Inducible DNA polymerase I synthesis in a UV hyper-resistant mutant of Escherichia coli

A mutant of Escherichia coli which is more resistant to shortwave UV light than its wild-type parent strain and which can synthesise DNA polymerase I constitutively has been further analysed. It carries two mutational alleles which are located about 1.5 min apart and cotransducible by P1 with the argH locus. The two mutational alleles have been segregated and their analysis shows that one of them is responsible for UV hyper-resistance whereas the other mutation confers UV sensitivity. Recombinant plasmids carrying various sections of the polA regulatory region, linked to a galK gene, were introduced into the mutant strains. Analysis of galactokinase shows that the enzyme activity in the UV hyper-resistant mutant is increased. The results suggest that the synthesis of DNA polymerase I in E. coli is inducible.     

The adaptive response to mitomycin C exposure in the hyper-radioresistant mutant Escherichia coli Gamr444

Adaptive response to mitomycin C (MC) (lethal effect and recovery of molecular mass of DNA) in hyper-radioresistant mutant Escherichia coli Gamr444 have been investigated. This mutant is more resistant to MC than parent strain E. coli K12 AB1157. Adaptation of Gamr444 mutant to MC in nonlethal concentrations increases its resistance to MC in lethal concentrations with dose modification factor (DMF) 2.4 at the LD90 level. During the adaptation of this mutant to methyl-methane sulfonate (MMS) its resistance to this agent increases with DMF by 2.2 and resistance to MC with DMF by 1.5 times. During the adaptation of Gamr444 mutant to MC its resistance to MMS increases with DMF by 1.5 times. Adaptive response to MC abolishes by chloroamphenicol treatment during the adaptation. Adaptive response to nitrogen mustard (HN2) in E. coli Gamr444 is absent (HN2 induces cross-links in DNA as MC). Degradation of DNA following the formation of cross-links in DNA takes place. Adaptation to MC in Gamr444 mutant leads to restoration of DNA molecular mass which is more quicker than in the case without adaptation. Adaptive restoration of DNA molecular mass after the MC treatment is absent in E. coli K12 AB1157. The repair of cross-links in DNA after the treatment of HN2 in Gamr444 mutant takes place with equal rate both in the case of adaptation to HN2 and in the case without adaptation. It is proposed, that under the treatment of MC in E. coli Gamr444 the ada-alkA-dependent adaptive response takes place. This adaptive response is connected with alkylation of O6-guanine and elimination of the product by O6-alkyl-DNA-alkyltransferase. Partial recA-dependency of the adaptive response to MC allows to suggest the participation of another inducible system. The nature of this system is unknown.     

DNA topoisomerase I mutants. Increased heterogeneity in linking number and other replicon-dependent changes in DNA supercoiling

Plasmid pBR322 DNA isolated from Salmonella typhimurium supX (topoisomerase I) mutants exhibits a novel supercoiling distribution characterized by extreme heterogeneity in linking number and the presence of highly negatively supercoiled topoisomers. The most negatively supercoiled topoisomers isolated from one supX mutant have more than twice the wild-type level of supercoiling; the distribution as a whole has a median superhelix density about 1.3 times that of wild type. Surprisingly, the supercoiling distribution of plasmid pUC9 DNA isolated from supX mutants differs from that of pBR322. Escherichia coli topoisomerase I mutants have been shown to acquire compensatory mutations that reduce bacterial chromosome supercoiling to below the wild-type level even in the absence of topoisomerase I. We find that such a compensatory mutation in an E. coli topoisomerase I deletion mutant does not reduce pBR322 DNA supercoiling to a level below that of wild type. Thus, the effects of topoisomerase mutations on supercoiling depend on the replicon.     

Isolation and analysis of a mutant of Escherichia coli hyper-resistant to near-ultraviolet light plus 8-methoxypsoralen

A mutant of Escherichia coli K12 was isolated which shows enhanced resistance towards near-ultraviolet (NUV) light plus 8-methoxypsoralen (MPS) compared with its wild-type parent strain. The PUVA (NUV + MPS)-resistant strain remains as sensitive for far-ultraviolet (FUV) light as its parent strain. A recA- derivative of this mutant strain was as sensitive to PUVA as its reca- parental strain. A polyacrylamide gel electrophoresis study of total cell lysates from the mutant bacteria showed that a protein of approximately 55 kd was synthesised in higher concentrations compared with its synthesis in the wild-type parent strain. Furthermore, synthesis of this protein was reduced in the recA- derivative of the mutant strain suggesting that the recA gene product might be acting as a regulator of the synthesis of the 55-kd protein. It is suggested that in E. coli damage to DNA by PUVA can be repaired by a specific RecA LexA-inducible repair system and the repair efficiency is enhanced if the 55-kd protein is present in concentrations higher than that synthesised by the wild-type parent E. coli.     

