Melibiose Transport System Research Articles

Melibiose transport system in Lactobacillus plantarum.

Lactobacillus plantarum ATCC 8014 grew on melibiose at 30 C, but not at 37 C, although it grew on galactose or lactose at either temperature. ATCC 8014 grown on lactose at 30 or 37 C accumulated melibiose slowly, suggesting that melibiose may partly be transported by a lactose transport system. A lactose-negative mutant, NTG 21, derived from ATCC 8014 was isolated. The mutant was totally deficient in lactose transport, but retained normal melibiose transport activity. In NTG 21, the melibiose transport activity was induced by melibiose at 30 C, but not at 37 C. The transport activity itself was found to be stable for at least 3 hr at 37 C, suggesting that the induction process in the cytoplasm rather than the inducer entrance is temperature-sensitive in the organism. The organism also failed to form alpha-galactosidase at 37 C when grown on melibiose. The enzyme synthesis, however, was induced by galactose in NTG 21 (and also by lactose in ATCC 8014) even at 37 C, indicating that the induction of the enzyme is essentially not temperature-sensitive. In NTG 21, melibiose transport system and alpha-galactosidase were induced by galactose, melibiose and o-nitrophenyl-alpha-D-galactopyranoside when the strain was grown at 30 C. Raffinose induced melibiose transport system only a little, while it was a good inducer for alpha-galactosidase. Inhibition studies revealed that galactose may be a weak substrate of the melibiose transport system; no inhibition was demonstrated with lactose and raffinose.

<i>Lactobacillus plantarum</i>におけるメリビオース代謝の高温感受性機構の解析

L. plantarum can grow on melibiose when incubated at 30°C but not when incubated at 37°C. In this study, the temperature sensitivity of melibiose metabolism was analyzed. The melibiose metabolism seemed to consist of the melibiose transport system and hydrolyzing enzyme, α-galactosidase. α-Galactosidase was induced with melibiose at 30°C but not at 37°C. This enzyme induced at 30°C hydrolyzed colorimetric substrate, α-ONPG at 30°C and 37°C. On the other hand, lactose induced α-galactosidase at both temperatures. These findings suggested that the temperature sensitivity of melibiose metabolism was not due to the induction and/or function of α-galactosidase.Thus, the effect of temperature on induction and function of the melibiose transport system was examined. Cells grown on melibiose at 30°C exhibited the rapid uptake of [3H] -melibiose at 30°C and 37°C, while [3H] -melibiose was not taken up by the cells grown at 37°C. The induction of the melibiose transport system seemed to be temperature-sensitive because the transport system was not inactivated by exposure of the cells to 37°C for 3 hours. In conclusion, the temperature sensitivity of melibiose metabolism was attributed to the temperature sensitivity of the induction of melibiose transport system.

Cation specificity for sugar substrates of the melibiose carrier in Escherichia coli

A study has been made of the sugar substrate specificities and the cation specificities of the melibiose transport system of Escherichia coli. The following β-galactosides were found to be transported: lactose, l-arabinose-β- d-galactoside, d-fructose-β- d-galactoside, o- and p- nitrophenyl-β- d-galactosides . These β-galactosides were cotransported with Na + but not with H +. The α-galactosides raffinose, melibiose and p-nitrophenyl- α-galactoside were transported with either H + or Na +. Of the monosaccharides tested d-galactose could use either Na + or H + for cotransport whereas d-fucose, l-arabinose and d-galactosamine could use only Na +. The sugar specificity requirements for H + cotransport are therefore more exacting than those for Na + cotransport.

30] Methods for the study of the melibiose carrier of Escherichia coli

This chapter describes methods used for measuring the transport of both the sugar and cation substrates. Some of these methods such as the preparation of radioactive melibiose and the construction of the Li+-selective electrode were devised specifically for studies of the melibiose transport system. The melibiose carrier of Escherichia coli is a representative of the class of carriers that require a cation for cotransport. An unusual feature of this transport system is that it is able to utilize Na + , Li + , or H + as the coupling cation depending on the conditions. Another interesting property of the carrier is that different sugar substrates show different specificities for the cation requirement. A variety of strains of E. coli has been isolated for use in experiments on the melibiose carrier. It is essential to start with a strain from which the lactose carrier (lac Y) has been deleted, as the lac carrier also is capable of transporting melibiose. The growth of normal E. coli cells on melibiose minimal medium is strongly inhibited by 10 mM Li + and it is therefore possible to isolate Li + -resistant mutants. This organism is dependent on Na + or Li + for growth on melibiose as it has lost the ability to transport the disaccharide with protons.

Cation coupling in the melibiose transport carrier.

