A ΔdinB mutation that sensitizes Escherichia coli to the lethal effects of UV- and X-radiation

Abstract

The DinB (PolIV) protein of Escherichia coli participates in several cellular functions. We investigated a dinB mutation, Δ(dinB-yafN)883(::kan) [referred to as ΔdinB883], which strongly sensitized E. coli cells to both UV- and X-radiation killing. Earlier reports indicated dinB mutations had no obvious effect on UV radiation sensitivity which we confirmed by showing that normal UV radiation sensitivity is conferred by the ΔdinB749 allele. Compared to a wild-type strain, the ΔdinB883 mutant was most sensitive (160-fold) in early to mid-logarithmic growth phase and much less sensitive (twofold) in late log or stationary phases, thus showing a growth phase-dependence for UV radiation sensitivity. This sensitizing effect of ