The nodI gene product of Rhizobium leguminosarum is closely related to ATP-binding bacterial transport proteins; nucleotide sequence analysis of the nodI and nodJ genes

The nucleotide sequence of a 2-kb fragment immediately downstream of the nodABC genes of the Rhizobium leguminosarum symbiotic plasmid pRL1JI has been determined. Genes corresponding to the two open reading frames identified are named nodI and nodJ. Tn 5 insertions into these genes result in a "nodulation-delayed" phenotype. The predicted amino acid sequence of the nodI gene shows considerable homology to inner-membrane-located gene products involved in active transport systems in Escherichia coli and Salmonella typhimurium. The predicted product of the nodJ gene is very hydrophobic, suggesting that it may be an integral membrane protein.     

Nucleotide sequence and genetic characterization reveal six essential genes for the LIV-I and LS transport systems of Escherichia coli

The nucleotide sequence of the genes encoding the high affinity, branched-chain amino acid transport systems LIV-I and LS has been determined. Seven genes are present on a 7568-base pair DNA fragment, six of which participate directly in branched-chain amino acid transport. Two periplasmic amino acid-binding proteins are encoded by the livJ (LIV-BP) and livK (LS-BP) genes. These two proteins confer specificity on the LIV-I and LS transport systems. livK is the first gene in a polycistronic message that includes four genes encoding membrane components, livHMGF. The protein products of the livHMGF genes are shared by the two systems. An analysis of the livH and livM DNA sequences suggests that they encode hydrophobic proteins capable of spanning the membrane several times. The LivG and LivF proteins are less hydrophobic, but are also tightly associated with the membrane. Both LivG and LivF contain the consensus sequence for adenine nucleotide binding observed in many other transport proteins. A deletion strain that does not express any of the liv genes was constructed. This strain was used to show that each of the membrane component genes is required for high affinity leucine transport, including two genes, livM and livF, for which no previous genetic evidence had been obtained.     

DiO-C3-(5) and DiS-C3-(5): Interactions with RBC,ghosts and phospholipid vesicles

Summary The transport of solutes by bacteria has been studied for about thirty years. Early experiments on amino acid entry and galactoside accumulation provided concrete evidence that bacteria possessed specific transport systems and that these were subject to regulation. Since then a large number of transport systems have been discovered and studied extensively. Many of these use entirely different strategies for capturing or accumulating substrates. This diversity reflects variation in the availability of nutrients and ions in the different environments tolerated and inhabited by microorganisms. Examination of a few bacterial transport systems provides an opportunity to gain insight into a wide range of topics in the area of membrane transport. These include: the identification of carrier proteins and their arrangement in the membrane, the regulation of transport protein synthesis by environmental factors, and the localization of transport proteins to their extracytoplasmic destinations.It has been possible to construct a number of bacterial strains in which the gene (lacZ) which codes for the cytoplasmic enzyme -galactosidase is fused to genes which code for transport proteins. The following article is intended to illustrate how these gene fusions have been used to study the regulation and structure of transport proteins inEscherichia coli.     

Cloning and nucleotide sequence of braC, the structural gene for the leucine-, isoleucine-, and valine-binding protein of Pseudomonas aeruginosa PAO.

The gene for the leucine-, isoleucine-, and valine-binding protein (LIVAT-BP) in Pseudomonas aeruginosa PAO was isolated, and its nucleotide sequence was determined. The gene consisted of 1,119 nucleotides specifying a protein of 373 amino acid residues. Determination of the N-terminal amino acid sequence of the LIVAT-BP purified from P. aeruginosa shock fluid suggested that the N-terminal 26 residues of the gene product are cleaved off posttranslationally, showing the characteristic features of procaryotic signal peptides. The amino acid composition of the mature product predicted from the nucleotide sequence was in good agreement with that of the purified LIVAT-BP. The plasmid carrying the LIVAT-BP gene restored the activity of the high-affinity branched-chain amino acid transport system (the leucine, isoleucine, valine [LIV-I] transport system) in the braC310 mutant of P. aeruginosa, confirming that braC is the structural gene for LIVAT-BP. The mutant LIVAT-BP lacking a 16-amino-acid peptide in the middle was found to be functional in the LIV-I transport system. LIVAT-BP showed extensive homology (51% identical) to the LIV- and leucine-specific-binding proteins of Escherichia coli K-12, which are coded for by the livJ and livK genes, respectively, suggesting that the role of the proteins in the LIV-I transport systems is analogous in both organisms.     

