Role of ATP in removal of psoralen cross-links from DNA of Escherichia coli permeabilized by treatment with toluene.

Removal of interstrand cross-linked from DNA was examined in Escherichia coli permeabilized by treatment with toluene. Under these conditions, the reaction requires ATP and Mg2+, and the mechanism appears to be similar to that occurring in whole cells. Under optimum conditions, the rate constant was 0.06 min-1. Genetical, physical, and biochemical analysis of the repair process suggest the following mechanism. In an ATP-dependent reaction, the uvrA and uvrB gene products cleave a phosphodiester bond on the 5' side of one arm of the cross-link, producing a 3'-OH terminus. Subsequently, DNA polymerase I (5'-3' exonuclease activity) makes a second strand cut on the 3' side of the cross-link in the same DNA strand, completing removal of the covalent link between complementary strands. The second reaction did not occur in a uvrD- strain, which had normal levels of DNA polymerizing activity. The uvrD gene may regulate the specificity or activity of the 5'-3' exonuclease of DNA polymerase I in vivo.     

Glutamine synthetase adenylylation in permeabilized cells of Escherichia coli

Following a freeze-thaw cycle, treatment of Escherichia coli with the nonionic detergent, Lubrol WX, renders the cells permeable to small molecules but not to cytosolic proteins. After such treatment, the permeabilized cell suspensions can be assayed directly by standard procedures both for intracellular levels of glutamine synthetase and the state of adenylylation (i.e. the average number, n, of adenylylated subunits/dodecameric molecule). Permeabilization of cells from cultures containing an adequate supply of glutamine as the sole nitrogen source led to complete retention of all protein components of the bicyclic cascade that regulates the interconversion of glutamine synthetase between adenylylated and unadenylylated forms; similar treatment of glutamine-starved cells leads to selective inactivation, only, of the uridylyltransferase. When suspended in buffers containing ATP and glutamine, the value of n in permeabilized cells increased to high values (n = 11), whereas in the presence of alpha-ketoglutarate, Pi, and ATP, the value of n decreased to approximately 2.0. Time-dependent changes in n that occur during incubations of permeabilized cells in buffers containing these effectors can be arrested either by sonication at 0-4 degrees C or by the addition of cetyltrimethylammonium bromide (to inactivate adenylyltransferase). It is thus evident that Lubrol-treated cells may be used to investigate the regulation of glutamine synthetase adenylylation in situ.     

Effects of toluene on Escherichia coli

Jackson, Robert W. (University of California, San Diego, La Jolla), and J. A. DeMoss. Effects of toluene on Escherichia coli. J. Bacteriol. 90:1420-1425. 1965.-When toluene is added at appropriate levels to exponentially growing cultures of Escherichia coli, a time-dependent loss of turbidity is observed which is concurrent with a loss of material to the medium and with unmasking of beta-galactosidase. In addition, the galactoside permease system is totally destroyed. Electron micrographs confirm the indications that the cells are not being lysed by toluene, although the cytoplasm collapses to the interior of the cell. Included in the material lost from the cell after toluene treatment is 85% of the total ribonucleic acid (RNA), the principal source of which appears to be the ribosomes. The loss of RNA is temperature-dependent. Protein is also lost to the medium as a function of both temperature and available toluene. Up to 25% of the total protein is found in the medium, the precise amount depending on the level of toluene employed. Zone centrifugation studies of extracts from treated cells indicate that toluene elicits a rapid disaggregation of ribosomes that is terminated, at any stage, by disruption of the cells. The disaggregation is temperature-dependent and does not occur at 4 C. It appears to be distinct from the actual degradation of ribosomal RNA and is accompanied by an accumulation of small particles during the initial phases of treatment at 21 C. Toluene added to crude extracts of normal E. coli cells is unable to cause detectable ribosome destruction.     

