Tomofusa Tsuchiya

Cation-sugar cotransport in the melibiose transport system of Escherichia coli.

The entry of Na+ or H+ into cells of Escherichia coli via the melibiose transport system was stimulated by the addition of certain galactosides. The principal cell used in these studies (W3133) was a lactose transport negative strain of E. coli possessing an inducible melibiose transport system. Such cells were grown in the presence of melibiose, washed, and incubated in the presence of 25 microM Na+. The addition of thiomethylgalactoside (TMG) resulted in a fall in Na+ concentration in the incubation medium. No TMG-stimulated Na+ movement was observed in uninduced cells. In an alpha-galactosidase negative derivative of W3133 (RA11) a sugar-stimulated Na+ uptake was observed in melibiose-induced cells on the addition of melibiose, thiodigalactoside, methyl-alpha-galactoside, methyl-beta-galactoside, and galactose, but not lactose. It was inferred from these studies that the substrates of the melibiose system enter the cell on the melibiose carrier associated with the simultaneous entry of Na+ when this cation is present in the incubation medium. Extracellular pH was measured in unbuffered suspensions of induced cells in order to study proton movement across the membrane of cells exposed to different galactosides. In the absence of external Na+ or Li+ the addition of melibiose or methyl-alpha-galactoside resulted in marked alkalinization of the external medium (consistent with H+-sugar cotransport). On the other hand TMG, thiodigalactoside, and methyl-beta-galactoside gave no proton movement under these conditions. When Na+ was present, the addition of TMG or melibiose resulted in acidification of the medium. This observation is consistent with the view that the entry of Na+ with TMG or melibiose carries into the cell a positive charge (Na+) which provides the driving force for the diffusion of protons out of the cell. It is concluded that the melibiose carrier recognition of cations differs with different substrates.

Co-transport of Na+ and methyl-β-D-thiogalactopyranoside mediated by the melibiose transport system of Escherichia coli

Na+-dependent transport of methyl-β-D-thiogalactopyranoside (TMG) mediated by the melibiose transport system was investigated in Escherichia coli mutants lacking the lactose transport system. When an inwardly-directed electro-chemical potential difference of Na+ was imposed across the membrane of energy depleted cells, transient uptake of TMG was observed. Addition of TMG to cell suspensions under anaerobic conditions caused a transient acidification of the medium. This acidification was observed only in the presence of Na+ or Li+. These results support the idea that TMG is taken up by a mechanism of Na+-TMG co-transport via the melibiose transport system in Escherichia coli.

A new Na+/H+ antiporter, NhaD, of Vibrio parahaemolyticus

A gene encoding an Na+/H+ antiporter was cloned from chromosomal DNA of Vibrio parahaemolyticus, a slightly halophilic bacterium, and expressed in Escherichia coli cells. The gene enabled mutant E. coli cells, which were unable to grow in the presence of 10 mM LiCl (or 0.2 M NaCl) because of the lack of major Na+(Li+)/H+ antiporters, to grow under such conditions. We detected Na+/H+ antiport activity due to the gene in membrane vesicles prepared from E. coli cells that harbored the plasmid carrying the gene. Li+ was also a substrate for this antiporter. Activity of this antiporter was pH-dependent with highest activity at pH 8.5 to 9 and no activity at 7.0 to 7.5. Restriction mapping and a Southern blot analysis revealed that the cloned gene was different from the nhaA and the nhaB of V. parahaemolyticus. We designated the gene nhaD. The gene was sequenced, and the amino acid sequence of the NhaD protein was deduced. The NhaD is a unique Na+/H+ antiporter with respect to the primary structure compared with known Na+/H+ antiporters.

