Excision Repair Characteristics of recB−res− and uvrC− Strains of Escherichia coli

An Escherichia coli strain carrying the recB21 and res-1 mutations showed an abnormally low level of colony-forming ability although it grew essentially normally in liquid medium. The recB21 res-1 strain showed little, if any, of the ultraviolet (UV)-induced deoxyribonucleic acid (DNA) breakdown characteristic of the res-1 mutant. Nevertheless, the double mutant was far more sensitive to UV than either the res-1 or the recB21 strain. When compared with a wild-type strain, the rate of release of dimers from UV-irradiated DNA was very slow in the recB21 res-1, but normal in the res-1 recB(+) or recB21 res(+) mutants. However, the ratio of dimer-to-thymine released into the acid-soluble fraction was three times higher than the wild type in recB21 res(+) and recB21 res-1 and only one-tenth as high as the wild type in res-1 rec(+). Alkaline sucrose gradient centrifugation revealed occurrence of single-strand incision of UV-irradiated DNA and the restitution of nicked DNA at a similar rate in the recB21 res-1 and recB21 res(+) strains. Mutants uvrC(-) showed increased amounts of nicks in their DNA with increasing incubation time after UV irradiation, although no detectable amounts of dimers were excised from UV-irradiated DNA. From these results, it is concluded that the increased sensitivity of the res-1 strain to UV light is due to a reduced ability to excise dimers from UV-irradiated DNA and that the high rate of UV-induced breakdown of DNA is not the primary cause. A possible role of uvrC gene in the excision repair is discussed.     

Excision Repair Properties of Isogenic rec− Mutants of Escherichia coli K-12

We have examined the excision repair properties of isogenic rec⁻ and uvr⁻ strains of Escherichia coli K-12. A recB⁻recC⁻ strain excises dimers at a rate nearly that of the rec⁺ parent, reaching the same extent of excision after a 1-hr postirradiation incubation. recA⁻ and recA⁻recB⁻ strains excise 75 to 80% of the dimers excised by their rec⁺ parent, whereas a uvrB⁻ strain excises no dimers during a 1-hr incubation. The doses of ultraviolet light (254 nm) required to reduce survival to 37% of the original population are 8 ergs/mm² for recA or recA recB mutants, 5 ergs/mm² for the uvrB⁻ strain, 30 ergs/mm² for the recB recC mutant, and 230 ergs/mm² for the wild-type parent. From these data one cannot account for the ultraviolet light sensitivity of rec⁻ strains on the basis of their excision repair properties. We conclude that rec gene products play no significant role in the early steps of excision repair. The assay we have used for excision of thymine dimers is a modification of the Carrier-Setlow technique, and is described in detail in the Appendix to this paper. To show the properties and validity of this method, results of experiments with thymine dimers formed in vitro and in vivo in E. coli K-12 are presented. These results show our method to be reproducible and sensitive to 0.005% of the total radioactive thymine present in thymine-containing dimers.     

Determination of Pyrimidine Dimers in Escherichia coli and Cryptosporidium parvum during UV Light Inactivation, Photoreactivation, and Dark Repair

UV inactivation, photoreactivation, and dark repair of Escherichia coli and Cryptosporidium parvum were investigated with the endonuclease sensitive site (ESS) assay, which can determine UV-induced pyrimidine dimers in the genomic DNA of microorganisms. In a 99.9% inactivation of E. coli, high correlation was observed between the dose of UV irradiation and the number of pyrimidine dimers induced in the DNA of E. coli. The colony-forming ability of E. coli also correlated highly with the number of pyrimidine dimers in the DNA, indicating that the ESS assay is comparable to the method conventionally used to measure colony-forming ability. When E. coli were exposed to fluorescent light after a 99.9% inactivation by UV irradiation, UV-induced pyrimidine dimers in the DNA were continuously repaired and the colony-forming ability recovered gradually. When kept in darkness after the UV inactivation, however, E. coli showed neither repair of pyrimidine dimers nor recovery of colony-forming ability. When C. parvum were exposed to fluorescent light after UV inactivation, UV-induced pyrimidine dimers in the DNA were continuously repaired, while no recovery of animal infectivity was observed. When kept in darkness after UV inactivation, C. parvum also showed no recovery of infectivity in spite of the repair of pyrimidine dimers. It was suggested, therefore, that the infectivity of C. parvum would not recover either by photoreactivation or by dark repair even after the repair of pyrimidine dimers in the genomic DNA.     