Lactose transport system of Streptococcus thermophilus: a hybrid protein with homology to the melibiose carrier and enzyme III of phosphoenolpyruvate-dependent phosphotransferase systems.

The gene responsible for the transport of lactose into Streptococcus thermophilus (lacS) was cloned in Escherichia coli as a 4.2-kilobase fragment from an EcoRI library of chromosomal DNA by using the vector pKK223-3. From deletion analysis, the gene for lactose transport mapped to two HindIII fragments with a total size of 2.8 kilobases. The gene was transcribed in E. coli from its own promoter. Functional expression of lactose transport activity was shown by assaying for the uptake and exchange of lactose both in intact cells and in membrane vesicles. The nucleotide sequence of lacS and 200 to 300 bases of 3' and 5' flanking regions were determined. The gene was 1,902 base pairs long, encoding a 69,454-dalton protein with an NH2-terminal hydrophobic region and a COOH-terminal hydrophilic region. The NH2-terminal end was homologous with the melibiose carrier of E. coli (23% similarity overall; greater than 50% similarity for regions with at least 16 amino acids), whereas the COOH-terminal end showed 34 to 41% similarity with the enzyme III (domain) of three different phosphoenolpyruvate-dependent phosphotransferase systems. Among the conserved amino acids were two histidyl residues, of which one has been postulated to be phosphorylated by HPr. Since sugars are not phosphorylated during translocation by the lactose transport system, it is suggested that the enzyme III-like region serves a regulatory function in this protein. The lacS gene also appears similar to the partially sequenced lactose transport gene of Lactobacillus bulgaricus (lacL; greater than 60% similarity). Furthermore, the 3' flanking sequence of the S. thermophilus lactose transport gene showed approximately 50% similarity with the N-terminal portion of the beta-galactosidase gene of L. bulgaricus. In both organisms, the lactose transport gene and the beta-galactosidase appear to be separated by a 3-base-pair intercistronic region.     

Investigating autocatalytic gene expression systems through mechanistic modeling.

A structured model of gene expression, which incorporates the stochastic behavior of cellular processes, was developed to examine the "all-or-none" phenomenon observed in autocatalytic systems (e.g. the lac operon). Autocatalytic expression systems typically have the genes encoding the inducer transport proteins controlled by internal inducer levels, so that transport of the inducer increases production of the transport protein. The model was able to predict the unique behaviors of autocatalytic expression systems that have been experimentally observed and provided valuable insight into the role of population heterogeneity in these systems. The simulations substantiate the importance of stochastic processes on induction of gene expression in autocatalytic systems. The simulation results show that the all-or-none phenomenon is governed largely by random cellular events, and that population-averaged variations in gene expression are due to changes in the frequency of full gene induction in individual cells rather than to uniform variations in gene expression across the entire population. In addition, the model shows how concentrations of inducer too low to induce expression in uninduced cells can maintain induction in pre-induced cultures. A comparison of induction behaviors from an autocatalytic system and a system having constitutive synthesis of the transport protein showed that transport protein levels must be decoupled from inducer control to achieve homogeneous expression of a gene of interest in all cells of a culture.     

Nucleotide sequence of the btuCED genes involved in vitamin B12 transport in Escherichia coli and homology with components of periplasmic-binding-protein-dependent transport systems.

