Reconstitution of the GalP galactose transport activity of Escherichia coli into liposomes made from soybean phospholipids

Galactose transport activity from Escherichia coli was solubilized with octyl glucoside, and reconstituted into liposomes made from soybean or E. coli lipid. Galactose counterflow in the proteoliposomes was inhibited by glucose, talose, 2-deoxygalactose and 6-deoxygalactose, confirming that it was due to GalP and not one of the other E. coli galactose transport systems.     

2-deoxygalactose, a specific substrate of the Salmonella typhiimurium galactose permease: its use for the isolation of galP mutants.

2-Deoxygalactose is a specific substrate of the galactose permease. The apparent Km is about 500 micron, compared to 45 micron for galactose, whereas the maximal rate of uptake is one-half to one-third of that of galactose. None of the other galactose transport systems, including methyl beta-D-thiogalactosides I and II, the beta-methyl-galactoside permease, and both arabinose systems, is able to catalyze transport of 2-deoxygalactose to a significant extent. 2-Deoxygalactose can also be used to isolate mutants defective in galactose permease, since it is bacteriostatic. Colonies that grow with lactate, malate, or succinate as a carbon source in the presence of 0.5 to 2 mM 2-doexygalactose were found to be mostly galP mutants, lacking galactose permease. Spontaneous 2-deoxygalactose-resistant strains arose with a frequency of about 2 X 10(-6). galP mutants have also been derived from pts deletion mutants that require galactose permease for growth on glucose. Revertants have been obtained that have acquired the parental phenotype.     

Lactose metabolism in Erwinia chrysanthemi.

Wild-type strains of the phytopathogenic enterobacterium Erwinia chrysanthemi are unable to use lactose as a carbon source for growth although they possess a beta-galactosidase activity. Lactose-fermenting derivatives from some wild types, however, can be obtained spontaneously at a frequency of about 5 X 10(-7). All Lac+ derivatives isolated had acquired a constitutive lactose transport system and most contained an inducible beta-galactosidase. The transport system, product of the lmrT gene, mediates uptake of lactose in the Lac+ derivatives and also appears to be able to mediate uptake of melibiose, raffinose, and galactose. Two genes encoding beta-galactosidase enzymes were detected in E. chrysanthemi strains. That mainly expressed in the wild-type strains was the lacZ product. The other, the lacB product, is very weakly expressed in these strains. These enzymes showed different affinities for the substrates o-nitrophenyl-beta-D-galactopyranoside and lactose and for the inhibitors isopropyl-beta-D-thiogalactopyranoside and galactose. The lmrT and lacZ genes of E. chrysanthemi, together with the lacI gene coding for the regulatory protein controlling lacZ expression, were cloned by using an RP4::miniMu vector. When these plasmids were transferred into Lac- Escherichia coli strains, their expression was similar to that in E. chrysanthemi. The cloning of the lmrT gene alone suggested that the lacZ or lacB gene is not linked to the lmrT gene on the E. chrysanthemi chromosome. One Lac+ E. chrysanthemi derivative showed a constitutive synthesis of the beta-galactosidase encoded by the lacB gene. This mutation was dominant toward the lacI lacZ cloned genes. Besides these mutations affecting the regulation of the lmrT or lacB gene, the isolation of structural mutants unable to grow on lactose was achieved by mutagenic treatment. These mutants showed no expression of the lactose transport system, the lmrT mutants, or the mainly expressed beta-galactosidase, lacZ mutants. The lacZ mutants retained a very low beta-galactosidase level, due to the lacB product, but this level was low enough to permit use of the lacZ mutants for the construction of gene fusions with the Escherichia coli lac genes.     

The association of proton movement with galactose transport into subcellular membrane vesicles of Escherichia coli.

1. Subcellular membrane vesicles were prepared from a strain of Escherichia coli constitutive for the GalP galactose-transport system. 2. The addition of substrates of the GalP transport system to vesicle suspensions promoted alkaline pH changes, which provided direct evidence for the coupling of sugar and proton transport. 3. Respiration-energized galactose transport was progressively inhibited at pH values above 6.0, and was abolished by agents that render the membrane permeable to protons. 4. The combined effects of valinomycin, the nigericin-like compound A217 and pH on galactose transport suggested that both delta pH and delta psi components of the protonmotive force contributed to energization of galactose transport. 5. These results substantiate the conclusion that the GalP transport system operates by a chemiosmotic mechanism.     

Galactose transport in Salmonella typhimurium.

