Mapping of mutations within the genes coding for enzyme I and Hpr proteins of the phosphoenolpyruvate-dependent phosphotransferase system of Escherichia coli K-12. I. Mapping of the mutations within the ptsI gene

Fine genetic mapping of the pts region coding for general components of PTS was performed. Over 30 spontaneous pts mutations were investigated. By means of the complementation test using the F'trp+ purC+ ptsI episome, both ptsI and ptsH mutations were revealed among them. With the help of reciprocal three point transduction crosses, 8 of them were situated in the pts region. Two of them were in ptsH gene, the rest being in ptsI gene. The lysogenic strain was obtained with lambda inserted in the pts region. Heat curing of the lysogene led to a number of deletions and insertions. Six of them were mapped with the help of the point mutations studied.     

Involvement of the histidine protein (HPr) of the phosphotransferase system in chemotactic signaling of Escherichia coli K-12.

It is known that in mutants of Escherichia coli lacking the histidine protein (HPr) of the carbohydrate: phosphotransferase system, all substrates of the system can be taken up in the presence of the fructose-regulated HPr-like protein FPr (gene fruF). Although this protein fully substituted for HPr in transport and phosphorylation, we found that it was not able to complement efficiently for HPr in mediating chemotaxis toward phosphotransferase system substrates. Furthermore, transport activity and chemotaxis could be genetically dissected by the exchange of single amino acids in HPr. The results suggest a specific role of HPr in chemotactic signaling. We propose a possible link of signal transduction pathways for phosphotransferase system- and methyl chemotaxis protein-dependent substrates via HPr.     

Dominant mutations of rifampicin resistance in Escherichia coli K-12

Significant portion (up to 20%) of dominant mutations (rifd mutations) was observed among spontaneous mutations of rifampicin resistance picked up in cells of haploid Escherichia coli strain. These mutations are similar to rifd mutations obtained earlier when selecting them in rif-s/rif-s merodiploids. On the basis of analysis of nucleotide substitutions taking place in formation of spontaneous and induced mutations, it is established that rifd mutations are caused by single nucleotide substitution. The majority of rifd mutations are localized in a small region of the central part of RNA polymerase beta-subunit gene covering less than 200 base pairs. A rifd mutant has been described which occurred as a result of micro-deletion in one of the "hot" spots of the central region of beta-subunit gene.     

The Escherichia coli phosphoenolpyruvate-dependent phosphotransferase system: observation of heterogeneity in the amino acid composition of HPr.

Resonances of the aromatic protons of tyrosine have been observed in the proton nuclear magnetic resonance (1H NMR) spectrum of purified HPr from Escherichia coli. Analysis of the NMR spectrum of native HPr suggests that the tyrosine is located in a single position in the secondary structure and that this position is on the interior of the molecule inaccessible to solvent. Previous reports suggested that E. coli HPr contained no tyrosine [Anderson, B., Weigel, N., Kundig, W., & Roseman, S. (1971) J. Biol. Chem. 246, 7023--7033]. In contrast, we find, by amino acid analysis and ultraviolet and NMR spectroscopy, that E. coli HPr does contain tyrosine but at a subintegral level of 0.5 +/- 0.1 mol of tyrosine per mol of HPr.     

The multifunctional fructose-specific component of the phosphoenolpyruvate-dependent phosphotransferase system of Escherichia coli K12--fruA gene product

Mutational damage of the ptsH gene leads to pleiotropic disturbance of sugar utilization in Escherichia coli K12. A fruS mutation suppresses the defect because of a constitutional expression of the fruB and fruA genes. FruB protein possessing a pseudo-HPr activity replaces the HPr. It was shown that wild type allele fruS+ dominates over the fruS1156 mutation in heterozygous merodiploid. The existence of thermosensitive mutations (fruS4 and fruS12) which repair the ptsH damage was also demonstrated. The fruS mutations were located in the fru operon. Fructose utilization was not disturbed in fruS1156 mutant, but fruS2 and fruS12 mutants were unable to utilize fructose. Spontaneous mutations (fruS6 and fruS13) possessing the same phenotype at any temperature similar to the thermosensitive ones under nonpermissive conditions were isolated. They were mapped using the P1vir transduction. The fruS mutations were found in the structural gene of the fructose operon. Presumably it is the fruA gene that cods for the fructose-specific multidomain protein IIB'Bc of the phosphoenolpyruvate-dependent phosphotransferase system.     

