录单位的转录。大肠杆菌aroP基因编码一种跨质膜主动运输芳香氨基酸的透性酶,其转录也受TyrR蛋白抑制。利用PCR反应从E.coli K12基因组中分别克隆了aroP(p)基因(携带自身的启动子)、aroP基因(不携带自身的启动子)和tyrR基因,并将它们导入苯丙氨酸生产菌E.coli WT5中。通过大细胞法检测到这3个基因的表达,并分别测定了相应的酶活力。结果:发现导入aroP(p)和aroP基因的大肠杆菌吸收苯丙氨酸的能力分别提高到原来的1．40和1．46倍,这表明利用pλPR质粒能够表达aroP基因,该质粒中的λ右向启动子(R)和aroP自身启动子(p)的表达效率几乎同样;导入tyrR基因的E．coli WT5 ATP酶活力提高到原来的1．69倍。将两种基因串联克隆在同一质粒中共表达时,携带aroP-tyr串联的大肠杆菌株运输苯丙氨酸的能力明显高于携带aroP(p)-tyrR串联的大肠杆菌株。以E．coli WT5为对照,其AroP的活性定为1,前者的Atop相对酶活力为1．31,后者为0．95,这一结果显示TyrR蛋白可能是通过与aroP基因自身启动子的结合作用来负调控aroP基因的表达。     

Derepressed leucine transport activity in Escherichia coli

Transport activity and synthesis of binding protein for the amino acids leucine, isoleucine and valine in E. coli are coordinately controlled by the level of leucine in the growth medium. Spontaneous mutants (dlu) which can utilize D-leucine as a source of L-leucine show derepressed transport activity for the three-branched chain amino acids. The increased transport activity is a result of an increase in the binding protein for these amino acids. Azaleucine-resistant mutants have been isolated which have a defect in leucine transport but normal levels of the binding protein for leucine.     

Osmoregulation by potassium transport in Escherichia coli

Abstract Cell turgor pressure determines the extent of K⁺ accumulation by Escherichia coli cells. K⁺ influx is mediated both by a constitutive system with a low affinity for K⁺ (Trk) and by an inducible high affinity system (Kdp). K⁺ efflux is controlled by as yet unidentified but independent systems. Cell K⁺ concentration may be the link between growth in media of high osmolarity and the concomitant accumulation of compatible solutes such as betaine.     

Cloning of the cDNA and mRNA expression of CLRP,a complex leucine repeat protein of the golgi apparatus expressed by specific neurons of the rat brain

We report the molecular cloning of one novel cDNA isolated from the rat brain. We have named the putative protein CLRP, for complex leucine‐repeat protein. The predicted CLRP amino acid sequence shares homology in the amino acid composition with the Galactose, N‐Acetylglucosamine, and Sialic acid transporters, and shows 91% identity with the sequence of one human chromosome 5 BAC clone. Expression of the CLRP cDNA tagged with GFP in COS‐7 cells was found in cell organelles that resemble the Golgi apparatus of the cytoplasm. In Northern blot, the CLRP probe labels a single band of 2.4 kb in the brain, kidney, lung, testis, and prostate. In the brain, CLRP mRNA is expressed by limited sets of neurons, such as the pyramidal cells of the cortex, the Purkinje cells of the cerebellum, and the motoneurons of the brainstem. In the brain, the CLRP mRNA is expressed at embryonic day 15; levels of expression are maintained until postnatal day 10 and decrease in adults. The results suggest that CLRP codes a novel member of the nucleotide–sugar family of proteins of the Golgi apparatus. © 2002 Wiley Periodicals, Inc. J Neurobiol 52: 166–173, 2002     

Orientation of the protonmotive force in membrane vesicles of escherichia coli

Membrane vesicles of Escherichia coli can be produced by 2 different methods: lysis of intact cells by passage through a French pressure cell or by osmotic rupturing of spheroplasts. The membrane of vesicles produced by the former method is everted relative to the orientation of the inner membrane in vivo. Using NADH, D-lactate, reduced phenazine methosulfate, or ATP these vesicles produce protonmotive forces, acid and positive inside, as determined using flow dialysis to measured the distribution of the weak base methylamine and the lipophilic anion thiocyanate. The vesicles accumulate calcium using the same energy sources, most likely by a calcium/proton antiport. Calcium accumulation, therefore, is presumably indicative of a proton gradient, acid inside. The latter type of vesicle, on the other hand, exhibits D-lactate-dependent proline transport but does not accumulate calcium with D-lactate as an energy source. NADH oxidation or ATP hydrolysis, however, will drive the transport of calcium but not proline in these vesicles. Oxidation of NADH or hydrolysis of ATP simultaneous with oxidation of D-lactate does not result in either calcium or proline transport. These results suggest that the vesicles are a patchwork or mosiac, in which certain enzyme complexes have an orientation opposite to that found in vivo, resulting in the formation of electrochemical proton gradients with an orientation opposite to that found in the intact cell. Other complexes retain their original orientation, making it possible to set up simultaneous proton fluxes in both directions, causing an apparent uncoupling of energy-linked processes. That the vesicles are capable of generating protonmotive forces of the opposite polarity was demonstrated by measurements of the distribution of acetate and methylamine (to measure the ΔpH) and thiocyanate (to measure the Δψ).     

A virtual high-throughput screening approach to the discovery of novel inhibitors of the bacterial leucine transporter,LeuT

Abstract

Membrane proteins are intrinsically involved in both human and pathogen physiology, and are the target of 60% of all marketed drugs. During the past decade, advances in the studies of membrane proteins using X-ray crystallography, electron microscopy and NMR-based techniques led to the elucidation of over 250 unique membrane protein crystal structures. The aim of the European Drug Initiative for Channels and Transporter (EDICT) project is to use the structures of clinically significant membrane proteins for the development of lead molecules. One of the approaches used to achieve this is a virtual high-throughput screening (vHTS) technique initially developed for soluble proteins. This paper describes application of this technique to the discovery of inhibitors of the leucine transporter (LeuT), a member of the neurotransmitter:sodium symporter (NSS) family.     

Regulation of Lactose Transport by the Phosphoenolpyruvate-Sugar Phosphotransferase System in Membrane Vesicles of Escherichia coli

Regulation of lactose uptake by the phosphoenolpyruvate-sugar phosphotransferase system (PTS) has been demonstrated in membrane vesicles of Escherichia coli strain ML308-225. Substrates of the phosphotransferase system inhibited D-lactate energized uptake of lactose but did not inhibit uptake of either L-alanine or L-proline. This inhibition was reversed by intravesicular (but not extravesicular) phosphoenolpyruvate. Lactose uptake was also inhibited by enzyme III^glc preparations that were shocked into the vesicles, and this inhibition was reversed by phosphoenolpyruvate. Intravesicular HPr and enzyme I stimulated methyl α-glucoside uptake but did not inhibit or stimulate lactose accumulation. Vesicles maintained at 0°C for several days partially lost 1) the ability to take up lactose, 2) the ability to accumulate PTS substrates, and 3) PTS-mediated regulation. Phosphoenolpyruvate addition restored all of these activities. These results support a mechanism in which the relative proportions of phosphorylated and nonphosphorylated forms of a phosphotransferase constituent regulate the activity of the lactose permcase.