The melibiose transport system in Escherichia coli shows versatility in cation coupling. Na+, H+ or Li+ couples to transport depending on substrate in this carrier. To elucidate the relationship between structure and function of the carrier we first determined the primary structure. The carrier consists of 469 amino acid residues with molecular weight of 52, 029. Hydropathy profile of the carrier suggests the presence of about 10 highly hydrophobic regions which probably traverse membrane. There are several hydrophilic regions which may be involved in transport of cations and substrates. We isolated mutants which showed altered cation specificity. We also determined the sequence of mutant carrier, and found replacement of one amino acid residue. This type of approach seems to be useful for understanding of the mechanism of cation coupling.

Lithium ion-sugar cotransport via the melibiose transport system in Escherichia coli. Measurement of Li+ transport and specificity.

A lithium ion-selective electrode was constructed using N,N'-diheptyl-N,N'-5,5-tetramethyl-3,7-dioxanonandiamid as a Li+ ionophore. Lithium ion-sugar cotransport via the melibiose transport system was measured with this electrode. Influx of methyl-beta-D-thiogalactoside, methyl-alpha-D-galactoside, methyl-beta-D-galactoside, and D-galactose elicited uptake of Li+. This Li+ uptake was not observed when the melibiose carrier was not present in the cells or the carrier was inactivated. Melibiose caused a small amount of Li+ uptake, indicating that Li+-melibiose cotransport proceeds inefficiently. Raffinose, another substrate, did not cause detectable Li+ transport. In mutant cells which showed altered cation coupling (Niiya, S., Yamasaki, K., Wilson, T. H., and Tsuchiya, T. (1982) J. Biol. Chem. 257, 8902-8906), Li+-melibiose cotransport was clearly demonstrated. Alteration in substrate specificity was also shown in the mutants.

Active transport of sugars coupled to Na+ in Escherichia coli

Cation-nutrient cotransport is ubiquitous in nature. For cotransport, coupling between H+ and substrate is common in microbial cells, and that between Na+ and substrate is common in animal cells. We have reported that the melibiose transport system of Escherichia coli could utilize Na+ as a coupling cation. This is the first example of Na+-substrate cotransport found in microbial cells. Versatility in the combination between cation and substrate was revealed. This system seems to be a suitable model system for the analysis of cation-substrate cotransport systems. Physiological, biochemical and genetical properties of this system are summarized.

Cation coupling to melibiose transport in Salmonella typhimurium.

Melibiose transport in Salmonella typhimurium was investigated. Radioactive melibiose was prepared and the melibiose transport system was characterized. Na+ and Li+ stimulated transport of melibiose by lowering the Km value without affecting the Vmax value; Km values were 0.50 mM in the absence of Na+ or Li+ and 0.12 mM in the presence of 10 mM NaCl or 10 mM LiCl. The Vmax value was 140 nmol/min per mg of protein. Melibiose was a much more effective substrate than methyl-beta-thiogalactoside. An Na+-melibiose cotransport mechanism was suggested by three types of experiments. First, the influx of Na+ induced by melibiose influx was observed with melibiose-induced cells. Second, the efflux of H+ induced by melibiose influx was observed only in the presence of Na+ or Li+, demonstrating the absence of H+-melibiose cotransport. Third, either an artificially imposed Na+ gradient or membrane potential could drive melibiose uptake in cells. Formation of an Na+ gradient in S. typhimurium was shown to be coupled to H+ by three methods. First, uncoupler-sensitive extrusion of Na+ was energized by respiration or glycolysis. Second, efflux of H+ induced by Na+ influx was detected. Third, a change in the pH gradient was elicited by imposing an Na+ gradient in energized membrane vesicles. Thus, it is concluded that the mechanism for Na+ extrusion is an Na+/H+ antiport. The Na+/H+ antiporter is a transformer which converts an electrochemical H+ gradient to an Na+ gradient, which then drives melibiose transport. Li+ was inhibitory for the growth of cells when melibiose was the sole carbon source, even though Li+ stimulated melibiose transport. This suggests that high intracellular Li+ may be harmful.

Mechanism of the melibiose porter in membrane vesicles of Escherichia coli.