We have studied the various systems by which galactose can be transported in Salmonella typhimurium, in particular the specific galactose permease (GP). Mutants that contain GP as the sole galactose transport system have been isolated, and starting from these mutants we have been able to select point mutants that lack GP. The galP mutation maps close to another mutation, which results in the constitutive synthesis of GP, but is not linked to galR. Growth of wild-type strains on glaactose induces GP but not the beta-methylgalactoside permease (MGP). Strains lacking GP are able to grow slowly on galactose, and MGP is induced; however, D-fucose is a much better inducer of MGP. Induction of GP or MGP is not prevented by a pts mutation, although this mutation changes the apparent Km of MGP for galactose. pts mutations have no effect on GP. GP has a rather broad specificity: galactose, glucose, mannose, fucose, 2-deoxygalactose, and 2-deoxyglucose are substrates, but only galactose and fucose can induce this transport system.     

Reconstitution of the GalP galactose transport activity of Escherichia coli into liposomes made from soybean phospholipids

Galactose transport activity from Escherichia coli was solubilized with octyl glucoside, and reconstituted into liposomes made from soybean or E. coli lipid. Galactose counterflow in the proteoliposomes was inhibited by glucose, talose, 2-deoxygalactose and 6-deoxygalactose, confirming that it was due to GalP and not one of the other E. coli galactose transport systems.     

Proton movements coupled to sugar transport via the galactose transport system in Salmonella typhimurium.

We have studied proton movements associated with substrate transport via the galactose transport system in Salmonella typhimurium. The addition of galactose to lightly buffered suspensions of anaerobic, non-metabolizing cells of Salmonella typhimurium, specifically induced for the galactose transport system, causes an increase in extracellularpH as galactose and protons enter the cell together. Other substrates for this transport system, D-fucose, 2-deoxygalactose, glucose and 2-deoxyglucose similarly cause an influx of protons when transported. In contrast, transport via the other major transport system for galactose, the methylgalactoside transport system, is not coupled to H+ influx. Comparison of kinetic data obtained from pH measurements with data obtained from measurement of active transport of galactose via the galactose transport system suggests that the apparent Km of the galactose transport system for this sugar differs under energized and non-energized conditions. At pH 7.2 the permeant anion SCN- increases both the rate and extent of galactose-induced proton influx; at pH 6 the rate, but not the extent is increased by SCN-.     

A functional genomics approach to establish the complement of carbohydrate transporters in Streptococcus pneumoniae

The aerotolerant anaerobe Streptococcus pneumoniae is part of the normal nasopharyngeal microbiota of humans and one of the most important invasive pathogens. A genomic survey allowed establishing the occurrence of twenty-one phosphotransferase systems, seven carbohydrate uptake ABC transporters, one sodium:solute symporter and a permease, underlining an exceptionally high capacity for uptake of carbohydrate substrates. Despite high genomic variability, combined phenotypic and genomic analysis of twenty sequenced strains did assign the substrate specificity only to two uptake systems. Systematic analysis of mutants for most carbohydrate transporters enabled us to assign a phenotype and substrate specificity to twenty-three transport systems. For five putative transporters for galactose, pentoses, ribonucleosides and sulphated glycans activity was inferred, but not experimentally confirmed and only one transport system remains with an unknown substrate and lack of any functional annotation. Using a metabolic approach, 80% of the thirty-two fermentable carbon substrates were assigned to the corresponding transporter. The complexity and robustness of sugar uptake is underlined by the finding that many transporters have multiple substrates, and many sugars are transported by more than one system. The present work permits to draw a functional map of the complete arsenal of carbohydrate utilisation proteins of pneumococci, allows re-annotation of genomic data and might serve as a reference for related species. These data provide tools for specific investigation of the roles of the different carbon substrates on pneumococcal physiology in the host during carriage and invasive infection.     

Galactokinase-deficient mutants of Tetrahymena thermophila: Selection and characterization

Summary We have isolated a series of mutants of Tetrahymena thermophila which are resistant to inhibition of growth by the galactose analog, 2-deoxygalactose. These mutants were obtained after mutagenesis with nitrosoguanidine and the induction of cytogamy to permit the recovery of recessive mutations induced in the germline micronucleus. Resistance to 2-deoxygalactose is correlated with a decreased rate of growth in galactose minimal medium and greatly reduced levels of galactokinase. The resistant phenotype of the mutants is apparently due to the galactokinase deficiency, which prevents the accumulation of toxic phosphorylated metabolites of 2-deoxygalactose. Genetic analyses reveal that the 2-deoxygalactose resistance alleles segregate as single Mendelian loci. The galactokinase-deficient strains described here represent the first mutants in this organism for which the biochemical basis of the mutant phenotype is known. These mutants, as well as others isolated similarly, should be of value in the elucidation of the mechanisms governing galactokinase gene regulation and in improving techniques of selection for other recessive mutations in Tetrahymena.     