Dominance studies with stable merodiploids in the D-serine deaminase system of Escherichia coli K-12

An episome, F32, which carries the genetic markers dsdA(+), the presumed structural gene for d-serine deaminase, dsdC(+), a regulatory locus governing the synthesis of d-serine deaminase, aroC(+), and purC(+) was obtained from strain AB311 of Escherichia coli K-12, and was used to construct appropriate merodiploids with dsdC markers. In all dsdC / dsdC(+) diploids examined, dsdC was found to be cis dominant, trans recessive, to dsdC(+). In two cases, however, the cis dominance was only partial. Moreover, complementation was observed between one of the dsdC markers which is fully cis dominant and one which is partially cis dominant. Because of the size of the dsdC region, the phenotypes of the mutants, and the partial trans dominance of dsdC(+) over some of the dsdC mutations, it is suggested that the dsdC region specifies a product, but that this product does not move with facility through the cytoplasm     

Integration and intramolecular transposition of the TnBP3 Bordetella pertussis transposon in the Escherichia coli K-12 cells -- mutant for the phosphoenolpyruvate-dependent phosphotransferase system Hpr protein

Integration of a plasmid carrying the TnBP3 transposon of Bordetella pertussis into the chromosome of Escherichia coli and transpositions of the integrated structure within a chromosome in the wild-type and mutant cells ptsH devoid of the major Hpr protein of the phosphoenolpyruvate-dependent phosphotransferase system were studied. When transposed to a new chromosome site, the integrated structure was precisely (or almost precisely) excised from the metY gene sequence, which resulted in restoration of the Met+ phenotype. The integration and transposition events were only observed in the E. coli cells carrying the ptsH+ allele. The ptsH mutations inhibited integration and intramolecular transposition, which were restored after phenotypic or genetic suppression of the ptsH mutation. The intensity of the processes studied were suggested to depend on the integrity of a chain that ensures transferring of the phosphoryl residue by proteins of the phosphotransferase system in E. coli K12. The results obtained indicate that the ptsH mutants of E. coli can serve as the optimal host for cloning of fragments carrying repeated sequences of B. pertussis, which may apply to the repeated sequences of other microorganisms.     

Insertion mutations in the dam gene of Escherichia coli K-12

The dam gene of E. coli can be inactivated by insertion of Tn9 or Mud phage. Strains bearing these mutations are viable indicating that the dam gene product is dispensable.     

大肠杆菌PTS系统改造及重组菌生长性能测定

利用Red重组系统对野生大肠杆菌Escherichia coli磷酸烯醇式丙酮酸-糖磷酸转移酶系统(Phosphoenolpyruvate:carbohydrate phosphotransferase system,PTS)进行修饰改造,敲除PTS系统中关键组分EⅡCBGlc的编码基因(ptsG),磷酸组氨酸搬运蛋白HPr的编码基因(ptsI),同时敲入来源于运动发酵单胞菌Zymomonas mobilis的葡萄糖易化体(Glucose facilitator)编码基因(glf),构建重组大肠杆菌,比较测定并系统评价了基因敲除和敲入对细胞的生长、葡萄糖代谢和乙酸积累的影响。敲除基因ptsG和ptsI造成大肠杆菌PTS系统部分功能缺失,细胞生长受到一定限制,敲入glf基因后,重组大肠杆菌能够利用Glf-Glk(葡萄糖易化体-葡萄糖激酶)途径,消耗ATP将葡萄糖进行磷酸化并转运进入细胞。通过该途径转运葡萄糖能够提高葡萄糖利用效率,降低副产物乙酸生成,同时能够使更多的碳代谢流进入后续相关合成途径,预期能够提高相关产物产量。     

Analysis of the ptsH-ptsI-crr region in Escherichia coli K-12: nucleotide sequence of the ptsH gene

The nucleotide sequence of an Escherichia coli DNA segment containing the ptsH gene and the first 162 nucleotides of the ptsI gene encoding, respectively, Hpr and enzyme I of the phosphoenolpyruvate-dependent glycose phosphotransferase system (PTS), was determined. The ptsH promoter was localized using the S1 mapping technique. A nucleotide sequence very similar to the consensus binding site for cAMP receptor protein was found in the -35 region of the ptsH promoter. The ptsH gene is transcribed in the same direction as the ptsI gene and the crr gene (encoding enzyme IIIGlc of the PTS). Analysis of the nucleotide sequence substantiates the notion that the ptsH-ptsI-crr genes constitute a polycistronic operon.     

Transport properties of merodiploids covering the dagA locus in Escherichia coli K-12.