Deoxyribonucleic Acid Synthesis and Cell Division in a lon− Mutant of Escherichia coli

The lon(-) mutants of Escherichia coli form long filamentous cells after temporary inhibition of deoxyribonucleic acid (DNA) synthesis by ultraviolet irradiation, treatment with nalidixic acid, or thymine starvation. The kinetics of DNA synthesis and cell division after a period of thymine starvation have been compared in lon(+) and lon(-) cells. After this treatment, both kinds of cells recover their normal DNA to mass ratio with the same kinetics. In contrast to previous reports, cell division is found to recommence in both lon(+) and in lon(-) cells after such a temporary period of inhibition of DNA synthesis. However, the delay separating the recommencement of DNA synthesis and of cell division is approximately three times as long in lon(-) as in lon(+) cells. Low concentrations of penicillin inhibit cell division in both lon(+) and lon(-) cells. In this case, cell division recommences with the same kinetics in both strains after the removal of penicillin. This suggests that different steps in the cell division process are blocked by inhibition of DNA synthesis and by penicillin treatment. The lon(-) mutation appears to affect the former of these steps.     

Excision of Thymine Dimers In Vitro by Extracts of Bacteriophage-Infected Escherichia coli

Extracts of DNA polymerase I defective Escherichia coli infected with phage T4 contain an exonuclease activity that removes thymine dimers from UV-irradiated DNA previously nicked with T4 UV endonuclease. This activity is not expressed if cells are infected in the presence of chloramphenicol. The enzyme has a requirement for divalent cation and is not affected by caffeine, but excision is inhibited in the presence of proflavine. The enzyme is present in all phage T4 mutants thus far examined, including 25 UV-sensitive mutants isolated during the course of the experiments, all of which are defective in the v gene. A similar activity can be detected in cells infected with phages T2, T3, and T6, but not in cells infected with phage T7.     

基因敲除提高大肠杆菌工程菌生产异丁醇产量的研究

探究pflB、frdAB、fnr和AdhE四基因缺失突变株对大肠杆菌工程菌发酵生产异丁醇的影响。运用Red重组系统敲除大肠杆菌BW25113的pflB、frdAB、fnr和AdhE基因,构建pflB、frdAB、fnr和AdhE四基因缺失突变株E.coliBW25113H,结合本实验室已经构建的表达质粒pSTV29-alsS-ilvC-ilvD-kdcA,并检测该工程菌在1L发酵罐的发酵过程中的生物量、突变菌株的稳定性、异丁醇产量及有机酸含量的变化情况。成功获得pflB、frdAB、fnr和AdhE四基因缺失突变株BW25113H。发酵结果表明,该工程菌能以较长时间,较高比生长速率保持对数生长期,其稳定性较好,异丁醇产量增加了40%。成功构建pflB、frdAB、fnr和AdhE四基因缺失突变株BW25113H,结合非自身发酵途径使异丁醇的产量由3 g/L提升至4.2 g/L。     

uvrC Gene Function in Excision Repair in Toluene-Treated Escherichia coli

We have examined the role of the uvrC gene in UV excision repair by studying incision, excision, repair synthesis, and DNA strand reformation in Escherichia coli mutants made permeable to nucleoside triphosphates by toluene treatment. After irradiation, incisions occur normally in uvrC cells in the presence of nicotinamide mononucleotide (NMN), a ligase-blocking agent, but cannot be detected otherwise. We conclude that repair incisions are followed by a ligation event in uvrC mutants, masking incision. However, a uvrC polA12 mutant accumulates incisions only slightly less efficiently than a polA12 strain without NMN. Excision of pyrimidine dimers is defective in uvrC mutants (polA(+) or polA12) irrespective of the presence or absence of NMN. DNA polymerase I-dependent, NMN-stimulated repair synthesis, which is demonstrable in wild-type cells, is absent in uvrC polA(+) cells, but the uvrC polA12 mutant exhibits a UV-specific, ATP-dependent repair synthesis like parental polA12 strains. A DNA polymerase I-mediated reformation of high-molecular-weight DNA takes place efficiently in uvrC polA(+) mutants after incision accumulation, and the uvrC polA12 mutant shows more reformation than the polA12 strain after incision. These results indicate that normal incision occurs in uvrC mutants, but there appears to be a defect in the excision of pyrimidine dimers, allowing resealing via ligation at the site of the incision. The lack of NMN-stimulated repair synthesis in uvrC polA(+) cells indicates that incision is not the only requirement for repair synthesis.     

