Photoreactivation in phr mutants of Escherichia coli K-12.

We have investigated the genetics of photoreactivation in Escherichia coli K-12. We found that strains with point mutations or deletions in the phr gene showed a significant residual level of photoreactivation after exposure to large fluences of photoreactivating light. It had been previously proposed that a gene in the gal-att lambda interval is also involved in photoreactivation and that the residual photoreactivating activity might be due to this so-called phrA gene located at this interval. We found that deletions of the gal-att lambda region had no effect on either the rate or the final extent of photoreactivation observed in phr+ cells or phr mutants; however strains carrying the delta (gal-att lambda) deletions displayed increased sensitivity to near-UV radiation.     

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.     

Synthesis of Ribonucleic Acid and Protein in Plasmid-Containing Minicells of Escherichia coli K-12

Unlike the deoxyribonucleic acid (DNA)-deficient minicells produced by F(-) parents, minicells produced by plasmid-containing strains contain significant amounts of plasmid DNA. We examined the ability of plasmid-containing minicells to synthesize ribonucleic acid (RNA) and protein. In vivo, minicells produced by F(-) parents are unable to incorporate radioactive precursors into acid-insoluble RNA or protein, whereas minicells produced by F', R(+), or Col(+) parents are capable of such synthesis. Using a variety of approaches, including polyacrylamide gel analysis of the RNA species produced and electron microscope autoradiography, we demonstrated that the synthesis observed in minicell preparations is a property of the plasmid-containing minicells and not a result of the few cells (approximately 1 per 10(6) minicells) contaminating the preparations. That the observed synthesis is of biological importance is suggested by the ability of plasmid-containing minicells to yield viable phage upon infection with T4.     

Excision and reintegration of the Escherichia coli K-12 chromosomal element e14.

The genetic element e14 is a natural component of the Escherichia coli K-12 chromosome. On induction of the SOS pathways, e14 excises as a 14.4-kilobase circle. We report here on the reintegration of e14 into the chromosome of cured (e14 degrees) E. coli K-12 derivatives. Using a Tn10 insertion mutant of e14, we found that reintegration occurred specifically at the locus originally occupied by e14 and with the same orientation. The reintegration event required neither the RecA nor the RecB functions. The attachment site of the free form was located within a 950-base-pair HindIII-AvaI fragment and shared sufficient homology with the host attachment site to form detectable DNA-DNA hybrids. Even though E. coli C and B/5 did not contain e14, they did possess a HindIII restriction fragment that hybridized to the free e14 attachment fragment. E. coli C could be transformed with e14-1272::Tn10, resulting in integration at this site of homology. The Tn10 mutants were also used in mapping the point of e14 attachment. We found the following sequence: fabD purB atte14 umuC. Furthermore, analysis of a recombinant plasmid that contained both the e14 attachment site and the purB locus showed that these two loci occur within 11 kilobases of each other.     

Abnormal Excision and Transfer of Chromosomal Segments by a Strain of Escherichia coli K-12

PB15 is an Hfr strain of Escherichia coli K-12. It arose from an F' strain carrying a temperature-sensitive F-gal by an event which blocked the detachment of F-gal in the normally reversible integration process. In PB15, the detachment of F-gal by a second mechanism can now be detected: this mechanism results in the excision and transfer of extended chromosomal segments which include the integrated F-gal; the excised segments are inferred to have circularized. Their excision, which is independent of the recA(+) allele, occurs at an unusually high rate during conjugation; a mutant F-initiator protein is suggested as the cause of this phenomenon. After their establishment in recipients, the enlarged F-genotes undergo further deletions of included donor genes by a process which is again recA(+)-independent. In Rec(+), but not in Rec(-), cells, a high proportion of the deleted fragments are rescued by integration into the recipient's chromosome.     

Conjugal Deoxyribonucleic Acid Replication by Escherichia coli K-12: Stimulation in dnaB(ts) Donors by Minicells

R64-11(+) donor cells that are thermosensitive for vegetative DNA replication will synthesize DNA at the restrictive temperature when recipient minicells are present. This is conjugal DNA replication because it is R64-11 DNA that is being synthesized and there is no DNA synthesis if minicells that cannot be recipients of R64-11 DNA are used. The plasmid DNA present in the donor cells before mating is transferred to recipient minicells within the first 20 min of mating, but additional copies of plasmid DNA synthesized during the mating continue to be transferred for at least 90 min. However, the transfer of R64-11 DNA to minicells is not continuous because the plasmid DNA in minicells is the size of one R64-11 molecule or smaller, and there are delays between the rounds of plasmid transfer. DNA is synthesized in minicells during conjugation, but this DNA has a molecular weight much smaller than that of R64-11. Thus, recipient minicells are defective and are not able to complete the synthesis of a DNA strand complementary to the single-stranded R64-11 DNA received from the donor cell.     