The products of the btuCED region of the Escherichia coli chromosome participate in the transport of vitamin B12 across the cytoplasmic membrane. The nucleotide sequence of the 3,410-base-pair HindIII-HincII DNA fragment carrying a portion of the himA gene and the entire btuCED region was determined. Comparison of the location of the open reading frames with the gene boundaries defined by transposon insertions allowed the assignment of polypeptide products to gene sequences. The btuC product is a highly nonpolar integral membrane protein of molecular weight 31,683. The distribution of hydrophobic regions suggests the presence of numerous membrane-spanning domains. The btuD product is a relatively polar but membrane-associated polypeptide of Mr 27,088 and contains segments bearing extensive homology to the ATP-binding peripheral membrane constituents of periplasmic binding protein-dependent transport systems. Other regions of this protein are similar to portions of the outer membrane vitamin B12 receptor. The btuE product (Mr 20,474) appears to have a periplasmic location. It has the mean hydropathy of a soluble protein but lacks an obvious signal sequence. The cellular locations and structural and sequence homologies of the Btu polypeptides point to the similarity of these three proteins to components of binding protein-dependent transport systems. However, the dependence on a periplasmic vitamin B12-binding protein has not yet been demonstrated.     

Escherichia coli possesses two homologous anaerobic C4-dicarboxylate membrane transporters (DcuA and DcuB) distinct from the aerobic dicarboxylate transport system (Dct).

The nucleotide sequences of two Escherichia coli genes, dcuA and dcuB (formerly designated genA and genF), have been shown to encode highly homologous products, M(r) 45,751 and 47,935 (434 and 446 amino acid residues) with 36% sequence identity (63% similarity). These proteins have a high proportion (approximately 61%) of hydrophobic residues and are probably members of a new group of integral inner membrane proteins. The locations of the dcu genes, one upstream of the aspartase gene (dcuA-aspA) and the other downstream of the anaerobic fumarase gene (fumB-dcuB), suggested that they may function in the anaerobic transport of C4-dicarboxylic acids. Growth tests and transport studies with mutants containing insertionally inactivated chromosomal dcuA and dcuB genes show that their products perform analogous and mutually complementary roles as anaerobic dicarboxylate carriers. The anaerobic dicarboxylate transport systems (Dcu) are genetically and functionally distinct from the aerobic system (Dct).     

CFTR in cystic fibrosis and cholera: from membrane transport to clinical practice

We have used a brief analysis of transport via cystic fibrosis (CF) transmembrane conductance regulators (CFTRs) in various organ systems to highlight the importance of basic membrane transport processes across epithelial cells for first-year medical students in physiology. Because CFTRs are involved in transport both physiologically and pathologically in various systems, we have used this clinical correlation to analyze how a defective gene leading to defective transport proteins can be directly involved in the symptoms of cholera and CF. This article is a "Staying Current" approach to transport via CFTRs including numerous helpful references with further information for a teaching faculty member. The article follows our normal presentation which begins with a discussion of the involvement of CFTR transport in the intestine and how cholera affects intestinal transport, extends to CFTR transport in various organ systems in CF, and concludes with the logic behind many of the treatments that improve CF. Student learning objectives are included to assist in assessment of student understanding of the basic concepts.     

Sequence of the mglB gene from Escherichia coli K12: comparison of wild-type and mutant galactose chemoreceptors

Summary The mglB gene of Escherichia coli codes for a galactose-binding protein (GBP) that serves both as the galactose chemoreceptor and as the recognition component of the -methylgalactoside transport system. The mglB551 mutation eliminates the chemotactic function of GBP without altering its transport or substrate-binding properties. To investigate the interaction between GBP and Trg, the chemotactic signal transducer for galactose, we sequenced the mglB genes from wild-type and mglB551 mutant strains. The mutation causes the replacement of Gly₇₄ of GBP by Asp. This residue is located in alpha-Helix III at the tip of the P domain in the GBP tertiary structure farthest removed from the substrate-binding cleft between the P and Q domains. We conclude that Helix III must be part of, or at least adjacent to, the recognition site for Trg. Our sequence also included part of the mglA gene, which is immediately distal to mglB. The amino acid sequence deduced for the beginning of the MglA protein showed homology with a family of polypeptides that contain an ATP-binding site and are components of binding-protein-dependent transport systems.     