Two outer membrane transport systems for vitamin B12 in Salmonella typhimurium.

The involvement of an outer membrane transport component for vitamin B12 uptake in Salmonella typhimurium, analogous to the btuB product in Escherichia coli, was investigated. Mutants of S. typhimurium selected for resistance to bacteriophage BF23 carried mutations at the btuB locus (butBS) (formerly called bfe, at the analogous map position as the E. coli homolog) and were defective in high-affinity vitamin B12 uptake. The cloned E. coli btuB gene (btuBE) hybridized to S. typhimurium genomic DNA and restored vitamin B12 transport activity to S. typhimurium btuBS mutants. An Mr-60,000 protein in the S. typhimurium outer membrane was repressed by growth with vitamin B12 and was eliminated in a btuBS mutant. The btuBS product thus appears to play the same role in vitamin B12 transport by S. typhimurium as does the E. coli btuBE product. A second vitamin B12 transport system that is not present in E. coli was found by cloning a fragment of S. typhimurium DNA that complemented btuB mutants for vitamin B12 utilization. In addition to this plasmid with a 6-kilobase insert of S. typhimurium DNA, vitamin B12 utilization by E. coli btuB strains required the btuC and btuD products, necessary for transport across the cytoplasmic membrane, but not the btuE or tonB product. The plasmid conferred low levels of vitamin B12-binding and energy-dependent transport activity but not susceptibility to phage BF23 or utilization of dicyanocobinamide. The cloned S. typhimurium DNA encoding this new transport system did not hybridize to the btuBE gene or to E. coli chromosomal DNA and therefore does not carry the S. typhimurium btuBS locus. Increased production of an Mr -84,000 polypeptide associated with the outer membrane was seen. The new locus appears to be carried on the large plasmid in most S. typhimurium strains. Thus S. typhimurium possesses both high- and low-affinity systems for uptake of cobalamins across the outer membrane.     

Mutations Affecting the Different Transport Systems for Isoleucine, Leucine, and Valine in Escherichia coli K-12

Uptake of isoleucine, leucine, and valine in Escherichia coli K-12 is due to several transport processes for which kinetic evidence has been reported elsewhere. A very-high-affinity transport process, a high-affinity transport process, and three different low-affinity transport processes were described. In this paper the existence of these transport processes is confirmed by the isolation and preliminary characterization of mutants altered in one or more of them. The very-high-affinity transport process is missing either in strains carrying the brnR6(am) mutation or in strains carrying the brn-8 mutation. This appears to be a pleiotropic effect since other transport systems are also missing. Mutant analysis shows that more than one transport system with high affinity is present. One of them, high-affinity 1, which needs the activity of a protein produced by the brnQ gene, transports isoleucine, leucine, and valine and is unaffected by threonine. The other, high-affinity 2, which needs the activity of a protein produced by the brnS gene, transports isoleucine, leucine, and valine; this uptake is inhibited by threonine which probably is a substrate. Another protein, produced by the brnR gene, is required for uptake through both high-affinity 1 and high-affinity 2 transport systems. The two systems therefore appear to work in parallel, brnR being a branching point. The brnQ gene is located close to phoA at 9.5 min on the chromosome of E. coli, the brnR gene is located close to lac at 9.0 min, and the brnS gene is close to pdxA at 1 min. A mutant lacking the low-affinity transport system for isoleucine was isolated from a strain in which the high-affinity system was missing because of a brnR mutation. This strain also required isoleucine for growth because of an ilvA mutation. The mutant lacking the low-affinity transport system was unable to grow on isoleucine but could grow on glycylisoleucine. This mutant had lost the low-affinity transport for isoleucine, whereas those for leucine and valine were unaffected. A pleiotropic consequence of this mutation (brn-8) was a complete absence of the very-high-affinity transport system due either to the alteration of a common gene product or to any kind of secondary interference which inhibits it. Mutants altered in isoleucine-leucine-valine transport were isolated by taking advantage of the inhibition that valine exerts on the K-12 strain of E. coli. Mutants resistant both to valine inhibition (Val(r)) and to glycylvaline inhibition are regulatory mutants. Val(r) mutants that are sensitive to glycylvaline inhibition are transport mutants. When the very-high-affinity transport process is repressed (for example by methionine) the frequency of transport mutants among Val(r) mutants is higher, and it is even higher if the high-affinity transport process is partially inhibited by leucine.     

Transport and utilization of the biosynthetic intermediate shikimic acid in Escherichia coli.