R Factor Proteins Synthesized in Escherichia coli Minicells: Membrane-Associated R Factor Proteins

R factor proteins are synthesized in R factor-containing Escherichia coli minicells. Half of this protein remained associated with the minicell membrane upon lysis of the minicells. Over 90% of the membrane-associated protein was extracted by sodium lauryl sarcosinate, suggesting a location of these proteins in the inner membrane. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of these membrane preparations demonstrated the presence of multiple peptides, including a prominent band with a molecular weight of 28,000 to 30,000. A polypeptide of similar size was seen in membrane preparations from minicells harboring R factors from five different compatibility types. This major R factor membrane peptide was seen with R factors repressed or derepressed for pilus synthesis, with and without antibiotic resistances. It was associated with R factor deoxyribonucleic acid in membrane-deoxyribonucleic acid complexes. Its possible role in R factor replication and/or transfer is being investigated.     

Repair and mutagenesis in Escherichia coli K-12 after exposure to various alkyl-nitrosoguanidines.

The mutagenic and toxic effects of a series of N-alkyl-N'-nitro-N-nitrosoguanidines were examined in Escherichia coli K-12. The role of nucleotide excision repair, the SOS response, and the adaptive response in both the reduction and the production of the biological effects of these chemicals was tested. The effects of ethyl-nitrosoguanidine are similar in nucleotide excision repair-proficient and -deficient strains, but both the mutagenicity and the toxicity of alkyl groups larger than two carbons are significantly reduced by the presence of this repair system. Similarly, when alkyl groups are larger than two carbons, the umuC gene product is essential for the production of a fraction of the mutations that these lesions produce. The induction of the adaptive response had a significant effect on the toxicity of all of the chemicals tested, but its effect on mutagenicity was less uniform, having a larger effect on ethylating and propylating agents than on butylating and amylating agents.     

Isolation, by tetracycline selection, of small plasmids derived from R-factor R12 in Escherichia coli K-12.

The examination, by agarose gel electrophoresis, of tetracycline-resistant colonies of Escherichia coli K-12 carrying R-factor R12 reveals the presence of smaller plasmid deoxyribonucleic acids (DNAs), incompatible with R12, in many of the clones. These plasmids are demonstrated to be homologous with R12 DNA by electron microscope heteroduplex experiments and by the production of consistent fragment patterns upon digestion with various restriction endonucleases. These autonomously replicating plasmids form a related series of covalently closed circular DNA molecules ranging in size from 3.6 X 10(6) to 61 X 10(6) daltons. Plasmids of molecular weight between 3.6 X 10(6) and 37 X 10(6) confer no antibiotic resistances, but when jointly present with R12 by nonetheless enhance the expression of the tetracycline resistance associated with this latter molecule.     

R Factors Improving Survival of Escherichia coli K-12 After Ultraviolet Irradiation

Two R factors which have the capacity to improve survival of some strains of Escherichia coli K-12 by approximately 60% after ultraviolet light have been identified and characterized. Both are fi(-), but neither produce colicins. The ability to enhance survival can be separated from all other identified R-factor functions. Improved survival does not result from improved excisional capacity, but does require an intact host capacity for genetic recombination. No effects on host cell growth or postirradiation lag were observed. A proposed mechanism of action is described.     

Repair of ultraviolet light-damaged deoxyribonucleic acid in sbc-A strains of Escherichia coli K-12.

An Escherichia coli strain carrying both rec+ and sbcA has been constructed. Repair of ultraviolet light-induced deoxyribonucleic acid damage was examined by measuring survival and thymine-dimer excision in the rec+ sbcA strain as well as rec+ sbcA+ and recB recC sbcA strains. The sbcA mutation restores normal survival in both recB recC uvrB and recB recC uvr+ strains. Excision of thymine-containing dimers does not occur in uvrB mutants, regardless of the rec or sbcA genotype. Survival, after ultraviolet-light damage, of a rec+ sbcA strain is quantitatively similar to rec+ sbcA+ and recB recC sbcA strains.     

