Search

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.

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.

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.

Strains of Escherichia coli devoid of systems for the active transport of galactose (galP mgl) still grow on galactose but at rates that are a function of the galactose concentration of the medium: half-maximal growth rates require more than 2 mM-galactose to be present. Evidence is presented that galactose is taken up by such strains by facilitated diffusion on a carrier specified by the umg gene (or by a gene highly co-transducible with it) which is thus a part of, or closely associated with, an enzyme II for glucose of the phosphoenolpyruvate-phosphotransferase system. However, the entry of galactose does not require phosphotransferase activity, and the sugar taken up appears in the cells as free galactose.

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.

A mutant, DAI, has been isolated from the Escherichia coli K12, strain K2. 1t, as a colony resistant to 2-deoxyglucose (DG) when growing on fructose but still sensitive to DG when growing on other sugars. The mutation in DAI specifically affects the catabolite inhibition of fructose utilization by glucose and glucose-6-phosphate; the affected gene (designated cif) is located at min 41 on the E. coli linkage map and is highly co-transducible with the genes that specify the uptake of fructose (ptsF) and enzymic conversion of fructose-1-phosphate to fructose-1,6-bisphosphate (fpk).

The Enzyme IIfru of the phosphoenolpyruvate- (PEP-) dependent phosphotransferase system (PTS), which catalyses the uptake of fructose and its concomitant phosphorylation to fructose 1-phosphate by Escherichia coli, is specified by a gene designated fruA. The nucleotide sequence of a 2.5 kb PvuII restriction fragment spanning fruA+, cloned on a plasmid, was determined. This fragment contained three open reading frames (ORFs) but only one complete ORF, 1689 base pairs long, which was preceded by a well-defined Shine-Dalgarno sequence and ended with a rho-independent transcription terminator. The amino acid sequence deduced from this DNA corresponds to that of a protein of 563 amino acids (57.5 kDa), which has the hydropathic profile expected of an integral membrane protein (average hydropathy = 0.40) and which is characterized by a number of well-marked hydrophobic loops that may correspond to membrane-spanning regions. There is relatively little overall homology between this protein and those of other Enzymes II of the PTS but there is considerable correspondence between the region surrounding one of the six histidine residues (His381) of Enzyme IIfru and those surrounding the particular histidines of other Enzymes II, and of HPr, known to be involved in phosphorylation. A plasmid carrying the complete fruA+ nucleotide sequence, but not that of any other functional protein, fully restored the ability of fruA mutants to grow on fructose and of extracts of fruA mutants to phosphorylate fructose, which confirms that the nucleotide sequence determined species Enzyme IIfru.

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.

Strains of Escherichia coli that are unable to grow on fructose because they lack the phosphoenolpyruvate: fructose phosphotransferases specified by ptsF and ptsX mutate to grow on media containing fructose as sole carbon source, but do not regain the function of either of the missing phosphotransferases. Instead, fructose is taken up and phosphorylated to fructose 6-phosphate by a phosphoenolpyruvate: sorbitol phosphotransferase which, in wild-type cells, is induced by sorbitol but not by fructose, but which is constitutively expressed in these mutants. The regulatory gene srlC controlling enzymes of sorbitol uptake and catabolism has been located on the E. coli genome as part of the linkage group cysI srlC attI86 pheA.

The isolation and properties of a mutant of Escherichia coli K12 that is totally unable to take up and utilize gluconate are described. Genetical analysis shows this phenotype to be associated with two lesions. One phenotype, designated GntM-, is the result of a mutation in a gene co-transducible with malA; the other, designated GNTS-, is the result of a mutation in a gene (GntS) co-transducible with fdp. The GntS--phenotype differs little from that of wild-type cells, but GntM- GntS+ organisms grow on gluconate only after a prolonged lag and form a gluconate uptake system that is strongly repressed by pyruvate. Moreover, such GntM- mutants readily give rise to further mutants that form a gluconate uptake system, gluconate kinase and 6-phosphogluconate dehydratase consititutively; in partial diploids, this constitutivity is recessive to the inducible character. It is postulated that the GntM- phenotype is due to malfunction of a negative control gene gntR, and that gntS+ specifies the activity of a gluconate uptake system.

