DNA supercoiling in a thermotolerant mutant ofEscherichia coli

A spontaneously occurring, nalidixic acid-resistant (Nal^R), thermotolerant (T/r) mutant ofEscherichia coli was isolated. Bacteriophage P1-mediated transduction showed that Nal^R mapped at or neargyr A, one of the two genes encoding DNA gyrase. Expression ofgyrA ⁺ from a plasmid rendered the mutant sensitive to nalidixic acid and to high temperature, the result expected for alleles mapping ingyrA. Plasmid linking number measurements, made with DNA from cells grown at 37° C or shifted to 48° C, revealed that supercoiling was about 12% less negative in the T/r mutant than in the parental strain. Each strain preferentially expressed two different proteins at 48° C. The genetic and supercoiling data indicate that thermo-tolerance can arise from an alteration in DNA gyrase that lowers supercoiling. This eubacterial study, when. coupled with those of archaebacteria, suggests that DNA relaxation is a general aspect of thermotolerance.     

Stability and folding of a mutant ribose-binding protein ofEscherichia coli

A mature mutant ribose-binding protein (RBP) ofEscherichia coli was obtained by site-directed mutagenesis, replacing Thr-3 in the N-domain of wild-type mature RBP (WT-mRBP) with a Trp residue (N-Trp-mRBP). The equilibrium unfolding properties and the refolding kinetics of this protein were monitored by fluorescence and circular dichroism (CD). The stability of N-Trp-mRBP appears to be the same as that of C-Trp-mRBP, another mutant obtained by replacing Phe-187 with a Trp, and lower than that of WT-mRBP. The overall refolding rate of N-Trp-mRBP is much smaller than that of C-Trp-mRBP, which, in turn, is similar to that of WT-mRBP. For the case of WT-mRBP, the rate constant obtained by Tyr fluorescence is identical to the value obtained by CD. But with C-Trp-mRBP, the rate constant from CD is smaller than the value from the Trp fluorescence and this difference in the rate constants is much greater with the N-TrpmRBP.     

Selenocysteine lyase activity in a cysteine-requiring mutant ofEscherichia coli K-12

Selenocysteine lyase activity was detected in crude extracts from a cysteine-requiring mutant ofEscherichia coli K-12. The level of activity was the same whether cells had been grown aerobically or anaerobically, with or without selenocysteine. Selenocysteine lyase catalyzes the conversion of selenocysteine to alanine and elemental Se, a reaction that is followed by a nonenzymatic reduction of the Se to hydrogen selenide. Both of these end products were identified in this study. With cysteine as the substrate, alanine and H₂S were formed, but only at levels 50% less than the products formed from selenocysteine. Selenocysteine lyase has been identified in a number of mammals and bacteria; its presence in a cysK mutant ofE. coli K-12 suggests a common route whereby hydrogen selenide, derived from selenocysteine, can then be assimilated into selenoproteins.     

Turnover of murein in a diaminopimelic acid dependent mutant ofEscherichia coli

Escherichia coli 173-25, whose cell wall was labelled with¹⁴C-diaminopimelic acid, was found to lose about 15% radioactivity during growth in a fresh medium, two thirds or more being lost during the first two generations. Degradation products of the cell wall were mostly of low-molecular type. About 5% of the cells lyzed as a result of transfer associated with filtration, washing and resuspension of the bacterial population in a diaminopimelic acid (DAP) deficient medium. The degradation was very low during the first 20 min. The amount of wall material released from the cells increased between 20–30 min and a sudden decrease of viability of the population was observed. The degradation of murein triggered by starvation for DAP continued when supplementing the deficient medium with DAP and when growth was resumed. About one-half of the cell wall material released into the medium under these conditions was macromolecular. However, lysis of the cells and release of proteins into the medium were rapidly interrupted after DAP was added to the starving culture and the differential rate of synthesis of the cell wall increased. Turnover of murein was not associated with protein turnover.     

Assay of lysine by means of a mutant ofEscherichia coli

Описывается простой и доступный турбидиметрический метод определения L-лизина с помощью мутанта Escherichia coli ATCC 12408, чувствительного к лизину. Это метод надежного определения количества лизина в пределах от 20 до 100 μг. Присутствие высоких концентраций аргинина или орнитина не оказывает влияния на точность метода.     

Metabolism of glycerol-3-phosphate and phospholipids in a temperature-sensitive lysis mutant ofEscherichia coli

Phospholipid metabolism in a temperature-sensitive lysis mutant ofEscherichia coli has been investigated. The incorporation of³²P into the phospholipids of this mutant was negligible, not only at the non-permissive temperature, as was demonstrated earlier, but also at 30 C. Furthermore, cultivation of the cells at the non-permissive temperature during 90 min, which is the time required to induce lysis, did not alter the amount of phospholipid per cell.In vitro experiments demonstrated that the enzymes involved in phospholipid biosynthesis are fully active at 42 C. Lysis therefore is not directly caused by a defect in phospholipid synthesis. Evidence is presented that the low³²P incorporation is the result of a defective glycerol-3-phosphate dehydrogenase which determines the turnover of the immediate lipid precursor, glycerol-3-phosphate.     