Release of the periplasmic penicillinases from Escherichia coli by toluene

Summary More than 80% of the chromosomally and R factor mediated, periplasmic penicillinases of Escherichia coli K-12, strains G11a1 and D1-R1, are released into the medium upon treatment with the membrane damaging agents toluene (50 l/ml) or polymyxin B (10 g/ml) within 10 min of incubation at 37°C. A concomitant release of protein (27 to 34%), but not of the cytoplasmic -galactosidase (<5%) occurs.     

Murein biosynthesis in ether permeabilized Escherichia coli starting from early peptidoglycan precursors

ETB, ether treated bacteria, from E. coli and other Gram-negative strains, contain in a cell-free system all enzymes necessary for murein biosynthesis. Starting with a variety of combinations of peptidoglycan precursors, high yields of sodium dodecylsulfate (SDS, 4%) insoluble murein or murein like material were synthesized. The amount of newly synthesized SDS insoluble material (NSM) was dependent upon the growing phase at which cells had been harvested for preparation of ETB. This data may provide some insight into the regulation of peptidoglycan biosynthesis.Starting from early peptidoglycan precursors, the cell-free synthesis of NSM was inhibited by specific inhibitors of murein synthesis, such as D-cycloserine, D-fluoroalanine, 2-amino-ethylphosphonate, analogues of D-alanyl-D-alanine and -lactam antibiotics at appropriate concentrations. Some D-alanyl-D-alanine analogues and 4-chlorodiaminopimelic acid were incorporated into NSM in place of their corresponding natural substrates.Abbreviations ETB ether treated bacteria (E. coli) - NSM newly synthesized SDS insoluble material - SDS sodium dodecylsulfate - UDP-MAG UDP-MurNAc-dipeptide, UDP-N-acetylmuramoyl-L-alanyl-D-glutamate - UDP-MAGD UDP-MurNAc-tripeptide, UDP-N-acetylmuramoyl-L-alanyl-D-glutamyl-meso-2,6-diaminopimelate - UDP-MAGDAA UDP-MurNAc-pentapeptide, UDP-N-acetylmuramoyl-L-alanyl-D-glutamyl-meso-2,6-diaminopimeloyl-D-alanyl-D-alanine - GINAc N-Acetylglucosamine Definitions Murein highly cross-linked bagshaped peptidoglycan (Weidel and Pelzer 1964)     

Immobilization of permeabilized Escherichia coli cells with penicillin acylase activity.

Escherichia coli cells with penicillin acylase activity were sequentially treated at pH 7.8 with aqueous solutions of N-cetyl-N,N,N-trimethylammonium bromide and glutaraldehyde and then immobilized within porous polyacrylamide beads. The immobilized whole cells showed enhanced hydrolysis rates in the conversion of benzylpenicillin to 6-aminopenicillanic acid (6-APA) compared to untreated cells immobilized and used under identical conditions. The immobilized system showed no apparent loss in enzyme activity when used repeatedly over 90 cycles for 6-APA production from 4% benzylpenicillin.     

Engineered citrate synthase alters Acetate Accumulation in Escherichia coli

Metabolic engineering is used to improve titers, yields and generation rates for biochemical products in host microbes such as Escherichia coli. A wide range of biochemicals are derived from the central carbon metabolite acetyl-CoA, and the largest native drain of acetyl-CoA in most microbes including E. coli is entry into the tricarboxylic acid (TCA) cycle via citrate synthase (coded by the gltA gene). Since the pathway to any biochemical derived from acetyl-CoA must ultimately compete with citrate synthase, a reduction in citrate synthase activity should facilitate the increased formation of products derived from acetyl-CoA. To test this hypothesis, we integrated into E. coli C ΔpoxB twenty-eight citrate synthase variants having specific point mutations that were anticipated to reduce citrate synthase activity. These variants were assessed in shake flasks for growth and the production of acetate, a model product derived from acetyl-CoA. Mutations in citrate synthase at residues W260, A267 and V361 resulted in the greatest acetate yields (approximately 0.24 g/g glucose) compared to the native citrate synthase (0.05 g/g). These variants were further examined in controlled batch and continuous processes. The results provide important insights on improving the production of compounds derived from acetyl-CoA.     

