Escherichia coli K-12 mutants that allow transport of maltose via the beta-galactoside transport system.
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We have isolated mutants of Escherichia coli that have an altered beta-galactoside transport system. This altered transport system is able to transport a sugar, maltose, that the wild-type beta-galactoside transport system is unable to transport. The mutation that alters the specificity of the transport system is in the lacY gene, and we refer to the allele as lacYmal. The lacYmal allele was detected originally in strains in which the lac genes were fused to the malF gene. Thus, as a result of gene fusion and isolation of the lacYmal mutation, a new transport system was evolved with regulatory properties and specificity similar to those of the original maltose transport system. Maltose transport via the lacYmal gene product is independent of all of the normal maltose transport system components. The altered transport system shows a higher affinity than the wild-type transport system for two normal substrates of the beta-galactoside transport system, thiomethyl-beta-D-galactoside and o-nitrophenyl-beta-D-galactoside. (+info)
Energy coupling to K+ uptake via the Trk system in Escherichia coli: the role of ATP.
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The dual energy requirement (protonmotive force and ATP) of the Escherichia coli Trk potassium transport system has been investigated. Using inhibitors and unc mutants we show that Trk is not an ATPase but may be regulated by ATP. Possible mechanisms of energy coupling to Trk are discussed. (+info)
Characterization of lactose carrier mutants which transport maltose.
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Brooker and Wilson (Brooker, R. J., and Wilson, T. H. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 3959-3963) previously isolated lactose carrier mutants which were able to transport maltose. All of the mutants were found to be single amino acid substitutions for alanine 177 or for tyrosine 236. In the present study, we have examined the ability of these mutants to transport maltose, lactose, o-nitrophenyl-beta-D-galactopyranoside, methyl-beta-D-thiogalactopyranoside, and H+. Both the position 177 and 236 mutants have enhanced rates of maltose transport and exhibit apparent Km values for maltose which are substantially less than that of the wild-type strain. The position 177 mutants transport lactose and other galactosides at a normal rate and with normal affinity during downhill transport and show counterflow transport rates which are faster than the wild-type strain. Interestingly, these mutants are markedly defective in accumulating substrates against a concentration gradient, yet retain a normal H+:galactoside stoichiometry. The position 236 mutants appear to be defective in the downhill, uphill, and counterflow transport of galactosides but exhibit a normal H+:galactoside stoichiometry. (+info)
Possible involvement of lipoic acid in binding protein-dependent transport systems in Escherichia coli.
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We describe the properties of the binding protein dependent-transport of ribose, galactose, and maltose and of the lactose permease, and the phosphoenolpyruvate-glucose phosphotransferase transport systems in a strain of Escherichia coli which is deficient in the synthesis of lipoic acid, a cofactor involved in alpha-keto acid dehydrogenation. Such a strain can grow in the absence of lipoic acid in minimal medium supplemented with acetate and succinate. Although the lactose permease and the phosphoenolypyruvate-glucose phosphotransferase are not affected by lipoic acid deprivation, the binding protein-dependent transports are reduced by 70% in conditions of lipoic acid deprivation when compared with their activity in conditions of lipoic acid supply. The remaining transport is not affected by arsenate but is inhibited by the uncoupler carbonylcyanide-m-chlorophenylhydrazone; however the lipoic acid-dependent transport is completely inhibited by arsenate and only weakly inhibited by carbonylcyanide-m-chlorophenylhydrazone. The known inhibitor of alpha-keto acid dehydrogenases, 5-methoxyindole-2-carboxylic acid, completely inhibits all binding protein-dependent transports whether in conditions of lipoic supply or deprivation; the results suggest a possible relation between binding protein-dependent transport and alpha-keto acid dehydrogenases and shed light on the inhibition of these transports by arsenicals and uncouplers. (+info)
Catabolite inhibition and sequential metabolism of sugars by Streptococcus lactis.