A membrane componenet of the dag transport system which serves for glycine, D-alanine, and D-serine is coded for by the dagA gene at minute 83 of the Escherichia coli chromosome. Merodiploid strains (dagA+/dagA+) show two to three times the transport activity for only those amino acids that are substrates of the dag transport system. The increased transport activity is a result of a two-to threefold increase in Vmax for amino acid uptake with little or no change in the Km value. The two- to threefold gene dose effect of the merodiploid strains is maintained even during carbon starvation, eliminating the possibility that a greater energy supply for transport activity may account for the effect. Since merodiploids which carry more than one copy of the dagA allele show a gene dose response for transport activity, we conclude that the membrane componenet of the dag transport system which is coded for by the dagA allele is present in limiting amounts.     

Analysis of mutations that uncouple transport from phosphorylation in enzyme IIGlc of the Escherichia coli phosphoenolpyruvate-dependent phosphotransferase system.

Mutations that uncouple glucose transport from phosphorylation were isolated in plasmid-encoded Escherichia coli enzyme IIGlc of the phosphoenolpyruvate-dependent phosphotransferase system (PTS). The uncoupled enzymes IIGlc were able to transport glucose in the absence of the general phosphoryl-carrying proteins of the PTS, enzyme I and HPr, although with relatively low affinity. Km values of the uncoupled enzymes IIGlc for glucose ranged from 0.5 to 2.5 mM, 2 orders of magnitude higher than the value of normal IIGlc. Most of the mutant proteins were still able to phosphorylate glucose and methyl alpha-glucoside (a non-metabolizable glucose analog specific for IIGlc), indicating that transport and phosphorylation are separable functions of the enzyme. Some of the uncoupled enzymes IIGlc transported glucose with a higher rate and lower apparent Km in a pts+ strain than in a delta ptsHI strain lacking the general proteins enzyme I and HPr. Since the properties of these uncoupled enzymes IIGlc in the presence of PTS-mediated phosphoryl transfer resembled those of wild-type IIGlc, these mutants appeared to be conditionally uncoupled. Sequencing of the mutated ptsG genes revealed that all amino acid substitutions occurred in a hydrophilic segment within the hydrophobic N-terminal part of IIGlc. These results suggest that this hydrophilic loop is involved in binding and translocation of the sugar substrate.     

Nucleotide sequence of the gene ompA coding the outer membrane protein II of Escherichia coli K-12.

A nucleotide sequence of 2271 basepairs has been determined from cloned E. coli DNA which contains ompA. Withing that sequence, starting at nucleotide 1037, an open translational reading frame encodes a protein of 367 amino acids which starting with amino acid 22 agrees with the primary structure of protein II. The preceeding 21 amino acids constitute a typical signal sequence. There is a non-translated region of 360 nucleotides in front of the translational start. The insertion point of an IS1 element 110 nucleotides upstream from the start codon and an amber codon at the position of amino acid residue 28 have been localized in the DNA from two ompA mutants.     

Nucleotide sequence of the melA gene, coding for alpha-galactosidase in Escherichia coli K-12.

Melibiose uptake and hydrolysis in E.coli is performed by the MelB and MelA proteins, respectively. We report the cloning and sequencing of the melA gene. The nucleotide sequence data showed that melA codes for a 450 amino acid long protein with a molecular weight of 50.6 kd. The sequence data also supported the assumption that the mel locus forms an operon with melA in proximal position. A comparison of MelA with alpha-galactosidase proteins from yeast and human origin showed that these proteins have only limited homology, the yeast and human proteins being more related. However, regions common to all three proteins were found indicating sequences that might comprise the active site of alpha-galactosidase.     

Hyper-recombination in Escherichia coli K-12 mutants constitutive for protein X synthesis.

Genetic recombination was studied in Escherichia coli F- strains in which synthesis of the recA gene product protein X is increased due to mutation in either recA (tif-1) or lexA (spr). When a single donor marker was selected, the recombination proficiency of these strains was not significantly altered in Hfr crosses. However, linkage of unselected, proximal Hfr markers was found to be much reduced among the progeny tested, and more of the progeny showed evidence of multiple exchanges between donor and recipient DNA. These effects were much more apparent when the recipient carried both tif-1 and spr mutations, but in this case recombination proficiency was reduced compared with those strains carrying either mutation alone, particularly in crosses with Hfr Cavalli. A lexA mutation was found to suppress the effect of tif-1 on the recombinant genotype.