CLONING AND SEQUENCING OF A NOVEL NA+/H+ ANTIPORTER GENE FROM PSEUDOMONAS AERUGINOSA

We cloned a gene for Na+/H+ antiporter from chromosomal DNA of Pseudomonas aeruginosa. Introduction of the gene into host Escherichia coli mutant cells lacking all of the major Na+/H+ antiporters enabled the cells to grow in the presence of 0.2 M NaCl, although the original host cells could not. Membrane vesicles prepared from cells of the transformant possessing the cloned gene showed Na+/H+ antiport activity. As a result of DNA sequencing, we found one open reading frame (nhaP). The deduced amino acid sequence suggests that the Na+/H+ antiporter (NhaP) of P. aeruginosa consists of 424 amino acid residues with molecular mass of 45486 Da, and hydropathy analysis suggested the presence of 12 putative transmembrane domains. We found no bacterial Na+/H+ antiporter which showed significant sequence similarity with the NhaP in the protein sequence database. The NhaP showed partial sequence similarity with animal Na+/H+ exchangers. Thus, the NhaP of P. aeruginosa is unique among bacterial antiporters.

Suppression of ethanol-induced apoptotic DNA fragmentation by geranylgeranylacetone in cultured guinea pig gastric mucosal cells

The purpose of this study was to elucidate themolecular mechanism of action of geranylgeranylacetone,an antiulcer drug. Treatment with ethanol for 8 hr atthe optimum concentration (7.5%) caused apoptotic DNA fragmentation in cultured guinea piggastric mucosal cells. Pretreatment of cells withgeranylgeranylacetone suppressed the DNA fragmentationin a dose-dependent manner. The maximum effect wasachieved at 10-6 M, at which concentration the drug waspreviously shown to induce heat-shock proteins. Thesuppression required an incubation period longer than 1hr. Pretreatment of cells with low concentrations of ethanol also prevented DNAfragmentation.

Effect of lithium ion on melibiose transport inEscherichia coli

SummaryBoth Li+ and Na+ stimulated the uptake of thiomethylgalactoside by the melibiose transport system ofEscherichia coli. On the other hand, Li+ inhibited the growth of cells on melibiose as a sole source of carbon. This inhibition was specific for melibiose, and Li+ had no effect on growth of cells on glucose, galactose, lactose, or glycerol. The effect of the cation on melibiose transport was investigated in a mutant which cannot utilize glucose. After entry into this cell, melibiose is cleaved into glucose and galactose by α-galactosidase, and the resulting glucose is excreted. Since the entry step was found to be rate-limiting, glucose production could be taken as a measure of melibiose transport. Li+ inhibited the transport of melibiose, but not the induction of the melibiose operon nor the activity of α-galactosidase. Li+ was found to inhibit the entry ofp-nitrophenyl-α-d-galactoside, but notp-nitrophenyl-β-d-galactoside entry. Thus, the cation specificity for the melibiose membrane carrier varies with different transport substrates.

Identification of amino acids involved in the functional interaction between DnaA protein and acidic phospholipids

DnaA protein, the initiator of chromosomal DNA replication in Escherichia coli, seems to be regulated through its binding to acidic phospholipids, such as cardiolipin. In our previous paper (Hase, M., Yoshimi, T., Ishikawa, Y., Ohba, A., Guo, L., Mima, S., Makise, M., Yamaguchi, Y., Tsuchiya, T., and Mizushima, T. (1998) J. Biol. Chem. 273, 28651–28656), we found that mutant DnaA protein (DnaA431), in which three basic amino acids (Arg360, Arg364, and Lys372) were mutated to acidic amino acids showed a decreased ability to interact with cardiolipin in vitro, suggesting that DnaA protein binds to cardiolipin through an ionic interaction. In this study, we construct three mutant dnaA genes each with a single mutation and examined the function of the mutant proteins in vitro and in vivo. All mutant proteins maintained activities for DNA replication and ATP binding. A mutant protein in which Lys372 was mutated to Glu showed the weakest interaction with cardiolipin among these three mutant proteins. Thus, Lys372 seems to play an important role in the interaction between DnaA protein and acidic phospholipids. Plasmid complementation analyses revealed that all these mutant proteins, including DnaA431 could function as an initiator for chromosomal DNA replication in vivo.