Functional expression of the CMP-sialic acid transporter in Escherichia coli and its identification as a simple mobile carrier

The architectural conservation of nucleotide sugar transport proteins (NSTs) enabled the theoretical prediction of putative NSTs in diverse gene databases. In the human genome, 17 NST sequences have been identified but only six have been unequivocally characterized with respect to their transport specificities. Defining transport characteristics of recombinant NSTs has become a major challenge because true zero background systems are widely absent. Production of recombinant NSTs in heterologous systems has developed multifunctionality for some NSTs leading to a novel level of complexity in the field. Assuming that (1) the specificity of NSTs is determined at the primary sequence level and (2) the proteins are autonomously functional units, final definition of the substrate specificity will depend on the use of isolated transport proteins. Herein, we describe the first report of the functional expression of mouse CMP-sialic acid transporter (CST) in Escherichia coli and thus provide significant progress towards the production of transporter proteins in quantities suitable for functional and structural analyses. Recovery of the active NST from inclusion bodies was achieved after solubilization with 8 M urea and stepwise renaturation. After reconstitution into phospholipid vesicles, the recombinant protein demonstrated specific transport for CMP-N-acetylneuraminic acid (CMP-Neu5Ac) with no transport of UDP-sugars. Kinetic studies carried out with CMP-Neu5Ac and established CMP-Neu5Ac antagonist's evaluated natural conformation of the reconstituted protein and clearly demonstrate that the transporter acts as a simple mobile carrier.     

Plasma membrane transport systems in higher plants: From black boxes to molecular physiology

Considerable progress in identifying transport systems of the plant plasma membrane has been made recently. The putative systems cloned to date comprise H⁺-ATPases, potassium, chloride and water channels, and carriers involved in the transport of glucose, sucrose, amino acids, peptides, potassium, nitrate, ammonium, phosphate, sulfate, iron and copper. Most of these systems were identified first in Arabidopsis thaliana . Successful cloning strategies have involved the following variety of techniques: complementation of yeast mutants, screening of Arabidopsis mutants, immunoscreening of a cDNA expression library expressed in mammalian cells, screening of genomic and cDNA libraries with probes (or degenerate oligonucleotides) derived from yeast and/or animal genes, or database screening for sequence similarity to eukaryotic counterparts. Many related transport systems have subsequently been identified either by screening libraries directly, or by systematic cDNA sequencing programs. Surprisingly large gene families have been revealed. Heterologous expression systems, such as yeast, Xenopus oocytes or insect cells, provide tools for studying the transport activities, biochemical properties and structure-function relationships of these systems. Their expression and functions in planta are investigated using northern blot analysis, in situ hybridization, and transgenic approaches. Individual systems encoded by the same gene family can differ in their transport properties and have distinct tissue expression patterns. Such diversity might be central to the integration of solute transport at the whole plant level, allowing the differential expression of sets of transport systems specifically tailored to the requirements of each tissue.     

Structure,function and regulation of the tricarboxylate transport protein from rat liver mitochondria

Recent progress is summarized on the structure, function, and regulation of the tricarboxylate (i.e., citrate) transport protein (CTP) from the rat liver mitochondrial inner membrane. The transporter has been purified and its reconstituted function characterized. A cDNA clone encoding the CTP has been isolated and sequenced, thus enabling a deduction of the complete amino acid sequence of this 32.6 kDa transport protein. Dot matrix analysis and sequence alignment indicate that based on structural considerations the CTP can be assigned to the mitochondrial carrier family. Hydropathy analysis of the transporter sequence indicates six putative membrane-spanning -helices and has permitted the development of an initial model for the topography of the CTP within the inner membrane. The questions as to whether more than one gene encodes the CTP and whether more than one isoform is expressed remain unanswered at this time. Studies documenting a diabetes-induced alteration in the function of several mitochondrial anion transporters, which can be reversed by treatment with insulin, provide a physiologically/pathologically relevant experimental system for studying the molecular mechanism(s) by which mitochondrial transporters are regulated. Potential future research directions are discussed.     