Auxotrophic mutants of Escherichia coli W or K12 blocked before shikimic acid in the aromatic biosynthetic pathway grew poorly on shikimic acid as sole aromatic supplement. This poor growth response was correlated with a relatively poor ability to transport shikimic acid. If citrate was present in the growth medium (as it is in some commonly used basal media) the growth of some of the E. coli K12 mutants on shikimate was further reduced. Mutants were derived from pre-shikimate auxotrophs which grew rapidly on media containing shikimic acid. These derivatives all had an increased ability to transport shikimic acid. Thus, it is proposed that the growth on shikimate observed in the parent cells is restricted by their relatively poor uptake of shikimate from the medium and that this restriction may be removed by a mutation which enhances shikimate transport. Transduction analysis of the mutations which enhanced utilization and transport of shikimic acid by E. coli K12 strains indicated at least two classes. Class 1 was about 20% cotransduced with the histidine region of the E. coli K12 chromosome and appeared to be coincident with a known shikimate transport locus, shiA. Class 2 was not cotransduced with his. The locus (or loci) of this class is unknown. Kinetic measurements suggested that both classes had shikimate uptake systems derived from the wild-type system. Two class 1 mutants had increased levels of otherwise unaltered wild-type transport while one class 2 mutant had an altered Michaelis constant (Km) for shikimate transport.     

Genetic and biochemical studies of transport systems for branched-chain amino acids in Escherichia coli.

Mutants of Escherichia coli K-12 requiring high concentrations of branched-chain amino acids for growth were isolated. One of the mutants was shown to be defective in transport activity for branched-chain amino acids. The locus of the mutation (hrbA) was mapped at 8.9 min on the E. coli genetic map by conjugational and transductional crosses. The gene order of this region is proC-hrbA-tsx. The hrbA system was responsible for the uptake activity of cytoplasmic membrane vesicles. It was not repressed by leucine. The substrate specificities and kinetics of the uptake activities were studied using cytoplasmic membrane vesicles and intact cells of the mutants grown in the presence or absence of leucine. Results showed that there are three transport systems for branched-chain amino acids, LIV-1, -2, and -3. The LIV-2 and -3 transport systems are low-affinity systems, the activities of which are detectable in cytoplasmic membrane vesicles. The systems are inhibited by norleucine but not by threonine. The LIV-2 system is also repressed by leucine. The LIV-1 transport system is a high-affinity system that is sensitive to osmotic shock. When the leucine-isoleucine-valine-threonine-binding protein is derepressed, the high-affinity system can be inhibited by threonine.     

A process related to membrane potential involved in bacterial chemotaxis to galactose

Attractants, in the presence of respiration and ATPase inhibitors, stimulate a hyperpolarization in Escherichia coli [Eisenbach, M. (1982) Biochemistry 21, 6818-6825]. In order to examine whether this hyperpolarization is correlated with chemotaxis, the effect of the attractant D-galactose and its analogues on the membrane potential of wild-type E. coli strains and some of their mutants was studied. The main observations were the following: (i) Wild-type cells became hyperpolarized by either galactose or its nonmetabolizable analogues, D-fucose and L-sorbose. (ii) A mutant defective in galactose metabolism became hyperpolarized by galactose. (iii) Inhibiting the galactose permease system did not prevent the hyperpolarization, rather it facilitated the observation of the hyperpolarization. (iv) Mutants unable to transport galactose via the methyl beta-galactoside (Mgl) transport system but having normal chemotaxis to galactose became normally hyperpolarized by D-fucose. (v) Mutants which cannot bind galactose were not hyperpolarized by galactose. (vi) The hyperpolarization in flaI mutants, in which the whole chemotaxis machinery is repressed, was reduced to 12-15% of the hyperpolarization in the parent strains. (vii) Nonattractant sugars did not stimulate hyperpolarization. It is concluded that the hyperpolarization is the consequence of neither galactose metabolism nor transport but rather is correlated with galactose taxis.     

Genetic evidence for substrate and periplasmic-binding-protein recognition by the MalF and MalG proteins, cytoplasmic membrane components of the Escherichia coli maltose transport system

We isolated mutants of Escherichia coli in which the maltose-binding protein (MBP) is no longer required for growth on maltose as the sole source of carbon and energy. These mutants were selected as Mal+ revertants of a strain which carries a deletion of the MBP structural gene, malE. In one class of these mutants, maltose is transported into the cell independently of MBP by the remaining components of the maltose system. The mutations in these strains map in either malF or malG. These genes code for two of the cytoplasmic membrane components of the maltose transport system. In some of the mutants, MBP actually inhibits maltose transport. We demonstrate that these mutants still transport maltose actively and in a stereospecific manner. These results suggest that the malF and malG mutations result in exposure of a substrate recognition site that is usually available only to substrates bound to MBP.     