The uhp gene, which specifies the uptake of hexose phosphates, and several other genes in the vicinity of minute 81 on the E. coli linkage map have been located by phage-mediated transductions. The order found is mtl-gpsA-pyre-gltc-uhp-tna-dnaa. Alleles specifying the Uhp- and Uhp+ characters were separated from that specifying constitutivity of hexose phosphate uptake (Uhpc). Although cotransduction frequencies between gltC and uhp as high as 90%, and between uhp and tna as high as 80%, were observed, these frequencies were unusually strongly dependent on which marker was selected. This may be due to the proximity of the uhp region to the point of origin of chromosome replication.

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.

Procedures are described for the selection of Escherichia coli mutants that constitutively take up and phosphorylate fructose, and convert it to fructose 1,6-bisphosphate. The phenotype of such mutants is described. The altered regulatory gene, fruC, is highly co-transducible with leu and other markers located at min 2 on the genome. In merozygotes, fruC+ is dominant to fruC. Mutants can be readily isolated that are fruC at 42 degrees C but fruC+ at 30 degrees C; moreover, the integration of a Tn10 transposon in the genome at min 2 converts fruC+ strains to fruC. It is therefore likely that the fruC+ regulatory gene specifies a repressor protein.

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.

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.

Mutants of Escherichia coli K12 have been isolated that grow on media containing pyruvate of proline as sole carbon sources despite the presence of 10 or 50 mM-sodium fluoroacetate. Such mutants lack either acetate kinase [ATP: acetate phosphotransferase; EC 2.7.2.1] or phosphotransacetylase [acetyl-CoA: orthophosphate acetyltransferase; EC 2.3.1.8] activity. Unlike wild-type E. coli, phosphotransacetylase mutants do not excrete acetate when growing aerobically or anaerobically on glucose; their anaerobic growth on this sugar is slow. The genes that specify acetate kinase (ack) and phosphotransacetylase (pta) activities are cotransducible with each other and with purF and are thus located at about min 50 on the E. coli linkage map. Although Pta- and Ack- mutants are greatly impaired in their growth on acetate, they incorporate [2-14C]acetate added to cultures growing on glycerol, but not on glucose. An inducible acetyl-CoA synthetase [acetate: CoA ligase (AMP-forming); EC 6.2.1.1] effects this uptake of acetate.

The EnzymeIIbgl of the phosphoenolpyruvate- (PEP-) dependent phosphotransferase system catalyses the uptake and concomitant phosphorylation of beta-glucosides by Escherichia coli; it is specified by the gene bglC. The nucleotide sequence of a 3.6 kb HindIII restriction fragment spanning bglC, cloned on a plasmid, was determined. DNA analysis strongly suggests that the published order of this and other genes involved in beta-glucoside utilization, bgl C, S, B, is incorrect, and that the regulatory gene bglS may be located upstream of the structural genes bglC and bglB. From the deduced amino acid sequence it is predicted that the membrane protein specified by bglC consists of 625 amino acid residues (66.48 kDa). The protein has the hydropathic profile expected of an integral membrane protein (average hydropathy = 0.62). Comparisons between the amino acid sequences deduced for the EnzymeIIbgl and for the mannitol-specific EnzymeIImtl show that these proteins are related, and a little direct homology is apparent. A 2.3 kb AluI fragment spanning bglC was subcloned into an expression vector which carries the lambda PL promoter and then transformed into a host strain which produces thermolabile cI857 repressor and the anti-terminator N; thermoinduction resulted in the overproduction of a membrane protein and the appearance of Bgl activity.