Effect of translesion DNA polymerases, endonucleases and RpoS on mutation rates in Salmonella typhimurium

Abstract

It has been suggested that bacteria have evolved mechanisms to increase their mutation rate in response to various stresses and that the translesion DNA polymerase Pol IV under control of the LexA regulon and the alternative sigma factor RpoS are involved in regulating this mutagenesis. Here we examined in Salmonella enterica serovar Typhimurium LT2 the rates for four different types of mutations (rifampicin, nalidixic acid, and chlorate resistance and Lac(+) reversion) during various growth conditions and with different levels of four translesion DNA polymerases (Pol II, Pol IV, Pol V, and SamAB) and RpoS. Constitutive derepression of the LexA regulon by a lexA(def) mutation had no effect on Lac(+) reversion rates but increased the other three mutation rates up to 11-fold, and the contribution of the translesion DNA polymerases to this mutagenesis varied with the type of mutation examined. The increase in mutation rates in the lexA(def) mutant required the presence of the LexA-controlled UvrB protein and endonucleases UvrC and Cho. With regard to the potential involvement of RpoS in mutagenesis, neither an increase in RpoS levels conferred by artificial overexpression from a plasmid nor long-term stationary phase incubation or slow growth caused an increase in any of the four mutation rates measured, alone or in combination with overexpression of the translesion DNA polymerases. In conclusion, mutation rates are remarkably robust and no combination of growth conditions, induction of translesion DNA polymerases by inactivation of LexA, or increased RpoS expression could confer an increase in mutation rates higher than the moderate increase caused by derepression of the LexA regulon alone.

Figure 1.—

Relative mutation rates for four different types of mutations, rifampicin resistance, nalidixic acid resistance, chlorate resistance, and Lac⁺ reversion. Rates are given relative to the mean mutation rate of the wild type for each type of mutation in minimal M9 glucose medium (Table 1). Error bars are SEM.

Figure 2.—

Relative mutation rates for chlorate resistance mutations. Rates are given relative to the mean mutation rate of the wild type in minimal M9 glucose medium (Table 1). Error bars are SEM.

Figure 3.—

Relative mutation rates for three types of mutations, rifampicin resistance, nalidixic acid resistance, and chlorate resistance. Rates are given relative to the mean mutation rate of the wild type in minimal M9 glucose medium for each type of mutation (Table 1). Error bars are SEM.

Figure 4.—

(A) Relative mutation rates for four different types of mutations, rifampicin resistance, nalidixic acid resistance, chlorate resistance, and Lac⁺ reversion in the presence or absence of the arabinose inducible plasmid pBAD30∷rpoS. Rates are given relative to the mean mutation rate of the wild type for each type of mutation in LB medium (Table 1). +, presence of pBAD30∷rpoS in strain DA14346 and lexA(def) mutant DA14400 induced with the indicated concentration of L-arabinose (0.01–0.2%) (top panel). –, mutation rate in wild-type DA10212 and lexA(def) DA10598 without the plasmid. The bottom panel shows a Western blot with commercial mouse anti-RpoS antibodies for the respective strain in the same experiment. Error bars are SEM. (B) Relative mRNA levels measured by real-time PCR in cells grown with or without induction of the pBAD30∷rpoS plasmid by 0.1% L-arabinose. mRNA levels are given relative to the wild-type level set to 1. Error bars are SEM.

Figure 5.—

(A) Relative mutation rates for four different types of mutations, rifampicin resistance, nalidixic acid resistance, chlorate resistance, and Lac⁺ reversion in different media: LB, minimal M9 glucose, and minimal M9 glycerol. Rates are given relative to the mean mutation rate of the wild type for each type of mutation in minimal M9 glucose medium (Table 1). (B) Relative Pol IV (dinB)-expression measured as β-galactosidase activity (dinB promoter–lacZ fusion on pRS551 plasmid, see materials and methods). Rates are given relative to the wild-type Pol IV expression in each experiment in LB medium. Error bars are SEM.

Figure 6.—

(A and B) Relative mutation rates for four different types of mutations, rifampicin resistance, nalidixic acid resistance, chlorate resistance, and Lac⁺ reversion for wild-type and mutant cells grown in minimal M9 glucose medium. Rates are given relative to the mean mutation rate of the wild-type for each type of mutation in minimal M9 glucose medium at day 1 (Table 1). Error bars are SEM.