UV irradiation alters deoxynucleoside triphosphate pools in Escherichia coli

UV irradiation of exponentially growing Escherichia coli increased intracellular concentration of dATP and DTTP without significantly changing the concentrations of dGTP and dCTP. These selective increases in dATP and dTTP pools are seen in wild-type E. coli K12 and AB1157, as well as in recA and umuC strains, and are proportional to UV dose. The possible significance of these findings with respect to induction of the SOS response and nontargeted mutagenesis are discussed.     

Excision repair of ultraviolet radiation damage in toluene treated Escherichia coli

ATP independent excision repair of UV damage has been studied in $\text{E. coli}$ made permeable to nucleotides by treatment with toluene. In using this system, separation of the first step from the subsequent steps in the repair process is achieved. It was found that completion of repair is observed only in strains that have normal levels of DNA polymerase I.     

Regulated toluene cis-glycol production by recombinant Escherichia coli strains constructed by PCR amplification of the toluene dioxygenase genes from Pseudomonas putida

The toluene dioxygenase genes from Pseudomonas putida NCIMB 11767 were isolated by PCR amplification from recombinant plasmid, p1/1. The genes were subcloned into pUC18 and pKK223-3 and expressed under the lac and tac promoters, respectively. In both cases, toluene cis-glycol was produced, with higher levels of product formation when the genes were expressed from the tac promoter.     

Reprogramming of sugar transport pathways in Escherichia coli using a permeabilized SecY protein-translocation channel

In the initial step of sugar metabolism, sugar-specific transporters play a decisive role in the passage of sugars through plasma membranes into cytoplasm. The SecY complex (SecYEG) in bacteria forms a membrane channel responsible for protein translocation. The present work shows that permeabilized SecY channels can be used as nonspecific sugar transporters in Escherichia coli. SecY with the plug domain deleted allowed the passage of glucose, fructose, mannose, xylose, and arabinose, and, with additional pore-ring mutations, facilitated lactose transport, indicating that sugar passage via permeabilized SecY was independent of sugar stereospecificity. The engineered E. coli showed rapid growth on a wide spectrum of monosaccharides and benefited from the elimination of transport saturation, improvement in sugar tolerance, reduction in competitive inhibition, and prevention of carbon catabolite repression, which are usually encountered with native sugar uptake systems. The SecY channel is widespread in prokaryotes, so other bacteria may also be engineered to utilize this system for sugar uptake. The SecY channel thus provides a unique sugar passageway for future development of robust cell factories for biotechnological applications.     

Novobiocin—A specific inhibitor of semiconservative DNA replication in permeabilized Escherichia coli cells

The effect of novobiocin on macromolecule synthesis was investigated in Escherichia coli cells permeabilized by treatment with toluene or 2 m-sucrose. It was found that (1) semiconservative DNA replication is strongly and immediately inhibited and (2) ATP-independent DNA repair as well as RNA and protein synthesis are not affected.     

rpoB mutation in Escherichia coli alters control of ribosome synthesis by guanosine tetraphosphate.

An isogenic pair of relA+ and relA strains of Escherichia coli B/r with a mutation in the RNA polymerase subunit gene rpoB (Rifr) was isolated in which the relationship between guanosine tetraphosphate (ppGpp) concentration and stable RNA (rRNA, tRNA) gene activity was altered. The RNA polymerase in the rpoB strains was found to be about 20-fold more sensitive to ppGpp with respect to its stable RNA promoter activity than was the wild-type enzyme. The existence of such mutants is consistent with the idea that ppGpp interacts with the RNA polymerase enzyme and thereby alters its promoter selectivity, i.e., reduces its affinity for the stable RNA promoters. Under most conditions, the rpoB mutants had a reduced rate of growth and about a 10-fold-reduced intracellular concentration of ppGpp compared with the rpoB wild-type strains. The reduction of the level of ppGpp in the rpoB mutants during exponential growth was presumably a reflection of an indirect effect of the rpoB mutation on the control of relA-independent ppGpp metabolism.