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Growth of galactose-adapted cells of Streptococcus lactis ML(3) in a medium containing a mixture of glucose, galactose, and lactose was characterized initially by the simultaneous metabolism of glucose and lactose. Galactose was not significantly utilized until the latter sugars had been exhausted from the medium. The addition of glucose or lactose to a culture of S. lactis ML(3) growing exponentially on galactose caused immediate inhibition of galactose utilization and an increase in growth rate, concomitant with the preferential metabolism of the added sugar. Under nongrowing conditions, cells of S. lactis ML(3) grown previously on galactose metabolized the three separate sugars equally rapidly. However, cells suspended in buffer containing a mixture of glucose plus galactose or lactose plus galactose again consumed glucose or lactose preferentially. The rate of galactose metabolism was reduced by approximately 95% in the presence of the inhibitory sugar, but the maximum rate of metabolism was resumed upon exhaustion of glucose or lactose from the system. When presented with a mixture of glucose and lactose, the resting cells metabolized both sugars simultaneously. Lactose, glucose, and a non-metabolizable glucose analog (2-deoxy-d-glucose) prevented the phosphoenolpyruvate-dependent uptake of thiomethyl-beta-d-galactopyranoside (TMG), but the accumulation of TMG, like galactose metabolism, commenced immediately upon exhaustion of the metabolizable sugars from the medium. Growth of galactose-adapted cells of the lactose-defective variant S. lactis 7962 in the triple-sugar medium was characterized by the sequential metabolism of glucose, galactose, and lactose. Growth of S. lactis ML(3) and 7962 in the triple-sugar medium occurred without apparent diauxie, and for each strain the patterns of sequential sugar metabolism under growing and nongrowing conditions were identical. Fine control of the activities of preexisting enzyme systems by catabolite inhibition may afford a satisfactory explanation for the observed sequential utilization of sugars by these two organisms. (+info)
A lectin which binds specifically to beta-D-galactoside groups is present at the earliest stages of chick embryo development.
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Extracts obtained from chick embryos at the pre-gastrula and gastrula stages are able to agglutinate trypsinized rabbit erythrocytes fixed with glutaraldehyde. Agglutination is inhibited by saccharides sharing a beta-D-galactopyranoside configuration. Agglutinin activity is also inhibited by desialysed fetuin which bears terminal galactose residues but not by native fetuin, desialysed agalacto-fetuin, bovine submaxillary mucin and desialysed bovine submaxillary mucin. Lectin activity is present in extracts obtained from the embryonic area of the blastoderms as well as in extracts from extra-embryonic endoderm and ectoderm. In extracts subjected to gel filtration on ECD Sepharose, lectin activity eluted between ovalbumim (mol. wt 45 000) and chymotrypsinogen (mol. wt 25 000). Under some experimental conditions, thiodigalactoside, the most potent inhibitor of lectin activity, inhibited the aggregation of cells of the extra-embryonic endoderm of the primitive chick embryo. (+info)
Mechanism of inducer expulsion in Streptococcus pyogenes: a two-step process activated by ATP.
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The mechanism of methyl-beta-D-thiogalactoside-phosphate (TMG-P) expulsion from Streptococcus pyogenes was studied. The expulsion elicited by glucose was not due to exchange vectorial transphosphorylation between the expelled TMG and the incoming glucose since more beta-galactoside was displaced than glucose taken up, and the stoichiometry between TMG and glucose transport was inconstant. Instead, two distinct and sequential reactions, intracellular dephosphorylation of TMG-P followed by efflux of free TMG, mediated the expulsion. This was shown by temporary accumulation of free TMG effected by competitive inhibition of its efflux and by the aid of arsenate, which arrested dephosphorylation of TMG-P but did not affect efflux of free TMG formed intracellularly before arsenate addition. The competitive inhibition of TMG efflux by its structural analogs suggests that a transport protein facilitates the expulsion. Iodoacetate or fluoride prevented TMG-P dephosphorylation and its expulsion. However, provision of ATP via the arginine deiminase pathway restored these activities in the presence of the glycolytic inhibitors and stimulated expulsion in their absence. Other amino acids tested did not promote this restoration, and canavanine or norvaline severely inhibited it. Arginine without glucose neither elicited the dephosphorylation nor evoked the expulsion of TMG-P. Ionophores or ATPase inhibitors did not prevent the expulsion as elicited by glucose or its restoration by arginine. The results suggest that activation of the dephosphorylation-expulsion mechanism occurs independently of a functional glycolytic pathway, requires ATP provision, and is possibly due to protein phosphorylation controlled by a yet unknown metabolite. The in vivo phosphorylation of a protein (approximate molecular weight - 10,000) under the conditions of expulsion was demonstrated. (+info)
Polarity of Tn5 insertion mutations in Escherichia coli.
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We assessed the effect of insertions of the kanamycin resistance transposon Tn5 in the lac operon of Escherichia coli on the expression of distal genes lacY and lacA (melibiose fermentation at 41 degrees C and thiogalactoside transacetylase synthesis, respectively). Every insertion mutation tested (41 in lacZ and 23 in lacY) was strongly polar. However, approximately one-third of the insertion mutants expressed distal genes at low levels due to a promoter associated with Tn5. To localize this promoter, we (i) reversed the orientation of Tn5 at several sites and (ii) replaced wild-type Tn5 with several substitution derivatives which lack Tn5's central region. Neither alteration changed the expression of distal genes. Thus, in contrast to transposons IS2 and TnA. Tn5's ability to turn on distal gene expression is not due to a promoter in its central region and therefore is not dependent on the overall orientation of Tn5 in the operon. Our results suggest that the promoter is within 186 base pairs of the ends of Tn5. It is possible that the promoter is detected in only a fraction of insertions because it overlaps Tn5-target sequence boundary. (+info)