Competitive fitness during feast and famine: how SOS DNA polymerases influence physiology and evolution in Escherichia coli

Abstract

Escherichia coli DNA polymerases (Pol) II, IV, and V serve dual roles by facilitating efficient translesion DNA synthesis while simultaneously introducing genetic variation that can promote adaptive evolution. Here we show that these alternative polymerases are induced as cells transition from exponential to long-term stationary-phase growth in the absence of induction of the SOS regulon by external agents that damage DNA. By monitoring the relative fitness of isogenic mutant strains expressing only one alternative polymerase over time, spanning hours to weeks, we establish distinct growth phase-dependent hierarchies of polymerase mutant strain competitiveness. Pol II confers a significant physiological advantage by facilitating efficient replication and creating genetic diversity during periods of rapid growth. Pol IV and Pol V make the largest contributions to evolutionary fitness during long-term stationary phase. Consistent with their roles providing both a physiological and an adaptive advantage during stationary phase, the expression patterns of all three SOS polymerases change during the transition from log phase to long-term stationary phase. Compared to the alternative polymerases, Pol III transcription dominates during mid-exponential phase; however, its abundance decreases to <20% during long-term stationary phase. Pol IV transcription dominates as cells transition out of exponential phase into stationary phase and a burst of Pol V transcription is observed as cells transition from death phase to long-term stationary phase. These changes in alternative DNA polymerase transcription occur in the absence of SOS induction by exogenous agents and indicate that cell populations require appropriate expression of all three alternative DNA polymerases during exponential, stationary, and long-term stationary phases to attain optimal fitness and undergo adaptive evolution.

Keywords: Escherichia coli; GASP; Pol II (polB), Pol IV (dinB); Pol V (umuDC); SOS polymerase; alternative DNA polymerase; chemostat; stationary phase; translesion synthesis.

Figure 1

Type 1 and type 2 competition experiments between double-mutant strains. Representative type 1 low initial cell density (A–C) and type 2 high initial cell density (E–G) competitions between unaged polymerase-deficient strains: red lines, Pol II⁺-only; green lines, Pol IV⁺-only; blue lines, Pol V⁺-only. Color-coded Roman numerals in each panel refer to the polymerases expressed in each pair of mutant strain competitions. Three representative competitions are shown where squares, circles, and triangles indicate competition pairs. Competition data, summarized in D and H, reflect the results of nine competition experiments for each of the three pairings of mutant strains. The “>” sign indicates that the strain listed on the left was the “winner”; the “<” sign on the right indicates that the strain listed on the right was the “winner.” One strain outcompeting the other is defined by a >10-fold difference in cell yield on day 14. When final yields are within 10-fold, no winners were declared as reflected by the “=” sign. Asterisks indicate that cell titers were below the limit of detection (<1000 CFU/ml.)

Figure 2

GASP competitions between wild-type and double-mutant strains. Polymerase double-mutant strains were aged for 10 days and competed to determine their GASP phenotypes against unaged wild-type strains (A–C) or each unaged polymerase mutant strain (D–I) in all possible combinations. Solid symbols correspond to strains aged for 10 days; open symbols correspond to unaged strains. Strains are indicated by line color: wild type, black; Pol II⁺-only, red; Pol IV⁺-only, green; Pol V⁺-only, blue. Unaged wild-type cells were competed against 10-day-old (A) Pol II⁺-only, (B) Pol IV⁺-only, or (C) Pol V⁺-only. Aged Pol II⁺-only strains were competed against unaged (D) Pol IV⁺-only or (G) Pol V⁺-only; aged Pol IV⁺-only strains were competed against unaged (E) Pol II⁺-only or (H) Pol V⁺-only; and aged Pol V⁺-only strains were competed against unaged (F) Pol II⁺-only or (I) Pol IV⁺-only. Three representative competitions are shown for each pair where squares, circles, and triangles indicate competition pairs. Asterisks indicate that titers were below the limit of detection (<1000 CFU/ml.) Color-coded Roman numerals in each panel refer to the polymerases expressed in each pair of mutant strain competitions.

Figure 3

Increased relative fitness of Pol II⁺-only mutants following serial passage. Populations of unaged Pol IV⁺-only (green lines, A–E) or unaged Pol V⁺-only (blue lines, F–J) were each competed against the Pol II⁺-only strain (red lines) after one to five serial passages. (A and F) Competition between the Pol II⁺-only strain with no additional passage. (B and G) Competitions after one additional passage of Pol II⁺-only. (C and H) Two additional passages of Pol II⁺-only. (D and I) Three additional passages of Pol II⁺-only. (E and J) Five additional passages of Pol II⁺-only. The average relative log₁₀ ratio of final cell densities between Pol II⁺-only vs. Pol IV⁺-only (green bars) or Pol V⁺-only (blue bars) for all six conditions is plotted in K.

Figure 4

Relative fitness during chemostat competitions. The relative fitness of Pol II⁺-only compared to Pol IV⁺-only (in green) and Pol V⁺-only (in blue) during continuous culture competitions is shown for chemostats run with different dilution rates (volumes per hour).

Figure 5

Strain-specific Rif^R mutation frequency and spectrum. The frequency of spontaneous rifampicin resistance was determined for 159 independent replicates of the wild-type and each polymerase mutant strain. Mutation frequencies are presented in ascending order. (A) Mutation frequencies for the wild-type (black), all three double-mutant strains (Pol II⁺-only, red; Pol IV⁺-only, green; Pol V⁺-only, blue), and the triple mutant (gray). (B) Mutation frequencies for the wild-type (black), all three single-mutant strains (Pol II⁻, red; Pol IV⁻, green; Pol V⁻, blue), and the triple mutant (gray). (C) Average Rif^R frequencies for the wild type, all three single-mutant strains, all three single- and double-mutant strains, and the triple mutant. Error bars denote ±SEM. (D) For all eight strains, each class of mutation is presented as a percentage of all mutations observed. Detailed mutation data are presented in Table S1, Table S2, and Table S3.

Figure 6

Alternative polymerase transcript abundance changes over the cell cycle. Messenger RNA transcript abundance, in the absence of exogenous SOS inducers, was determined by qRT-PCR. (A) Transcript abundance for each gene relative to its concentration at 2 hr of incubation. Genes are identified as the following: polB, red, open circles; dinB, green, open triangles; umuC, blue, open squares; umuD, blue, solid squares; dnaE, black diamonds; sulA, purple, solid circles; and lexA, orange, solid triangles. (B) The proportion of polymerase transcripts over time, expressed as a percentage of total transcript abundance, is shown for four representative transcripts: Pol III, dnaE, black diamonds; Pol IV, dinB, green triangles; Pol II, polB, red circles; Pol V, umuC, blue squares.