COMPARACIÓN ENTRE CONDUCTORES DE COBRE Y ALUMINIO

OÏE. coli

5.1. Introduction

Previous results presented in this thesis have led to the suggestion that error-prone DNA repair might at least contribute to the mechanism o f adaptive mutation to nalidixic acid resistance. Strains o f E, coli deficient in individual DNA repair processes were therefore examined to test this hypothesis. It was predicted that strains deficient in error- prone repair would show reduced adaptive mutation to nalidixic acid resistance. This should be seen not only in strains deficient in umuDC but also in strains defective for SOS induction. For example, strains carrying defective recA or lexA genes, would be SOS uninducible and expected to show reduced frequencies of mutation to nalidixic acid resistance. A reduced mutation frequency to nalidixic acid resistance might also be expected in a strain carrying a defective recB gene. The latter encodes a subunit of exonuclease V, which is required for SOS induction by nalidixic acid (Gudas and Pardee, 1975; McPartland et al, 1980). As a control, it was predicted that a strain carrying a defective uvrA gene, which is deficient in error-free excision repair, might show increased frequencies of induced mutation since more damage could be processed via the error-prone repair pathway in the absence of excision repair.

5.2. Mutation to nalidixic acid resistance in repair-deficient strains of E» coli The MICs o f nalidixic acid for the DNA repair-deficient strains are shown in Table 19. Since 3 /^g mL * nalidixic acid, the sub-inhibitory concentration used to grow the wild-type repair-proficient ABl 157 strain, was supra-inhibitory for AB2463 recAlS and AB2470 recB21 (Table 19), it was decided to grow the repair-deficient strains in 0.75

times their respective MICs before plating on medium containing four times their respective MICs. The effect of plasmid R46 on the development o f nalidixic acid resistance was also examined in each repair-deficient background (Table 21). The presence o f the plasmid did not affect the MIC of any strain tested (results not shown). Selection-induced mutation frequencies to nalidixic acid resistance in the repair-deficient strains were determined after four days incubation. On continued incubation, the viability of strain AB2463 recA13 decreased, so affecting mutation frequencies and not allowing accurate comparisons to be made at later times.

Table 19. Minimum inhibitory concentrations (MICs) o f nalidixic acid (NA) for repair- deficient E. coli strains.

Strain and relevant genotype

DNA repair phenotype NAM IC

(//g m L ') ABl 157 Wild-type, fully repair-proficient 4

TK702 umuC36 SOS repair deficient 4

A B l886 uvrA6 Excision repair deficient 4

AB2463 recA13 Recombination repair deficient, SOS induction deficient

2

AB2470 recB21

Recombination repair deficient, lacks exonuclease V, SOS induction

deficient (by quinolones)

2

AB2494 lexA3 SOS induction deficient, recombination repair proficient

5

E. coli TK702 is deficient in SOS error-prone DNA repair due to a mutation in umuC. Compared with the fully repair-proficient AB 1157 control strain, TK702 exhibited a 1000-fold decrease in mutation frequency to nalidixic acid resistance after growth in 0.75 X MIC of nalidixic acid for four days (Table 20). However, as predicted, the

introduction o f plasmid R46 mucAB^ into this strain increased mutation 4.5 x 10^-fold, returning it to the level of the wild-type strain (Table 21).

As predicted, the 5-fold increase in mutation frequency to nalidixic acid resistance in strain A B l886 uvrA6 (Table 20) probably results from enhanced error-prone repair of DNA damage in the absence of error-free excision repair. Introduction of R46 into this strain gave an expected increase in mutation frequency of 36-fold, which was higher than that produced by the plasmid in the wild-type strain (Table 21). This increase could result from enhanced error-prone processing of DNA damage by both the mucAB gene products

Table 20. Mutation frequencies to nalidixic acid (NA) resistance (4 x MIC) in repair- deficient mutants of E. coli after four days growth in nutrient broth containing 0.75 x MIC nalidixic acid for each respective strain.

Strain Cone, o f NA in growth medium (/^gm L') (0.75 X MIC) Cone, of NA on which cultures plated (/^g mL-') (4 X MIC) Total viable count of cultures after 4 days incubation Number of NA"^ mutants after 4 days incubation Mutation frequency toN A resistance* ABl 157 3 16 1.3 X 10* 6.7 X 10* 5.3 X 10 * TK702 umuC36 3 16 8.5 X 10’ 4.4 X 10’ 5.2 X 10* ABl 886 uvrA6 3 16 2.0x10* 5.3 X 10* 2.7 X 10’ AB2470 recB21 1.5 8 8.5 X 10* 1.5 X 10* 1.8 X 10-* AB2494 lexAS 3.75 20 4.9 x 10* 1.8 X 10* 3.8 X 10-* AB2463 recAlS 1.5 8 6.7 X 10’ 7.1 X 10’ 1.1 X 10-*

*Mutation frequencies are expressed as the number of nalidixic acid-resistant mutants per viable organism.

of R46 and the chromosomally-encoded UmuDC proteins.

In line with the prediction, mutation frequencies to nalidixic acid resistance were reduced 2.9 x 10^- and 1.4 x 10^-fold respectively in the recB and lexA mutants compared to the repair-proficient strain (Table 20). The presence o f the recAlS mutation in E. coli AB2463 led to a 5.0 x 10^-fold reduction in mutation (Table 20). The latter result was expected since this strain is completely defective in inducible SOS functions (Howard- Flanders and Theriot, 1966).

Three results with plasmid-containing strains did not agree with the hypothesis that had been predicted. The presence o f R46 in the lexA mutant reduced the mutation frequency 5-fold, whereas it should have no effect since the plasmid mucAB genes should

Table 21. The effect of carriage of plasmid R46 on mutation frequencies to nalidixic acid (NA) resistance (4 x MIC) in repair-deficient mutants o f E. coli after four days growth in nutrient broth containing 0.75 x MIC nalidixic acid for each respective strain.

Strain Mutation frequency* to NA resistance in R strain Mutation frequency* to NA resistance in R46- containing strain Fold increase (+) or decrease (-) produced by presence o f R46 ABl 157 5.3 X 10'^ 1 . 1 X 1 0 - 2 + 2 . 1 TK702 umuC36 5.2 X 10'^ 2.3 X 10-2 +4.5 X 10^ ABl 8 8 6 uvrA6 2.7 X 10-2 9.7 X 10-' +36 AB2470 recB21 1 . 8 X 1 0 ’^ 1.9 X 10-^ + 1 0 AB2494 lexAS 3.8 X 10'^ 7.9 X 10-2 -4.8 AB2463 recAlS 1 . 1 X 1 0 ' ^ 9.6 X 10-^ +8.7 X 102 *Mutation frequencies are expressed as the number of nalidixic acid-resistant mutants per viable organism.

be uninduced. A 10-fold increase in mutation frequency produced by R46 in the recB mutant was also unexpected, since SOS function should not be induced in this recB- deficient mutant. Similarly, the increase in mutation to nalidixic acid resistance in the recA strain o f approximately 9 x 10^-fold was unexpected (Table 21). All these exceptions involve the plasmid-encoded mucAB operon, rather than expression of chromosomal umuDC genes, and it is possible that the R46-mediated nalidixic acid resistance-inducing function may bypass, at least to an extent, the requirement for RecA and RecB to initiate error-prone DNA repair.

5.3. Summary and conclusions

The results presented in this chapter demonstrate the involvement of error-prone DNA repair on nalidixic acid-directed adaptive mutation to nalidixic acid resistance in E. coli. Mutation frequencies were greatly reduced in strains defective in SOS induction, such as AB2463 recAlS, AB2470 recB21 and AB2494 lexA3, or expression, such as TK702 umuC36 (Table 20). Introduction o f plasmid R46, which codes for MucAB proteins that are homologous to UmuDC, into the UmuC-deficient strain returned its mutation frequency to the wild-type level (Table 21). These data are in agreement with the hypothesis that induction of the SOS response may be at least partly responsible for the development of mutations conferring nalidixic acid resistance in drug-exposed cultures.