Either bacteriophage T4 RNase H or Escherichia coli DNA polymerase I is essential for phage replication.

Bacteriophage T4 rnh encodes an RNase H that removes ribopentamer primers from nascent DNA chains during synthesis by the T4 multienzyme replication system in vitro (H. C. Hollingsworth and N. G. Nossal, J. Biol. Chem. 266:1888-1897, 1991). This paper demonstrates that either T4 RNase HI or Escherichia coli DNA polymerase I (Pol I) is essential for phage replication. Wild-type T4 phage production was not diminished by the polA12 mutation, which disrupts coordination between the polymerase and the 5'-to-3' nuclease activities of E. coli DNA Pol I, or by an interruption in the gene for E. coli RNase HI. Deleting the C-terminal amino acids 118 to 305 from T4 RNase H reduced phage production to 47% of that of wild-type T4 on a wild-type E. coli host, 10% on an isogenic host defective in RNase H, and less than 0.1% on a polA12 host. The T4 rnh(delta118-305) mutant synthesized DNA at about half the rate of wild-type T4 in the polA12 host. More than 50% of pulse-labelled mutant DNA was in short chains characteristic of Okazaki fragments. Phage production was restored in the nonpermissive host by providing the T4 rnh gene on a plasmid. Thus, T4 RNase H was sufficient to sustain the high rate of T4 DNA synthesis, but E. coli RNase HI and the 5'-to-3' exonuclease of Pol I could substitute to some extent for the T4 enzyme. However, replication was less accurate in the absence of the T4 RNase H, as judged by the increased frequency of acriflavine-resistant mutations after infection of a wild-type host with the T4 rnh (delta118-305) mutant.     

Biochemical characterization of mutant forms of DNA polymerase I from Escherichia coli. I. The polA12 mutation.

DNA polymerase I has been purified to greater than 90% homogeneity from a strain of Escherichia coli K12 that bears the temperature-sensitive DNA polymerase I mutatation, polA12. The mutant enzyme has a reduced electrophoretic mobility and sedimentation rate. It is abnormally thermolabile and is rapidly inactivated at low salt concentrations. Its polymerase and 5' leads to 3' exonuclease activities are not grossly defective at 30 degrees, yet its capacity to promote the concerted 5' leads to 3' polymerization and the 5' leads to 3' exonucleolytic hydrolysis of nucleotides at a nick ("nick translation") is decreased 10-fold. These effects are probably the result of a significant alteration in the tertiary structure of the enzyme.     

Interactions of subunits of DNA gyrase

Interaction of DNA gyrase A- and B-subunits during the process of DNA supercoiling was studied. For this purpose a E. coli Cour-1 mutant resistant to coumermycin and containing a mutation in the B-subunit of DNA gyrase was isolated and the influence of the DNA gyrase A-subunit specific inhibitor-nalidixic acid-on DNA supercoiling by wild-type and mutant enzymes was investigated. It turned out that the enzyme from the Cour-1 mutant strain was more sensitive to nalidixic acid than the DNA gyrase from the wild-type strain. Hence, the mutation affecting the B-subunit is capable to change A-subunit properties. That makes it possible to draw the conclusion about a close structural interaction of DNA gyrase subunits during DNA supercoiling.     

Methyl methane sulfonate-sensitive mutant of Escherichia coli deficient in an endonuclease specific for apurinic sites in deoxyribonucleic acid.

A methyl methane sulfonate (MMS)-sensitive mutant of Escherichia coli AB 1157 was obtained by N-methyl-N'-nitro-N-nitrosoguanidine treatment. The mutant strain, AB 3027, is defective both in endonuclease activity for apurinic sites in deoxyribonucleic acid (DNA) and in DNA polymerase I, as shown by direct enzyme assays. Derivative strains, which retained the deficiency in endonuclease activity for apurinic sties (approximately 10% of the wild-type enzyme level) but had normal DNA polymerase I activity, were obtained by P1-mediated transduction (strain NH5016) or by selection of revertants to decreased MMS sensitivity. These endonuclease-deficient strains are more MMS-sensitive than wild-type strains. Revertants of these deficients strains to normal MMS resistance were isolated. They had increased levels of the endonuclease activity but did not attain wild-type levels. The data suggest that endonuclease for apurinic sites is active in repair of lesions introduced in DNA as a consequence of MMS treatment. Two different endonucleases that specifically attack DNA containing apurinic sites arepresented in E coli K-12. A heat-labile activity, sensitive to inhibition by ethylenediaminetetraacetate, accounts for 90% of the total endonuclease activity for apurinic sties in crude cell extracts. The residual 10% is due to a more heat-resistant activity, refractory to ethylenediaminetetraacetate inhibition. The AB3027 and NH5016 strains have normal amounts of the latter endonuclease but no or very little of the former activity.     