Ribonucleic Acid Polymerase Mutant of Escherichia coli Defective in Flagella Formation

Escherichia coli K-12 mutants that are resistant to bacteriophage chi, defective in motility, and unable to grow at high temperature (42 degrees C) were isolated from among those selected for rifampin resistance at low temperature (30 degrees C) after mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine. Genetic analysis of one such mutant indicated the presence of two mutations that probably affect the beta subunit of ribonucleic acid (RNA) polymerase: one (rif) causing rifampin resistance and the other (Ts-74) conferring resistance to phage chi (and loss of motility) and temperature sensitivity for growth. Observations with an electron microscope revealed that the number of flagella per mutant cell was significantly reduced, suggesting that the Ts-74 mutation somehow affected flagella formation at the permissive temperature. When a mutant culture was transferred from 30 to 42 degrees C, deoxyribonucleic acid synthesis accelerated normally, but RNA or protein synthesis was enhanced relatively little. The rate of synthesis of beta and beta' subunits of RNA polymerase was low even at 30 degrees C and was further reduced at 42 degrees C, in contrast to the parental wild-type strain. Expression of the lactose and other sugar fermentation operons, as well as lysogenization with phage lambda, occurred normally at 30 degrees C, suggesting that the mutation does not cause general shut-off of gene expression regulated by cyclic adenosine 3',5'-monophosphate.     

大肠杆菌astE、rph基因敲除突变株的构建

目的:分别构建大肠杆菌astE、rph基因敲除突变株,并检测其异丁醇耐受性的变化。方法:利用Red重组系统分别敲除大肠杆菌的astE和rph基因,并对所获得的突变株进行异丁醇耐受性相关实验研究。结果:成功构建了astE基因缺失突变株△astE和rph基因缺失突变株Δrph,发现两种突变株的异丁醇耐受性均有所提高。结论:通过缺陷菌株的构建,为未来进一步代谢改造生产异丁醇和研究异丁醇耐受机制奠定了基础。     

Repair of Uv-Induced Pyrimidine Dimers in DROSOPHILA MELANOGASTER Cells in Vitro

Tissue culture cells of Drosophila melanogaster were given various doses of ultraviolet light. The results indicate that Drosophila cells do have a dark-repair excision mechanism which is not sensitive to caffeine. Pyrimidine dimers were destroyed by photoreactivating illumination in these cells and this destruction probably represents monomerization of the pyrimidine dimers.     

A Mutant Escherichia coli Primase Defective in Elongation of Primer RNA Chains

Earlier we showed by affinity cross-linking of initiating substrates to Escherichia coli primase that one or more of the residues Lys211, Lys229, and Lys241 were involved in the catalytic center of the enzyme (A. A. Mustaev and G. N. Godson, J. Biol. Chem. 270:15711-15718, 1995). We now demonstrate by mutagenesis that only Lys241 but not Lys211 and Lys229 is part of the catalytic center. Primase with a mutation of Arg to Lys at position 241 (defined as K241R-primase) is almost unable to synthesize primer RNA (pRNA) on the single-stranded DNA-binding protein (SSB)/R199G4oric template. However, it is able to synthesize a pppApG dimer plus trace amounts of 8- to 11-nucleotide (nt) pRNA transcribed from the 5' CTG 3' pRNA initiation site on phage G4 oric DNA. The amount of dimer synthesized by K241R-primase is similar to that synthesized by the wild-type primase, demonstrating that the K241R mutant can initiate pRNA synthesis normally but is deficient in chain elongation. In the general priming system, the K241R-primase also can synthesize only the dimer and very small amounts of 11-nt pRNA. The results of gel retardation experiments suggested that this deficiency in pRNA chain elongation of the K241R mutant primase is unlikely to be caused by impairment of the DNA binding activity. The K241R mutant primase, however, can still prime DNA synthesis in vivo and in vitro.     

Thymineless Death in Escherichia coli 15T− and Recombinants of 15T− and Escherichia coli K-12

Thymineless death was examined in Escherichia coli 15T(-) and recombinants of 15T(-) and E. coli K-12. Those strains that were very sensitive to thymine deprivation were also very sensitive to a variety of inducing agents (mitomycin C, ultraviolet light, hydroxyurea, and nalidixic acid). Those strains that were relatively resistant to thymineless death were also relatively resistant to the inducing agents. After exposure to thymineless death and the inducing agents, sensitive strains lysed, produced colicin, and had phage particles in their lysates. These strains also showed an increase in the 6-methyladenine content of their deoxyribonucleic acid (DNA) and an increase in the DNA methylase activity of their crude extracts under these conditions. None of these effects was noted in the strains relatively resistant to thymineless death and the inducing agents. These data indicate that there are two types of thymineless death. One is represented by the strains that are very sensitive to thymine deprivation and other inducing agents and is secondary to the induction of phage psi. The strains more resistant to thymine deprivation and the other inducing agents undergo a non-phage-mediated thymineless death. The mechanism of this latter process is currently under study.     

