The cloning, DNA sequence, and overexpression of the gene araE coding for arabinose-proton symport in Escherichia coli K12.

A lambda placMu1 insertion was made into araE, the gene for arabinose-proton symport in Escherichia coli. A phage containing an araE'-'lacZ fusion was recovered from the lysogen and its restriction map compared with that of the 61-min region of the E. coli genome to establish the gene order thyA araE orf lysR lysA galR; araE was transcribed toward orf. A 4.8-kilobase SalI-EcoRI DNA fragment containing araE was subcloned from the phage lambda d(lysA+ galR+ araE+) into the plasmid vector pBR322. From this plasmid a 2.8-kilobase HincII-PvuII DNA fragment including araE was sequenced and also subcloned into the expression vector pAD284. The araE gene was 1416-base pairs long, encoding a hydrophobic protein of 472 amino acids with a calculated Mr of 51,683. The amino acid sequence was homologous with the xylose-proton symporter of E. coli and the glucose transporters from a human hepatoma HepG2 cell line, human erythrocytes, and rat brain. The overexpressed araE gene product was identified in Coomassie-stained sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels of cell membranes as a protein of apparent Mr 35,000 +/- 1,150. Arabinose protected this protein against reaction with N-ethylmaleimide.

Location and direction of transcription of the ptsH and ptsI genes on the Escherichia coli K12 genome.

Recombinant plasmids were constructed that carried various fragments of the DNA specifying the Escherichia coli genes ptsH and (part of) ptsI, the genes for the common components of the phosphoenolpyruvate: sugar phosphotransferase. Expression of plasmid-specified functions in minicells showed that ptsH and ptsI were transcribed clockwise. Most of the transcription of ptsI was from the ptsH promoter, but some was from a second site within or after ptsH.

Location on the Escherichia coli genome of a gene specifying O-acetylserine (thiol)-lyase.

The plasmid pAB65, derived from a specialized transducing phage carrying DNA from about 52 min on the Escherichia coli genome, coded for two polypeptides of Mr approx. 34 000. The expression of one was regulated by cyst(e)ine and the cysB gene product and the other by the cysB gene product only. One of these polypeptides was a subunit of O-acetylserine (thiol)-lyase (EC 4.2.99.8); the other, associated with the E. coli membrane, was the N-terminus of the product of the lambda ben gene. The pattern of peptide synthesis directed by plasmids carrying smaller DNA fragments indicated that the gene for O-acetylserine (thiol)-lyase was transcribed clockwise. The spectrum, amino acid composition and subunit number of the enzyme were determined. The enzyme appears homologous with the Salmonella typhimurium cysK gene product. This provides further evidence for the inversion of this region of the genome.

Location of a structural gene for xylose-H+ symport at 91 min on the linkage map of Escherichia coli K12.

Mutations in the xylose-H+ transport activity of Escherichia coli K12 were isolated using Mud(ApRlac). The initial selection was for simultaneous acquisition of ampicillin and xylose resistance in an fda background. Colonies were then screened for xylose-inducible beta-galactosidase and for growth on xylose of their fda+ derivatives. Two of the xylose-positive derivatives were shown to be impaired in xylose-H+ symport in whole cells and in xylose transport into subcellular vesicles. Their xylose transport in whole cells showed increased sensitivity to arsenate. The site of prophage insertion was mapped to 91.4 min on the E. coli genome between pgi and malB. It is proposed that the gene for the xylose-H+ symport system be called xylE.

Identification of the GalP galactose transport protein of Escherichia coli.

Escherichia coli strains have been isolated with a transposon 10 insertion or an amber mutation inactivating the galP gene, which specifies the galactose-H+ (GalP) transport system. Comparison of the membrane proteins between these strains and their GalP+ parents by dual isotope analysis showed that a component of Mr = 34-39,000 was consistently absent from the GalP- mutants. Galactose, methyl-beta-D-galactoside, and talose protected the GalP transport system from inactivation by N-ethylmaleimide. A membrane protein of Mr = 34-38,000 was modified by N-([2-3H]ethyl)maleimide at the binding site of these sugars. Two-dimensional gel electrophoresis of the membrane proteins has resolved a component of Mr = 35-38,000 (average apparent pI = 5.7) present in parent strains (GalP+) but not in the GalP- mutants. These observations identified a protein of apparent Mr = 37,000 as the product of the galP gene of E. coli.

Phosphotransferase-mediated regulation of carbohydrate utilization in Escherichia coli K12: the nature of the iex (crr) and gsr (tgs) mutations.

