Stationary-phase mutation in the bacterial chromosome: recombination protein and DNA polymerase IV dependence

Abstract

Several microbial systems have been shown to yield advantageous mutations in slowly growing or nongrowing cultures. In one assay system, the stationary-phase mutation mechanism differs from growth-dependent mutation, demonstrating that the two are different processes. This system assays reversion of a lac frameshift allele on an F' plasmid in Escherichia coli. The stationary-phase mutation mechanism at lac requires recombination proteins of the RecBCD double-strand-break repair system and the inducible error-prone DNA polymerase IV, and the mutations are mostly -1 deletions in small mononucleotide repeats. This mutation mechanism is proposed to occur by DNA polymerase errors made during replication primed by recombinational double-strand-break repair. It has been suggested that this mechanism is confined to the F plasmid. However, the cells that acquire the adaptive mutations show hypermutation of unrelated chromosomal genes, suggesting that chromosomal sites also might experience recombination protein-dependent stationary-phase mutation. Here we test directly whether the stationary-phase mutations in the bacterial chromosome also occur via a recombination protein- and pol IV-dependent mechanism. We describe an assay for chromosomal mutation in cells carrying the F' lac. We show that the chromosomal mutation is recombination protein- and pol IV-dependent and also is associated with general hypermutation. The data indicate that, at least in these male cells, recombination protein-dependent stationary-phase mutation is a mechanism of general inducible genetic change capable of affecting genes in the bacterial chromosome.

Figure 1

Chromosomal Tet^R mutation during lactose starvation requires RecA and RuvC. Values are the averaged mutation frequencies from multiple independent experiments (n = 9, 5, and 5 for rec⁺, recA, and ruvC, respectively). Daily measurements of lac⁻ viable cells on the plates (below), shown normalized to the first day's count, show no net growth or death during the experiments (black, rec⁺; gray, recA; white, ruvC). Error bars represent one SEM. The strains are SMR4576, SMR4608, and SMR4822.

Figure 2

Chromosomal stationary-phase Tet^R mutation increases in recD and recG cells, RecA and RuvC dependently. (A) The strains are SMR4576, SMR4721, SMR4727, and SMR4823. (B) The strains are SMR4576, SMR4733, SMR5790, SMR4758, SMR6048, and SMR6047. radC102 is a point mutation in the recG gene (77, 78). Use of this allele was necessary to make the recG ruvC combination. Values are the averaged mutation frequencies from multiple independent experiments: (A) n = 9, 6, 3, and 3 for rec⁺, recD, recD recA, and recD ruvC, respectively; (B) n = 9, 4, 3, 2, 2, and 3 for rec⁺, recG263, radC102, recG263 recA, radC102 ruvC, and ruvC, respectively). The rec⁺ data are those shown in Figs. 1 and 4. Error bars represent one SEM except for those in which n = 2 in which the error bars show the range. Mutant frequencies were determined as described in Materials and Methods except for two differences for the radC102 ruvC strain. First, only five cultures were tested in each experiment rather than six, and second, no Tet^R colonies were observed in any of the five cultures tested on day 0 in one experiment. The same is true for days 1 and 3 in the other experiment. For those days the value was calculated as if a single colony had been observed in one of the five cultures and that value was used in calculating the average shown, which is thus a “less than” value. Daily measurements of lac⁻ viable cells on the plates (below), shown normalized to the first day's count, show no net growth or death during the experiments: (A) □, rec⁺; ◊, recD; ○, recD ruvC; ▵, recD recA. (B) □, rec⁺; ◊, recG263; ○, recG ruvA; ▵, ruvC53; crossed squares, radC102; X, radC102 ruvC53.

Figure 3

Tet^R and Lac⁺ stationary-phase mutant colonies are randomly distributed on plates. We find that Tet^R (white) colonies are not satellites of the Lac⁺ (blue) mutant colonies, as would have been predicted if the Tet^R mutations were formed during growth of cells next to Lac⁺ colonies (caused by cross-feeding, discussed in text). A and B are day 3 plates of rec⁺ (SMR4576) and recG (SMR4733), respectively.

Figure 4

SOS-inducible DNA polymerase IV is required for Tet^R stationary-phase mutation in the bacterial chromosome. The strains are SMR4576 and SMR6049. Values are the averaged mutation frequencies from multiple experiments (n = 9 and 4 for din⁺ and dinB10, respectively). The din⁺ data are those shown in Figs. 1 and 2 (rec⁺). Daily measurements of lac⁻ viable cells on the plates (below), shown normalized to the first day's count, show no net growth or death during the experiments (black, din⁺; gray, dinB10). Error bars represent one SEM.