Ultraviolet-Sensitive Mutator Strain of Escherichia coli K-12

An ultraviolet (UV)-sensitive mutator gene, mutU, was identified in Escherichia coli K-12. The mutation mutU4 is very close to uvrD, between metE and ilv, on the E. coli chromosome. It was recessive as a mutator and as a UV-sensitive mutation. The frequency of reversion of trpA46 on an F episome was increased by mutU4 on the chromosome. The mutator gene did not increase mutation frequencies in virulent phages or in lytically grown phage lambda. The mutU4 mutation predominantly induced transitional base changes. Mutator strains were normal for recombination and host-cell reactivation of UV-irradiated phage T1. They were normally resistant to methyl methanesulfonate and were slightly more sensitive to gamma irradiation than Mut(+) strains. UV irradiation induced mutations in a mutU4 strain, and phage lambda was UV-inducible. Double mutants containing mutU4 and recA, B, or C were extremely sensitive to UV irradiation; a mutU4 uvrA6 double mutant was only slightly more sensitive than a uvrA6 strain. The mutU4 uvrA6 and mutU4 recA, B, or C double mutants had mutation rates similar to that of a mutU4 strain. Two UV-sensitive mutators, mut-9 and mut-10, isolated by Liberfarb and Bryson in E. coli B/UV, were found to be co-transducible with ilv in the same general region as mutU4.     

Mutant of Escherichia coli K-12 Defective in the Transport of Basic Amino Acids

Escherichia coli K-12 possesses two active transport systems for arginine, two for ornithine, and two for lysine. In each case there is a low- and a high-affinity transport system. They have been characterized kinetically and by response to competitive inhibition by arginine, lysine, ornithine and other structurally related amino acids. Competitors inhibit the high-affinity systems of the three amino acids, whereas the low-affinity systems are not inhibited. On the basis of kinetic evidence and competition studies, it is concluded that there is a common high-affinity transport system for arginine, ornithine, and lysine, and three low-affinity specific ones. Repression studies have shown that arginine and ornithine repress each other's specific transport systems in addition to the repression of their own specific systems, whereas lysine represses its own specific transport system. The common transport system was found to be repressible only by lysine. A mutant was studied in which the uptake of arginine, ornithine, and lysine is reduced. The mutation was found to affect both the common and the specific transport systems.     

Isolation and Characterization of an Escherichia coli K-12 Mutant with a Temperature-Sensitive RecA− Phenotype

A mutation that causes a temperature-sensitive RecA(-) phenotype was identified in a derivative of a PolA(-) strain that failed to grow at high temperature. The mutant allele (recA200) was shown to be linked to cysC, conferred a sharply temperature-sensitive, ultraviolet-sensitive Rec(-) phenotype in the range 35 to 42 C, and in crosses failed to show complementation at 42 C with Hfr's that transferred recA(-). Double mutants that carried both recA200 and polA were examined for ability to grow and synthesize DNA at restrictive temperatures.     

Evidence for Excision of Ultraviolet-Induced Pyrimidine Dimers from the DNA of Human Cells In Vitro

Within 12-24 hr after human cells were irradiated with ultraviolet light, approximately 50% of the ultraviolet-induced pyrimidine dimers were lost from the DNA. Pyrimidine dimers were found in the TCA-soluble fraction of ultraviolet-irradiated cells at 24 hr. Excess thymidine, caffeine, or hydroxyurea had no effect on the loss of pyrimidine dimers from the DNA of ultraviolet-irradiated cells.     

Isolation and Properties of Mutant Strains of Escherichia coli Defective in Succinate Transport System

To investigate the stereo-specificity and the genetic control of a succinate transport system, mutants of Escherichia coli defective in the transport of succinate were isolated. The mutants showed no detectable growth on fumarate and malate, as well as on succinate. All of the revertant strains from one of the transport defective mutants, T5, could grow either on succinate, fumarate or malate. The T5 cells accumulated only a trace amount of ¹⁴C-succinate or ¹⁴C-fumarate. These results indicated that at least succinate, fumarate, and malate were transported by the system involving the same component. From the competition experiments, it was suggested that oxalacetate was also transported by the same system. A partial participation of this system for the transport of aspartate was suggested.     

Lipopolysaccharide Defective Mutant of Escherichia coli with Decreased Sensitivity to Colicin E2

Several colicin-sensitivity mutants were isolated from Escherichia coli K-12. The mutants could not form colonies in the presence of colicin E2, but recovered their colony-forming ability on trypsin treatment even after prolonged incubation with the colicin. They showed increased sensitivity to hydrophobic antibiotics and detergents, as well as resistance against P1 and T4 phages, both of which seemed due to structural changes of lipopolysaccharide (LPS). Quantitative analysis by gas-liquid chromatography revealed that the mutant-LPS contained a different stereoisomer of heptose with decreased amounts of neutral sugars (rhamnose, glucose and galactose). LPS extracted from the parental colicin-sensitive strain could neutralize the killing activity of colicin E2 in vitro, but the mutant-LPS could not. The mutant strains retained functional receptor proteins for colicin E2. These observations suggest that LPS plays an important role in the early stage of the interaction of colicin E2 with E. coli cells.