Ionophore activity of sarcotoxin I, a bactericidal protein of Sarcophaga peregrina.

When Escherichia coli was treated with sarcotoxin I, a potent bactericidal protein of Sarcophaga peregrina (fleshfly), K+ inside of the cells leaked out rapidly and the ATP pool of the cells rapidly decreased. These results suggested that the bactericidal effect of sarcotoxin I was due to its ionophore activity, and that it blocked the generation of ATP by inhibiting formation of the proton gradient essential for oxidative phosphorylation. This was confirmed by use of an uncA mutant, which was much less susceptible than the wild-type strain to sarcotoxin I under fixed ionic conditions.     

A grpE mutant of Escherichia coli is more resistant to heat than the wild-type

The grpE gene of Escherichia coli is essential for bacteriophage lambda DNA replication and is also necessary for host RNA and DNA synthesis at high temperature. A grpE mutant of E. coli was found to be substantially more resistant to 50 degrees C heat treatment than the wild-type. Upon receiving a 42 degrees C heat shock for 15 min, both the wild-type and the grpE mutant became more resistant to heat (i.e. they became thermotolerant). A grpE+ revertant behaved similarly to the wild-type in that it was more sensitive to heat than grpE cells. In addition, grpE cells had the same H2O2 and UV sensitivity as the wild-type. This implies that the conditions for which a grpE mutation is beneficial are unique to heat exposure and are not caused by H2O2 or UV exposure. Furthermore, synthesis of heat-shock proteins occurred sooner in the grpE mutant than in the wild-type, indicating that the grpE gene of E. coli may influence the regulation of the heat-shock response.     

Natural plasmid pSD89 (Cmr) from Salmonella derby K89, which increases the radiation tolerance of Escherichia coli strain K-12]

Several plasmids with molecular mass of 1.3-9 MDa were found in a clinical isolate of Salmonella derby K89 by electrophoresis in agarose gel. One of these plasmids, designated pSD89 (Cmr), was derived from the K89 strain via transformation of the plasmidless recipient S. derby K82 to chloramphenicol resistance. The plasmid-carrying strain K89 and the K82 strain completely cured of plasmids were equally sensitive to the lethal action of UV light, whereas the plasmid-carrying strain was even more sensitive to ionizing radiation than the plasmidless variant. Nevertheless, transformants carrying only plasmid pSD89 (Cmr) were found to be more resistant to gamma-rays and UV light than the recipient. By using an intermediate host Escherichia coli Z80 (r-m+), plasmid pSD89 (Cmr) was introduced into different E. coli K-12 strains: polA-, recA-, uvrA-, umuC-, and the wild-type strain. A slight increase in radioresistance of E. coli wild-type cells and a significant complementation of a repair defect in recA and polA mutants, but not in uvrA and umuC, were observed.     

Effects of Escherichia coli ribosomal protein S12 mutations on cell-free protein synthesis.

We examined the effects of Escherichia coli ribosomal protein S12 mutations on the efficiency of cell-free protein synthesis. By screening 150 spontaneous streptomycin-resistant isolates from E. coli BL21, we successfully obtained seven mutants of the S12 protein, including two streptomycin-dependent mutants. The mutations occurred at Lys42, Lys87, Pro90 and Gly91 of the 30S ribosomal protein S12. We prepared S30 extracts from mutant cells harvested in the mid-log phase. Their protein synthesis activities were compared by measuring the yields of the active chloramphenicol acetyltransferase. Higher protein production (1.3-fold) than the wild-type was observed with the mutant that replaced Lys42 with Thr (K42T). The K42R, K42N, and K42I strains showed lower activities, while the other mutant strains with Lys87, Pro90 and Pro91 did not show any significant difference from the wild-type. We also assessed the frequency of Leu misincorporation in poly(U)-dependent poly(Phe) synthesis. In this assay system, almost all mutants showed higher accuracy and lower activity than the wild-type. However, K42T offered higher activity, in addition to high accuracy. Furthermore, when 14 mouse cDNA sequences were used as test templates, the protein yields of nine templates in the K42T system were 1.2-2 times higher than that of the wild-type.     