Phosphorylation of the Periplasmic Binding Protein in Two Transport Systems for Arginine Incorporation in Escherichia coli K-12 Is Unrelated to the Function of the Transport System

In Escherichia coli K-12, the accumulation of arginine is mediated by two distinct periplasmic binding protein-dependent transport systems, one common to arginine and ornithine (AO system) and one for lysine, arginine, and ornithine (LAO system). Each of these systems includes a specific periplasmic binding protein, the AO-binding protein for the AO system and the LAO-binding protein for the LAO system. The two systems include a common inner membrane transport protein which is able to hydrolyze ATP and also phosphorylate the two periplasmic binding proteins. Previously, a mutant resistant to the toxic effects of canavanine, with low levels of transport activities and reduced levels of phosphorylation of the two periplasmic binding proteins, was isolated and characterized (R. T. F. Celis, J. Biol. Chem. 265:1787–1793, 1990). The gene encoding the transport ATPase enzyme (argK) has been cloned and sequenced. The gene possesses an open reading frame with the capacity to encode 268 amino acids (mass of 29.370 Da). The amino acid sequence of the protein includes two short sequence motifs which constitute a well-defined nucleotide-binding fold (Walker sequences A and B) present in the ATP-binding subunits of many transporters. We report here the isolation of canavanine-sensitive derivatives of the previously characterized mutant. We describe the properties of these suppressor mutations in which the transport of arginine, ornithine, and lysine has been restored. In these mutants, the phosphorylation of the AO- and LAO-binding proteins remains at a low level. This information indicates that whereas hydrolysis of ATP by the transport ATPase is an obligatory requirement for the accumulation of these amino acids in E. coli K-12, the phosphorylation of the periplasmic binding protein is not related to the function of the transport system.     

Targeting DNA with triplex-forming oligonucleotides to modify gene sequence

Molecules that interact with DNA in a sequence-specific manner are attractive tools for manipulating gene sequence and expression. For example, triplex-forming oligonucleotides (TFOs), which bind to oligopyrimidine.oligopurine sequences via Hoogsteen hydrogen bonds, have been used to inhibit gene expression at the DNA level as well as to induce targeted mutagenesis in model systems. Recent advances in using oligonucleotides and analogs to target DNA in a sequence-specific manner will be discussed. In particular, chemical modification of TFOs has been used to improve binding to chromosomal target sequences in living cells. Various oligonucleotide analogs have also been found to expand the range of sequences amenable to manipulation, including so-called "Zorro" locked nucleic acids (LNAs) and pseudo-complementary peptide nucleic acids (pcPNAs). Finally, we will examine the potential of TFOs for directing targeted gene sequence modification and propose that synthetic nucleases, based on conjugation of sequence-specific DNA ligands to DNA damaging molecules, are a promising alternative to protein-based endonucleases for targeted gene sequence modification.     

The TonB protein of Yersinia enterocolitica and its interactions with TonB-box proteins

Summary The tonB gene is required for energy-dependent transport processes across the outer membrane of gram-negative bacteria. Using the antibiotics albomycin and ferrimycin, a tonB mutant of Yersinia enterocolitica was isolated. Comparison of the tonB mutant with the parent strain revealed that in Y. enterocolitica the uptake of ferrioxamine, ferrichrome, pesticin and heme is TonB-dependent. The tonB gene from Y. enterocolitica was sequenced and found to be similar to those of other Enterobacteria. The Y. enterocolitica tonB gene complemented a Y. enterocolitica tonB mutant. In contrast, some Tong functions of an Escherichia coli tonB mutant were not restored by the tonB gene of Y. enterocolitica. The observed differences in the ability to complement E. coli Tong functions correlated with the degree to which the Tong boxes of the receptors and colicins differed from the TonB box consensus sequence. Furthermore, the N-terminal membrane anchor of the TonB proteins and the TolA protein are likely to form an -helix with an identical sequence motif (SHLS) located at one face of the a-helix, suggesting this region to be involved in the functional cross-talk between the TonB-ExbBD-and TolABQR-dependent transport systems across the outer membrane.