A temperature-sensitive mutant ofEscherichia coli with an alteration in ribosomal protein L22

A temperature-sensitive, protein synthesis-defective mutant ofEscherichia coli exhibiting an altered ribosomal protein L22 has been investigated. The temperature-sensitive mutation was mapped to therplV gene for protein L22. The genes from the wild type and mutant strains were amplified by the polymerase chain reaction and the products were sequenced. A cytosine to thymine transition at position 22 of the coding sequence was found in the mutant DNA, predicting an arginine to cysteine alteration in the protein. A single cysteine residue was found in the isolated mutant protein. This amino acid change accounts for the altered mobility of the mutant protein in two-dimensional gels and during reversed-phase HPLC. The temperature-sensitive phenotype was fully complemented by a plasmid carrying the wild type L22 gene. Ribosomes from the complemented cells showed only wild type protein L22 by two dimensional gel analysis and were as heat-resistant as control ribosomes in a translation assay. The point mutation in the L22 gene is uniquely responsible for the temperature-sensitivity of this strain.     

Transport by reconstituted lactose carrier from parental and mutant strains ofEscherichia coli

Summary The lactose transport carrier from parental (X71/F'W3747) and mutant cells (54/F'5441) was reconstituted into proteoliposomes. Transport by the counterflow assay showed slightly greater activity in proteoliposomes prepared from extracts of the mutant membranes compared with that for the parental cell. The mutant carrier showed a threefold lowerK _m but similarV _max compared to the parent. On the other hand proteoliposomes from the mutant showed a defect in protonmotive force-driven accumulation, compared with the parent. With a pH gradient (inside alkaline) plus a membrane potential (inside negative) the parental proteoliposomes accumulated lactose 25-fold over the medium concentration while the mutant proteoliposomes accumulated sixfold. In a series of experiments proteoliposomes were exposed to proteolytic enzymes. Chrymotrypsin treatment resulted in 30% inhibition of counterflow activity for the reconstituted carrier from both parent and mutant. Papain produced 84% inhibition of transport by the reconstituted parental carrier but only 41% of that of the mutant. Trypsin and carboxypeptidase Y treatment had no effect on counterflow activity of either parent or mutant. Exposure of purified lactose carrier in proteoliposomes to carboxypeptidase Y resulted in the release of alanine and valine, the two C-terminal amino acids predicted from the DNA sequence.     

Tus-mediated arrest of DNA replication in Escherichia coli is modulated by DNA supercoiling

In the absence of RecA, expression of the Tus protein of Escherichia coli is lethal when ectopic Ter sites are inserted into the chromosome in an orientation that blocks completion of chromosome replication. Using this observation as a basis for genetic selection, an extragenic suppressor of Tus-mediated arrest of DNA replication was isolated with diminished ability of Tus to halt DNA replication. Resistance to tus expression mapped to a mutation in the stop codon of the topA gene (topA869), generating an elongated topoisomerase I protein with a marked reduction in activity. Other alleles of topA with mutations in the carboxyl-terminal domain of topoisomerase I, topA10 and topA66, also rendered recA strains with blocking Ter sites insensitive to tus expression. Thus, increased negative supercoiling in the DNA of these mutants reduced the ability of Tus-Ter complexes to arrest DNA replication. The increase in superhelical density did not diminish replication arrest by disrupting Tus-Ter interactions, as Tus binding to Ter sites was essentially unaffected by the topA mutations. The topA869 mutation also relieved the requirement for recombination functions other than recA to restart replication, such as recC, ruvA and ruvC, indicating that the primary effect of the increased negative supercoiling was to interfere with Tus blockage of DNA replication. Introduction of gyrB mutations in combination with the topA869 mutation restored supercoiling density to normal values and also restored replication arrest at Ter sites, suggesting that supercoiling alone modulated Tus activity. We propose that increased negative supercoiling enhances DnaB unwinding activity, thereby reducing the duration of the Tus-DnaB interaction and leading to decreased Tus activity.     

A histidine binding protein ofEscherichia coli: a component of cystine binding protein ofEscherichia coli

Summary Commercially obtained cystine binding protein (CBP), an osmotic shock protein ofEscherichia coli, was studied in an effort to determine its binding characteristics. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS/PAGE) analysis of commercially obtained CBP showed three protein bands. N-terminal amino acid microsequencing and subsequent computer search revealed that the sequence of one of these proteins (25-kDa) was nearly identical to histidine binding protein (HisJ) ofSalmonella typhimurium. Purification of CBP by HPLC yielded four protein peaks, of which one bound histidine exclusively. Binding was maximal at pH 5.0 to 6.0, at 4°C, did not require calcium or magnesium ions and was not inhibited by reduction of CBP disulfide bonds. Amino acids other than histidine or cystine did not bind to CBP. These data show that commercially available CBP is not a homogenous protein; it contains a histidine as well as a cystine binding component.