Melibiose carrier of Escherichia coli: Use of cysteine mutagenesis to identify the amino acids on the hydrophilic face of transmembrane helix 2

The melibiose carrier from Escherichia coli is a galactoside-cation symporter. Based on both experimental evidence and hydropathy analysis, 12 transmembrane helices have been assigned to this integral membrane protein. Transmembrane helix 2 contains several charged and polar amino acids that have been shown to be essential for the cation-coupled transport of melibiose. Starting with the cysteine-less melibiose carrier, we have individually substituted cysteine for amino acids 39-66, which includes the proposed transmembrane helix 2. In the resulting derivative carriers, we measured the transport of melibiose, determined the effect of the hydrophilic sulfhydryl reagent, p-chloromercuribenzenesulfonic acid (PCMBS), on transport in intact cells and inside out vesicles, and examined the ability of melibiose to protect the carrier from inactivation by the sulfhydryl reagent. We found a set of seven positions in which the reaction with the sulfhydryl reagent caused partial or complete loss of carrier function measured in intact cells or inside-out vesicles. The presence of melibiose protected five of these positions from reaction with PCMBS. The reaction of two additional positions with PCMBS resulted in the partial loss of transport function only in inside-out vesicles. Melibiose protected these two positions from reaction with the reagent. Together, the PCMBS-sensitive sites and charged residues assigned to helix 2 form a cluster of amino acids that map in three rows with each row comprised of every fourth residue. This is the pattern expected of residues that are part of an alpha-helical structure and thus the rows are tilted at an angle of 25 degrees to the helical axis. We suggest that these residues line the path of melibiose and its associated cation through the carrier.

SEQUENCE OF A NA+/GLUCOSE SYMPORTER GENE AND ITS FLANKING REGIONS OF VIBRIO PARAHAEMOLYTICUS

The nucleotide sequence of an approximately 6 kbp segment of chromosomal DNA of Vibrio parahaemolyticus was determined. The nucleotide sequence revealed four open reading frames (ORFs) in this region. Hydropathy profiles of the deduced amino acid sequence of the ORFs indicate that ORF1 encodes a hydrophobic polypeptide with typical characteristics of a membrane transport protein. All other ORFs encode hydrophilic polypeptides. ORF1 showed significant amino acid sequence similarity to proteins of the SGLT (Na+/glucose symporter) family, and the amino acid sequence of ORF4 showed very high similarity to several bacterial transcriptional repressor proteins (GalR-LacI family). We observed elevated glucose transport activity in cells harboring a plasmid carrying the DNA region corresponding to ORF1, and the glucose transport was greatly stimulated by Na+. Thus, we believe that ORF1 encodes a Na+/glucose symporter.

Involvement of Arg-328, Arg-334 and Arg-342 of DnaA protein in the functional interaction with acidic phospholipids

We reported previously that three basic amino acids (Arg-360, Arg-364 and Lys-372) of DnaA protein are essential for its functional interaction with cardiolipin. In this study, we examined the effect of mutation of some basic amino acids in a potential amphipathic helix (from Lys-327 to Ile-345) of DnaA protein on this interaction. ATP binding to the mutant DnaA protein, in which Arg-328, Arg-334 and Arg-342 were changed to acidic amino acids, was less inhibited by cardiolipin than that of the wild-type protein, as was the case for mutant DnaA protein with mutations of Arg-360, Arg-364 and Lys-372. A mutant DnaA protein with mutations of all six basic amino acids showed the most resistance to the inhibition of ATP binding by cardiolipin. These results suggest that Arg-328, Arg-334 and Arg-342, like Arg-360, Arg-364 and Lys-372, are also involved in the functional interaction between DnaA protein and acidic phospholipids.