Glutamate transport in wild-type and mutant strains of Escherichia coli

Halpern, Yeheskel S. (Hebrew University-Hadassah Medical School, Jerusalem, Israel), and Meir Lupo. Glutamate transport in wild-type and mutant strains of Escherichia coli. J. Bacteriol. 90:1288-1295. 1965.-Mutants of Escherichia coli able to grow on glutamate as their source of carbon showed glutamate dehydrogenase and glutamate-oxaloacetate transaminase activities similar to those possessed by the parent strain. The mutants took up glutamate at a much faster rate and showed a several-fold greater capacity for concentrating the amino acid than did the corresponding parent strains. Curvilinear double reciprocal plots of velocity of uptake versus glutamate concentration were obtained with the E. coli H strains. A break in the curve of glutamate uptake was observed with the E. coli K-12 strains when incubated in a glucose medium. It is suggested that these findings may be due to allosteric activation of glutamate permease by its substrate.     

Selection procedure for mutants defective in the beta-methylgalactoside transport system of Escherichia coli utilizing the compound 2R-glyceryl-beta-D-galactopyranoside

A procedure has been devised that allows selection of mutants defective in the beta-methylgalactoside transport system (mgl) of Escherichia coli. This procedure utilizes the compound 2R-glyceryl-beta-d-galactopyranoside (glycerylgalactoside), which is known to be transported by only two transport system in E. coli, namely, the lactose and the beta-methylgalactoside transport systems. Mutants lacking glycerol-3-phosphate dehydrogenase (glpD) are sensitive to glycerol. Similarly, mutants lacking uridine diphosphate-galactose-4-epimerase (galE) are sensitive to galactose. Glycerylgalactoside is an inducer of the lactose operon and also a substrate for beta-galactosidase. Thus, a mgl(+)glpD galE lacY strain will not grow in the presence of glycerylgalactoside owing to accumulated glycerol-3-phosphate, galactose-1-phosphate, and uridine diphosphate-galactose. We have constructed such a strain and shown that mgl mutants can be obtained by selecting for those that grow in the presence of glycerylgalactoside.     

Protein II influences ferrichrome-iron transport in Escherichia coli K12.

Ferrichrome-promoted iron uptake in Escherichia coli K12 is strictly dependent upon the tonA gene product, a 'minor' outer membrane protein. By selection for mutants of E. coli resistant to phages which require 'major' outer membrane proteins as receptors, strains with pronounced protein deficiencies were constructed. Such strains were tested for anomalous behaviour of ferrichrome transport. No significant differences in iron uptake were detected in E. coli K12 strains with markedly reduced amounts of protein I. However, a reduction in the initial velocity (up to 40%) was observed in E. coli deficient in outer membrane protein II. This difference was only evident when cells were grown under iron-starvation conditions; it was abolished when cells were grown in rich medium. Kinetic parameters for ferrichrome transport were determined for maximum velocity but for Km; double reciprocal plots showed a biphasic nature, probably attributable to a limited number of outer membrane binding sites and to the multi-component nature of the ferrichrome-iron transport system.     

Metabolic analysis of galactose toxicity in Escherichia coli with 2-deoxygalactose as the probe.

Biochemical basis of galactose toxicity has been studied in gal T mutants (CGSC 4974) using 2-deoxygalactose, a non-metabolizable analogue of galactose, as the probe. It is found that biochemical features of toxicity in wild type cells either with 2-deoxygalactose or with 2-deoxyglucose are very similar to the picture obtained with gal T mutants and the observed bacteriostasis is probably due to futile phosphorylation and not due to any specific inhibitory effect of phosphorylated galactose.     

Galactokinase Mutants of Chinese Hamster Somatic Cells Resistant to 2-Deoxygalactose

The growth of Chinese hamster somatic cells was inhibited by 0.2 mg/cc of 2-deoxygalactose. Mutants partially or fully resistant to 2-deoxygalactose were isolated in a single-step or two-step selection. Some of them did not grow as well as the wild type; one of them which lacked galactokinase(EC.2.7.1.6) activity did not grow at all in galactose medium. The galactokinase kinetic properties (Vmax & kmax of the other mutants and of the wild type were different. Therefore resistance resulted either from the possible absence of galactokinase synthesis or from a structural mutation, possible a missence mutation, in the galactokinase gene.- A simple diagnostic test for juvenile cataract is proposed.