Escherichia coli K-12 mutants with enhanced resistance to ionizing radiation. III. The effect of rec and lexA mutations on radioresistance

Lethal action of gamma-rays on derivatives of the wild-type strain AB1157 and of two radiation-resistant mutants (Gamr444 and Gamr445) containing additional mutations dnaA46, recB21, recF143, recA56, recA430, lexA3, lexA102 or lexA3 recAo98, was studied. When the mean number of genomes per cell was reduced by means of pre-incubation at 43 degrees C, radioresistance of the strains AB1157 dnaA46 and Gamr445 dnaA46 was not changed, and that of the strain Gamr444 dnaA46 was reduced to the level of the Gamr445 dnaA46 strain. Introduction of additional mutations recB21, recA56 or lexA3 (lexA102) into the genome of the strains Gamr444 or Gamr445 made them as radiosensitive as the corresponding variants of AB1157. Additional mutations recF143 or recA430 (lexB30) significantly decreased the radioresistance of Gamr444 and Gamr445 mutants, although did not level them to corresponding derivatives of AB1157. Operator-constitutive mutation recAo98 enhanced radioresistance of all lexA3 derivatives tested but not to the level of the corresponding lexA+ strains. The role of recombinational repair and the inducible SOS system in enhanced radioresistance of Gamr mutants is discussed. The data of post-irradiation DNA degradation in various derivatives of the strains AB1157 and Gamr suggest that Gamr mutants have a constitutive inhibitor of degradation which does coincide with RecA protein.     

Escherichia coli K-12 mutants with increased resistance to ionizing radiation. V. The effect of mutations on spontaneous and radiation mutagenesis

The frequencies of spontaneous mutations (reversions his-4----His+ and forward mutations to rifampicin-, nalidixic acid- or valine-resistance) in radiation-resistant mutants Gamr444 and Gamr445 are much lower than in the wild-type strain AB1157. His+ revertants and rifampicin-resistant mutants Rifr are induced by low doses of gamma-rays more efficiently than in the wild-type. Low doses of UV light only enhanced mutagenic activity in Gamr strains for induction of His+ reversions but not for Rifr mutations. For the wild-type strain the frequencies of His+ and Rifr mutations increase proportionally to the square of dose both of UV light and gamma-rays. For the most radioresistant Gamr444 mutant the frequencies of UV- and gamma-rays-induced Rifr mutations and of gamma-rays-induced reversions increase linearly with the dose. Possible reasons for these anomalies of radiation-induced mutagenesis in Gamr mutants are discussed.     

Mutants of Escherichia coli K-12 with increased resistance to ionizing radiation. VI. Increased radioresistance and heat shock proteins

By means of one-dimensional electrophoresis, it is shown that in radiation-resistant Gamr444 and Gamr445 mutants of Escherichia coli K-12 high-molecular weight heat shock proteins are hyperproduced at 32-37 degrees C and are induced more intensively during heat shock (in comparison to the parental wild-type strain AB1157). When the missense htpR15 mutation of the positive regulatory htpR gene for heat shock proteins was introduced by transduction into the genome of the Gamr444 mutant, its enhanced radiation-resistance disappeared but could be restored upon introduction of pKV3 plasmid bearing the htpR+ gene. These data show that heat shock proteins are participating in the enhanced radioresistance of Gamr mutants.     

Potassium-dependant mutants of Escherichia coli K-12

Mutants of Escherichia coli K-12 that grow more slowly in media containing low concentrations of K have been isolated. All independent mutants of this type which have been studied carry a mutation in a small region of the bacterial chromosome between the supE and gal loci. The growth rate of the mutants is the same as that of the parental strains in medium containing more than 1 mm K, but is only 50% that of the parent when the K concentration is reduced to 0.1 mm. The mutants do not appear to have a primary alteration in K transport, and are therefore referred to as K-dependent. The abbreviation kdp is proposed for this class of mutant.     

Flavodoxin mutants of Escherichia coli K-12

The flavodoxins are flavin mononucleotide-containing electron transferases. Flavodoxin I has been presumed to be the only flavodoxin of Escherichia coli, and its gene, fldA, is known to belong to the soxRS (superoxide response) oxidative stress regulon. An insertion mutation of fldA was constructed and was lethal under both aerobic and anaerobic conditions; only cells that also had an intact (fldA(+)) allele could carry it. A second flavodoxin, flavodoxin II, was postulated, based on the sequence of its gene, fldB. Unlike the fldA mutant, an fldB insertion mutant is a viable prototroph in the presence or absence of oxygen. A high-copy-number fldB(+) plasmid did not complement the fldA mutation. Therefore, there must be a vital function for which FldB cannot substitute for flavodoxin I. An fldB-lacZ fusion was not induced by H(2)O(2) and is therefore not a member of the oxyR regulon. However, it displayed a soxS-dependent induction by paraquat (methyl viologen), and the fldB gene is preceded by two overlapping regions that resemble known soxS binding sites. The fldB insertion mutant did not have an increased sensitivity to the effects of paraquat on either cellular viability or the expression of a soxS-lacZ fusion. Therefore, fldB is a new member of the soxRS (superoxide response) regulon, a group of genes that is induced primarily by univalent oxidants and redox cycling compounds. However, the reactions in which flavodoxin II participates and its role during oxidative stress are unknown.     

L-Serine-sensitive mutants of Escherichia coli K-12

While attempting to isolate d-serine-sensitive mutants of Escherichia coli K-12, we found a class of mutants sensitive to low concentrations of l-serine (10 to 25 mug/ml).     

Assembly-defective OmpC mutants of Escherichia coli K-12.

Novel ompC(Dex) alleles were utilized to isolate mutants defective in OmpC biogenesis. These ompC(Dex) alleles also conferred sensitivity to sodium dodecyl sulfate (SDS), which permitted the isolation of SDS-resistant and OmpC-specific phage-resistant mutants that remained Dex+. Many mutants acquired resistance against these lethal agents by lowering the OmpC level present in the outer membrane. In the majority of these mutants, a defect in the assembly (metastable to stable trimer formation) was responsible for lowering OmpC levels. The assembly defects in various mutant OmpC proteins were caused by single-amino-acid substitutions involving the G-39, G-42, G-223, G-224, Q-240, G-251, and G-282 residues of the mature protein. This assembly defect was correctable by an assembly suppressor allele, asmA3. In addition, we investigated one novel OmpC mutant in which an assembly defect was caused by a disulfide bond formation between two nonnative cysteine residues. The assembly defect was fully corrected in a genetic background in which the cell's ability to form disulfide bonds was compromised. The assembly defect of the two-cysteine OmpC protein was also mended by asmA3, whose suppressive effect was not achieved by preventing disulfide bond formation in the mutant OmpC protein.     

Characterization of lipopolysaccharides from Escherichia coli K-12 mutants.

Chemical analyses of the carbohydrate composition of lipopolysaccharides (LPS) from a number of LPS mutants were used to propose a schematic composition for the LPS from Escherichia coli K-12. The formula contains four regions: the first consists of lipid A, ketodeoxyoctonoic acid, and a phosphorous component; the second contains only heptose; the third only glucose; and the fourth additional glucose, galactose, and rhamnose. LPS from E. coli B may have a similar composition but lacks the galactose and rhamnose units. A set of LPS-specific bacteriophages were used for comparing three mutants of Salmonella with a number of LPS mutants of E. coli K-12. The results confirm that there are basic similarities in the first and second regions of the LPS structure; they also support the four region divisions of the LPS formula. Paper chromatography was used for characterization of 32-P-labeled LPS from different strains of E. coli and Salmonella. The Rf values for LPS varied from 0.27 to 0.75 depending on the amounts of carbohydrates in the molecule. LPS from all strains studied was homogenous except for strain D31 which produced two types of LPS. Mild acid hydrolysis of labeled LPS liberated lipid A and two other components with phosphate, one of which was assigned to the first region. It is suggested that paper chromatography can be used in biosynthetic studies concerning regions 2 to 4.     

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.     

Actinomycin sensitive mutants of Escherichia coli K-12

Summary Actinomycin sensitive mutants of E. coli K12 have been isolated and shown to have pleiotropic defects in the fermentation of sugars. The locus of a gene controlling actinomycin resistance is very close to that of the lactose gene.     

Suppression by thymidine-requiring mutants of Escherichia coli K-12.

Thymidine-requiring strains of Escherichia coli isolated by trimethoprim selection often simultaneously acquire the ability to suppress bacteriophage T4 nonsense mutations. Suppression is lost in Thy+ revertants and recombinants, but is sometimes retained in thyA plasmid-bearing transformants. Suppression is restricted in Strr derivatives of the Thy- mutants, indicating that suppression occurs at the level of translation.     

Recombination and the Escherichia coli K-12 sex factor F.

Recombination between two Flac tra minus elements to give Flac tra plus recombinants was measured in Rec plus and Rec minus strains of Escherichia coli K-12. Polar tra mutations were used to increase the proportion of tra plus recombinants among the parental Flac tra minus elements transferred by complementation. The kinetics, measured in a rec plus strain, showed that recombination began about 1 h after the initiation of mating and was completed about 1 h later. Recombination was abolished in a recA minus strain, reduced by two-thirds in a recF minus strain, and unaffected in recB minus and recC minus strains. It is proposed that the part not due to the RecF pathway results from a RecBC- and RecF-independent system for formation of single-stranded joins. One such join could be followed either by transfer and a site-specific recombination event, or by a second single-stranded join and then transfer: in either case replication and inheritance of the recombinant molecule would be dependent upon the F transfer replication system. Chromosome mobilization by an F' element was normal in a recB plus recF minus strain, and was reduced only fourfold in a recB minus recF plus strain: in the latter strain, both the RecF pathway and the system for single-stranded joins may have contributed to mobilization. Measurement of post-conjugational chromosomal recombination in exponential-phase recipient cells carrying surface exclusion-deficient Flac mutants indicated that F does not itself determine a generalized recombination system able to replace the RecA plus product or the RecBC and RecF pathways.     

Escherichia coli K-12 mutants deficient in uracil-DNA glycosylase.

A new assay specific for uracil-DNA glycosylase is described, Escherichia coli mutants partially and totally deficient in uracil-DNA glycosylase activity have been isolated by using this assay in mass-screening procedures. These have been designated ung mutants. The ung gene maps between tyrA and nadB on the E. coli chromosome. T4 phage containing uracil in their DNA grow on the most glycosylase-deficient hosts but are unable to grow on wild-type bacteria. This provides a simple spot test for the ung genotype. The ung mutants show slightly higher rates of spontaneous mutation to antibiotic resistance. Taken together, these results suggest a central role for uracil-DNA glycosylase in the initiation of an excision repair pathway for the exclusion of uracil from DNA.     

Aggregation deficient Escherichia coli K-12 female mutants with altered lipopolysaccharide.

Two F- mutants deficient in conjugation with F-donors have been characterized. They map at about 83 minut position, show resistance to T3 and T7 bacteriophages, and form mating aggregates in the liquid medium with lowered efficiency. Mutants have no detectable alterations in their outer membrane protein composition.     

Recipient ability of bacteriophage-resistant mutants of Escherichia coli K-12.

The ability of a wide range of bacteriophage-resistant mutants to act as recipients in conjugation with F'lac pro and R100-1 donors has been studied. A number of mutant types defective in recipient ability with F'lac pro, as well as mutants which were hyperrecptive with R100-1, have been detected.