Mutants of Escherichia coli K12 defective in the gene iex (crr) no longer utilize glucose or N-acetylglucosamine in preference to lactose, but competition between either of these sugars and another that also enters by a phosphotransferase (PT) mechanism is not affected. In this they differ from gsr (tgs) mutants. In gsr mutants, glucose does not exclude any other sugar, though N-acetylglucosamine still does so. In gsr mutants that are also ptsM the phosphoenolpyruvate-dependent phosphorylation of glucose or methyl alpha-glucoside is reduced by 90%: N-acetylglucosamine phosphorylation is not affected. The iex mutation does not affect the phosphorylation of either of these compounds. The wild-type alleles iex+ and gsr+ are dominant in lambda heterozygotes. Glucose inhibits the lactose permease of wild-type cells, but only when the permease is present in low amounts. The inhibition is also relieved (1) by induction of another transport system that is subject to regulation by the iex system or (2) by an iex mutation. We suggest that the iex gene specifies a protein that, in cells transporting certain sugars by a PT mechanism, acts to inhibit active transport systems. The protein is present in limiting concentration in the cell, sufficient only to inhibit the basal, uninduced, level of the active transport systems. In consequence the inducer (or its precursor) may be excluded from the cell and induction thus prevented.

Phosphotransferase-mediated regulation of carbohydrate utilization in Escherichia coli K12: location of the gsr (tgs) and iex (crr) genes by specialized transduction.

A lysogen of Escherichia coli K12 with lambda cI857 S7 xis6 nin5 b515 b519 integrated into ptsI was induced and the lysates plated on a Pel- host [on which lambda strains with less than the wild-type amount of DNA form plaques at low frequency (Cameron et al., 1977)]. All of the 40 plaques examined contained phage able to transduce at least two of the genes known from bacteriophage P1 transduction experiments to be closely linked to ptsI. Assuming that each specialized transducing phage arose by a single illegitimate recombination event, the distribution of phage types showed that the gene order is cysA gsr ptsI (ptsH, iex) cysZ lig; both gsr+ and iex+ were dominant. Analysis of restriction endonuclease digests of the transducing phage confirmed that no unexpected DNA rearrangements had taken place and allowed the construction of a map of the sites of action of the restriction endonucleases EcoRI, HindIII, BamI and Kpn for over 20 kilobases of E. coli DNA. In an Appendix, we show cysA and cysZ mutants to be deficient in sulphate assimilation.

Isolation and mapping of glutathione reductase-negative mutants of Escherichia coli K12.

Two independent mutants defective in glutathione reductase (EC 1.6.4.2) were isolated in an Escherichia coli K12 strain lysogenized with bacteriophage Mu. The prophage was lost (and the ability to reduce glutathione regained) by 32% of the xylose-positive transductants when T4GT7 was used as the vector, but the markers were not cotransduced by P1. Similarly, the prophage site and malA were cotransduced by T4GT7 but not by P1. The gor gene maps between min 77 and 78 on the E. coli genome, and the mutation causes no growth defect.

A specialized transducing phage, lambda psrlA, for the sorbitol phosphotransferase of Escherichia coli K12.

A specialized transducing phage for the srlA gene, specifying the sorbitol-specific Enzyme II of the phosphoenolpyruvate:sugar phosphotransferase system, was constructed and its DNA was analysed by restriction endonuclease digestion. Phage construction involved four steps: (1) integration of lambda into the srlA gene; (2) selection of phage carrying (a) the left and (b) the right end of the srlA gene by means independent of the function of the new DNA acquired; (3) reconstitution of the srlA gene in a dilysogen of these two phage; and (4) the excision, using the heteroimmune lambdoid phage 21, of a plaque-forming srlA+ phage from the dilysogenic chromosome. Comparison of the DNA restriction digests of the transducing phage with those of its parents and of wild-type lambda revealed fragments consisting partly of lambda and partly of Escherichia coli DNA. The junction points in the intermediate phage define a site that must lie within the reconstituted gene of the final phage. This technique should be of general application in relating genes, cloned by our method, to DNA sequences.

A mutant inducible for galactitol utilization in Escherichia coli K12.

Galactitol-positive strains of Escherichia coli K12 are inhibited by the galactitol analogues L-fucitol and 2-deoxy-D-galactitol, but not by D-fucitol; Salmonella typhimurium LT2 is not inhibited by these compounds. Most mutants selected as resistant to either toxic compound are unable to utilize galactitol as carbon source, but a relatively rare class is inducible for the Enzyme II of the galactitol:phosphoenolpyruvate phosphotransferase system, the product of which is D-galactitol 6-phosphate. The lesion in one such mutant maps near metG at about min 45 on the E. coli genome.

Phosphotransferase-mediated regulation of carbohydrate utilisation in Escherichia coli K12: identification of the products of genes on the specialised transducing phages lambda iex (crr) and lambda gsr (tgs).