Comparison of the Escherichia coli umu+-encoded function with plasmid R46-mediated error-prone repair in DNA-damaged cells

Escherichia coli strain TK701 umu+ was more resistant than strain TK702 umu when tested against bleomycin (BLM), cis-platinum(II) diamminodichloride (PDD), ultraviolet light and methyl methanesulphonate (MMS), which produce single-strand DNA damage. However, the umu mutant was no more sensitive to mitomycin C (MTC) or proflavine (PF), which cause double-strand DNA binding. Strain TK702 umu was nonmutable by any of the agents, whereas mutations were induced in the wild-type strain by PDD, UV, MMS and MTC. The E. coli umu+ function therefore mimics plasmid R46-mediated error-prone repair in protecting only against single-strand DNA damage, whilst enhancing mutagenesis by both single- and double-strand damaging agents. Comparison of plasmid R46-mediated protection and mutagenesis in umu+ and umu strains indicated that the plasmid confers a greater error-prone DNA-repair activity in the mutant. Results are discussed in terms of analogy between host umu+ and plasmid muc+ functions.     

Multiple pathways for repair of oxidative DNA damages caused by X rays and hydrogen peroxide in Escherichia coli.

The responses of Escherichia coli to X rays and hydrogen peroxide were examined in mutants which are deficient in one or more DNA repair genes. Mutant cells deficient in either exonuclease III (xthA) or endonuclease IV (nfo) had normal resistance to X rays, but an xthA-nfo double mutant showed a sensitivity increased over that of either parental strain. A DNA polymerase I mutant (polA) was more sensitive than the xthA-nfo mutant. Cells bearing mutations in all of the polA, xthA, and nfo genes were more sensitive to X rays than polA and xthA-nfo mutants. Similar repair responses were obtained by exposing these mutant cells to hydrogen peroxide, with the exception of the xthA mutant, which was hypersensitive to this agent. The DNA polymerase III mutant (polC(Ts)) was slightly more sensitive to the agents than the wild-type strain at the restrictive temperature. The sensitivity of the polC-xthA-nfo mutant to X rays and hydrogen peroxide was greater than that of polC but almost the same as that of the xthA-nfo mutant. From these results it appears that there are at least four repair pathways, the DNA polymerase I-, exonuclease III/endonuclease IV and DNA polymerase I-, exonuclease III/endonuclease IV and DNA polymerase III-, and exonuclease III/endonuclease IV-dependent pathways, for the repair of oxidative DNA damages in E. coli.     

DNA degradation in wild-type and repair-deficient strains of salmonella typhimurium exposed to ultraviolet light of photodynamic treatment.

Five mutants of Salmonella typhimurium strain LT2 trp DI (ColEI)+, initiallly detected because they released little or no colicin when tested on solid medium, proved to be sensitive to ultraviotet light (u.v.). Further testing indicated that one of the mutants was deficient in genetic recombination and was probably a recA-type mutant, while three of the others were deficient in DNA polymerase activity and appeared to be typical polA mutants. The fifth mutant was less sensitive than the others to methyl methanesulphonate, showed reduced proficiency in genetic recombination, and was of approximately normal u.v. mutability. This mutant may be a counterpart of the class known as uvrD in Escherichia coli. All five mutants degraded significantly more of their DNA following exposure to u.v. than did the wild-type strain. The recA-type mutant and the possible uvrD mutant also degraded significantly more of their DNA spontaneously than did the wild-type. Treatment with visible light and acridine orange (photodynamic treatment) cause no significant degradation of DNA in the wild-type strain, a highly significant increase in the extent of DNA degradation in a polA mutant, and a decrease in the extent of degradation in the recA-type mutant.     

Phage Growth and Deoxyribonucleic Acid Synthesis in Escherichia coli Infected by a Thymine-Requiring Bacteriophage.

Mathews, Christopher K. (Yale University, New Haven, Conn.). Phage growth and deoxyribonucleic acid synthesis in Escherichia coli infected by a thymine-requiring bacteriophage. J. Bacteriol. 90:648-652. 1965.-Cultures of Escherichia coli B infected with a mutant strain of phage T4 which cannot induce the formation of thymidylate synthetase produce deoxyribonucleic acid (DNA) at about two-thirds the rate of cultures infected with the parent strain. Under certain conditions the yield of viable phage observed with the mutant is one-third of that brought about by the wild-type strain. Addition of thymine increases both DNA synthesis and phage production in cells infected by the mutant. It is suggested that the ability to induce thymidylate synthetase formation in infected cells confers a selective advantage on the wild-type strain.     