The melibiose transport system of Escherichia coli catalyzes sodium--methyl 1-thio-beta-D-galactopyranoside (TMG) symport, and the cation is required not only for respiration-driven active transport but also for binding of substrate to the carrier in the absence of energy and for carrier-mediated TMG efflux. As opposed to the proton--beta-galactoside symport system [Kaczorowski, G. J., & Kaback, H. R. (1979) Biochemistry 18, 3691], efflux and exchange of TMG occur at the same rate, implying that the rates of the two processes are limited by a common step, most likely the translocation of substrate across the membrane. Furthermore, the rate of exchange, as well as efflux, is influenced by imposition of a membrane potential (delta psi; interior negative), suggesting that the ternary complex between sodium, TMG, and the porter may bear a net positive charge. Consistently, energization of the vesicles leads to a large increase in the Vmax for TMG influx, with little or no change in the apparent Km of the process. It is proposed that the sodium gradient (Na+out < Na+in) and the delta psi (interior negative) may affect different steps in the overall mechanism of active TMG accumulation in the following manner: the sodium gradient causes an increased affinity for TMG on the outer surface of the membrane relative to the inside and the delta psi facilitates a reaction involved with the translocation of the positively charged ternary complex to the inner surface of the membrane.

Membrane transport of p-nitrophenyl-alpha-galactoside by the melibiose carrier of Escherichia coli.

p-Nitrophenyl-alpha-galactoside (alpha-pNPG) was found to be a substrate for the melibiose transport system of Escherichia coli. This sugar enters induced cells via the carrier and is split by alpha-galactosidase to galactose and p-nitrophenol. In mutant cells lacking the alpha-galactosidase [3H]-alpha-pNPG accumulated to concentrations 15 times higher than the external medium. The transport of alpha-pNPG is inhibited by both Na+ and Li+. Na+ (10 mM) reduced the Km for alpha-pNPG from 0.45 to 0.18 mM and reduced the Vmax from 6.7 nmoles/min/mg dry wt to a value of 3.0.

H + -substrate cotransport by the melibiose membrane carrier in Escherichia coli.

Proton entry into anaerobic Escherichia coli in response to the addition of HCl was measured by monitoring pH changes in the external solution. Preincubation of cells in a Na+ -free medium containing melibiose or methyl-alpha-galactoside (alpha MG) stimulated the rate of H+ entry in response to the acid pulse. This melibiose- or alpha MG-dependent proton pathway appeared to be identical to the melibiose carrier, since the channel was only observed in melibiose-induced cells. Furthermore, this membrane pathway for protons showed the same temperature sensitivity as the melibiose carrier (active at 30 degrees but inactive at 37 degrees). These observations are consistent with the idea that the melibiose transport system provides a pathway for protons in the presence of appropriate substrates, but that the pathway is closed to protons in the absence of the sugar. Such observations indicate that there is an obligatory coupling between H+ flux and melibiose or alpha MG flux through the carrier when Na+ is omitted from the incubation medium.

Extrusion of Sodium Ions Energized by Respiration and Glycolysis in &lt;italic&gt;Escherichia coli&lt;/italic&gt;&lt;xref ref-type="fn" rid="fn1"&gt;&lt;sup&gt;1&lt;/sup&gt;&lt;/xref&gt;

Extrusion of sodium ion from cells of Escherichia coli was measured using an Na+ electrode. When oxygen was supplied to an anaerobic cell suspension, extrusion of Na+ was observed. The addition of glucose under anaerobic conditions also caused Na+ efflux. The extrusion of Na+ energized by respiration and glycolysis was completely inhibited by a proton conductor, carbonyl cyanide m-chlorophenylhydrazone. These observations are consistent with the view that Na+ transport occurs secondarily to H+ circulation. Interestgly, induction of the melibiose transport system, which is coupled to Na+, greatly enhanced Na+ transport activity.

Role of Na+ and Li+ in thiomethylgalactoside transport by the melibiose transport system of Escherichia coli.

Thiomethyl-beta-galactoside (TMG) accumulation via the melibiose transport system was studied in lactose transport-negative strains of Escherichia coli. TMG uptake by either intact cells or membrane vesicles was markedly stimulated by Na+ or Li+ between pH 5.5 and 8. The Km for uptake of TMG was approximately 0.2 mM at an external Na+ concentration of 5 mM (pH 7). The alpha-galactosides, melibiose, methyl-alpha-galactoside, and o-nitrophenyl-alpha-galactoside had a high affinity for this system whereas lactose, maltose and glucose had none. Evidence is presented for Li+-TMG or Na+-TMG cotransport.

Effect of lithium ion on melibiose transport inEscherichia coli

Both Li+ and Na+ stimulated the uptake of thiomethylgalactoside by the melibiose transport system of Escherichia coli. On the other hand, Li+ inhibited the growht 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 alpha-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 melibose transport. Li+ inhibited the transport of melibiose, but not the induction of the melibiose operon nor the activity of alpha-galactosidase. Li+ was found to inhibit the entry of p-nitrophenyl-alpha-D-galactoside, but not p-nitrophenyl-beta-D-galactoside entry. Thus, the cation specificity for the melibiose membrane carrier varies different transport substrates.

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 [formula omitted

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 .