The expression of genes adjacent to ptsI was investigated using a series of specialised transducing phages carrying different, overlapping, segments of the cysA-gsr-ptsI-ptsH- iex - cysZ -lig region of the genome of Escherichia coli. The polypeptides were synthesised following the infection of u.v.-irradiated lysogenic and non-lysogenic uvrA recA hosts or a uvrA recA host carrying the lambda cI+ plasmid pKB280 . The polypeptides were identified by SDS-polyacrylamide gel electrophoresis and fluorography. The gsr gene product had a mol. wt. of 23 000. The product of the iex gene was tentatively identified as a protein of mol. wt. of either 33 000 or 21 000. Hpr, the product of the gene ptsH, had a mol. wt. of 9000. The gsr gene appeared to be expressed at a higher level in a non-immune host, which suggests that it was transcribed from lambda promoters. A new lambda host strain, suitable for the detection of small polypeptides (mol. wt. less than 30 000) is described.

L-Sorbose phosphorylation in Escherichia coli K-12.

L-Sorbose is phosphorylated by Escherichia coli by two distinct Enzymes II of the phosphoenolpyruvate-dependent phosphotransferase system. The glucose Enzyme II (specified by the gene ptsG) phosphorylates L-sorbose with an apparent Km of 0.08 +/- 0.03 mM and V of 31.8 +/- 3.5 nmol . mg-1 . min-1 whilst the fructose Enzyme II (specified by the gene ptsF) phosphorylates it with an apparent Km of 28.9 +/- 2.7 mM and V of 20.2 +/- 0.8 nmol . mg-1 . min-1. L-Sorbose induces neither of these Enzymes II, but sorbose inhibits the growth of strains expressing either of these functions constitutively. Mutants that have lost their sensitivity to L-sorbose are found to have lost either the glucose or the fructose phosphotransferase Enzyme II.U

Identification of the AraE transport protein of Escherichia coli.

1. Two arabinose-inducible proteins are detected in membrane preparations from strains of Escherichia coli containing arabinose-H+ (or fucose-H+) transport activity; one protein has an apparent subunit relative molecular mass (Mr) of 36 000-37 000 and the other has Mr 27 000. 2. An araE deletion mutant was isolated and characterized; it has lost arabinose-H+ symport activity and the arabinose-inducible protein of Mr 36 000, but not the protein of Mr 27 000. 3. An araE+ specialized transducing phage was characterized and used to re-introduce the araE+ gene into the deletion strain, a procedure that restores both arabinose-H+ symport activity and the protein of Mr 36,000. 4. N-Ethylmaleimide inhibits arabinose transport and partially inhibits arabinose-H+ symport activity. 5. N-Ethylmaleimide modifies an arabinose-inducible protein of Mr 36 000-38 000, and arabinose protects the protein against the reagent. 6. These observations identify an arabinose-transport protein of Escherichia coli as the product of the araE+ gene. 7. The protein was recognized as a single spot staining with Coomassie Blue after two-dimensional gel electrophoresis.

Proton-linked D-xylose transport in Escherichia coli.

The addition of xylose to energy-depleted cells of Escherichia coli elicited an alkaline pH change which failed to appear in the presence of uncoupling agents. Accumulation of [14C]xylose by energy-replete cells was also inhibited by uncoupling agents, but not by fluoride or arsenate. Subcellular vesicles of E. coli accumulated [14C]xylose provided that ascorbate plus phenazine methosulfate were present for respiration, and this accumulation was inhibited by uncoupling agents or valinomycin. Therefore, the transport of xylose into E. coli appears to be energized by a proton-motive force, rather than by a phosphotransferase or directly energized mechanism. Its specificity for xylose as inducer and substrate and the genetic location of a xylose-H+ transport-negative mutation near mtl showed that the xylose-H+ system is distinct from other proton-linked sugar transport systems of E. coli.

Two genes affecting glucarate utilization in Escherichia coli K12.

D-Glucarate is transported into Escherichia coli K12 by an inducible system at an apparent rate of 7 to 15 nmol min-1 (mg dry mass)-1. The apparent Km for uptake is 16 muM. The system is induced by growth on glucarate or glycollate. Galactarate competes with glucarate for the uptake system. A mutation (gar A) was isolated in which activities of glucarate transport and glucarate dehydratase and the ability to grow on glucarate or galactarate are all impaired. The mutation maps at min 16. Another mutation of indistinguishable phenotype is probably a deletion of the genes garB and tonA at min 3.5.

Amino-sugar transport systems of Escherichia coli K12.

Glucosamine, mannose and 2-deoxyglucose enter Escherichia coli by the phosphotransferase system coded for by the gene ptsM. The glucosamine- and mannose-negative, deoxyglucose-resistant phenotype of ptsM mutants can be suppressed by a mutation mapping near ptsG that allows constitutive expression of the glucose phosphotransferase coded for by the gene ptsG. N-Acetylglucosamine enters E. coli by two distinct phosphotransferase systems (White, 1970). One of these is the PtsM system, the other is coded for by a gene which maps near the nagA,B genes at about min 15 on the E. coli chromosome. We propose that this gene be designated ptsN. Strains with either of these components of the phosphotransferase system will utilize N-acetylglucosamine as sole carbon source.