Cloning of the gene for Escherichia coli glutamyl-tRNA synthetase

The structural gene for the glutamyl-tRNA synthetase of Escherichia coli has been cloned in E. coli strain JP1449, a thermosensitive mutant altered in this enzyme. Ampicillin-resistant and tetracycline-sensitive thermoresistant colonies were selected following the transformation of JP1449 by a bank of hybrid plasmids containing fragments from a partial Sau3A digest of chromosomal DNA inserted into the BamHI site of pBR322. One of the selected clones, HS7611, has a level of glutamyl-tRNA synthetase activity more than 20 times higher than that of a wild-type strain. The overproduced enzyme has the same molecular weight and is as thermostable as that of a wild-type strain, indicating that the complete structural gene is present in the insert. These characteristics were lost by curing this clone of its plasmid with acridine orange, and were transferred with high efficiency to the mutant strain JP1449 by transformation with the purified plasmid. A physical map of the plasmid, which contains an insert of about 2.7 kb in length, is presented.     

Cofactor requirements of BamHI mutant endonuclease E77K and its suppressor mutants

A mutant BamHI endonuclease, E77K, belongs to a class of catalytic mutants that bind DNA efficiently but cleave DNA at a rate more than 10(3)-fold lower than that of the wild-type enzyme (S. Y. Xu and I. Schildkraut, J. Biol. Chem. 266:4425-4429, 1991). The preferred cofactor for the wild-type BamHI is Mg2+. BamHI is 10-fold less active with Mn2+ as the cofactor. In contrast, the E77K variant displays an increased activity when Mn2+ is substituted for Mg2+ in the reaction buffer. Mutations that partially suppress the E77K mutation were isolated by using an Escherichia coli indicator strain containing the dinD::lacZ fusion. These pseudorevertant endonucleases induce E. coli SOS response (as evidenced by blue colony formation) and thus presumably nick or cleave chromosomal DNA in vivo. Consistent with the in vivo result, the pseudorevertant endonucleases in the crude cell extract display site-specific partial DNA cleavage activity. DNA sequencing revealed two unique suppressing mutations that were located within two amino acid residues of the original mutation. Both pseudorevertant proteins were purified and shown to increase specific activity at least 50-fold. Like the wild-type enzyme, both pseudorevertant endonucleases prefer Mg2+ as the cofactor. Thus, the second-site mutation not only restores partial cleavage activity but also suppresses the metal preference as well. These results suggest that the Glu-77 residue may play a role in metal ion binding or in enzyme activation (allosteric transition) following sequence-specific recognition.     

Effect of the deletion of the σ32-dependent promoter (P1) of the Escherichia coli topoisomerase I gene on thermotolerance

Topoisomerase I and DNA gyrase are the major topoisomerase activities responsible for the regulation of DNA supercoiling in the bacterium Escherichia coli . The P1 promoter of topA has previously been shown to be a σ³²-dependent heat-shock promoter. A mutant strain with a deletion of P1 was constructed. This mutant is >10-fold more sensitive to heat treatment (52°C) than the wild type. After brief treatment at 42°C, wild-type Escherichia coli acquires an enhanced resistance to the effects of a subsequent 52°C treatment. This is not the case for the P1 deletion mutant, which, and under these conditions, is about 100-fold less thermotolerant than the wild type. The presence of a plasmid expressing topoisomerase I restored the heat-survival level of the mutant to that of the wild type. During heat shock, the superhelical density of a plasmid with the heat-inducible rpoD promoter is increased in the P1 deletion mutant. We also note that the pulse-labelling pattern of proteins at 42°C (displayed on SDS–polyacrylamide gels) is different in the mutant, and, most notably, the amounts of DnaK and of GroEL protein are reduced. A model is proposed in order to unify these observations.     

Evidence that discontinuous DNA replication in Escherichia coli is primed by approximately 10 to 12 residues of RNA starting with a purine

Intact primer RNA for discontinuous DNA replication of Escherichia coli has been detected by specific labeling in vitro of its 5'-terminal tri- (or di-) phosphate group with vaccinia guanylyltransferase and [alpha-32P]GTP. A mutant defective either in RNase H or in both RNase H and DNA polymerase I accumulated about 10 or 30 times more intact primer RNA, respectively, than wild-type cells. The primers started with purine in an A to G ratio of 5 and the most abundant 5'-terminal dinucleotide sequence was (p)ppA-Pu. The chain length of the intact primer RNA was approximately 10 to 12 nucleotide residues. The structural properties of the E. coli primer RNa resemble those of the eukaryotic primer RNA.