Effect of Thymine Starvation on Deoxyribonucleic Acid Repair Systems of Escherichia coli K-12

Thymine starvation of Escherichia coli K-12 results in greatly increased sensitivity to ultraviolet light (UV). Our studies, using isogenic strains carrying rec and uvr mutations, have shown the following. (i) Common to all strains tested is a change from multihit to single-hit kinetics of survival to UV after 60 min of thymine starvation. However, the limiting slope of UV survival curves decreases in the rec(+)uvr(+) strain and changes very little in several rec mutant strains and one uvrB mutant strain. Thus, when either the rec or uvr system is functioning alone, the limiting slopes of the UV survival curves are relatively unaffected by thymine starvation. (ii) Thymine starvation does not significantly inhibit repair processes carried out by either repair system alone; i.e., host cell reactivation of irradiated phage (carried out by the uvr system), excision of thymine dimers (uvr), or X-ray repair (rec). (iii) In a rec(+)uvr(+) strain, repair appears to be a synergistic rather than additive function of the two systems. However, after thymine starvation, repair capacity is reduced to about the sum of the repair capacities of the independent systems. (iv) The kinetics of thymineless death are not changed by rec and uvr mutations. This indicates that the lesions responsible for thymineless death are not repaired by rec or uvr systems. (v) Withholding thymine from thy rec(+)uvr(+) bacteria not undergoing thymineless death has no effect on UV sensitivity. Under these conditions one sees higher than normal UV resistance in the presence or absence of thymine. This is due to increased repair carried out by the uvr system. To explain these results we postulate that thymine starvation does not inhibit either the rec or uvr repair pathway directly. Rather it appears that thymine starvation results in increased UV sensitivity in part by inhibiting a function which normally carries out efficient coordination of rec and uvr pathways.     

Separate Branches of the uvr Gene-Dependent Excision Repair Process in Ultraviolet-Irradiated Escherichia coli K-12 Cells; Their Dependence upon Growth Medium and the polA, recA, recB, and exrA Genes

The extent of repair of single-strand breaks (incision breaks) induced in the deoxyribonucleic acid (DNA) of Escherichia coli K-12 cells by the uvr gene-dependent excision repair process after ultraviolet (UV) radiation was determined in the wild-type, polA1, recA56, recB21, and exrA strains. The wild-type strain repaired all incision breaks after incident doses of UV radiation (254 nm) of approximately 60 J m(-2) or less when incubated in growth medium, or approximately 15 J m(-2) or less when incubated in buffer. The polA1 strain repaired the incision breaks completely after incident doses of approximately 12 J m(-2) or less when incubated in growth medium, or after approximately 4 J m(-2) when incubated in buffer. The recA13, recB21, and exrA strains showed essentially complete repair after incident doses of 10 to 15 J m(-2) whether the cells were incubated in buffer or growth medium. These results suggest that the uvr gene-dependent excision repair process may be divided into two branches, one which is dependent on the presence of growth medium and also the rec(+)exr(+) genotype, and a second which can occur in buffer (growth medium-independent) and is largely dependent on DNA polymerase I. The presence of chloramphenicol in the growth medium resulted in an inhibition of the growth medium-dependent repair occurring in wild-type and polA1 cells and had little or no effect on the extent of repair observed in recA56, recB21, or exrA cells. The similarities between the growth medium-dependent and -independent branches of excision repair and two known processes for the repair of X-ray-induced single-strand breaks are discussed.     

Random replication of the stringent plasmid R1 in Escherichia coli K-12.

The R-factor R1 is present in a low number of copies per genome (near unity, so-called stringent control of replication). The replication of R1 was studied in a density-shift experiment. One generation after the shift about 20% of the R1 copies had not replicated, whereas about 20% had replicated at least twice. The results are in quantitative accordance with a random replication of R1 in which the replicating molecules are taken from a cytoplasmic plasmid pool and transferred back to the pool after replication. This is analogous to the results obtained by Bazaral and Helinski (1970) and Rownd (1969) for plasmids that are present in 10 to 20 copies per genome (so-called relaxed control of replication). Hence, there seem to be no difference between stringent and relaxed plasmids with respect to selection of plasmid molecules for replication. However, we cannot tell whether all R1 copies in a cell replicate during a fraction of or throughout the cell cycle. The random selction of plasmid copies for replication has to be considered when models for control of replication are constructed.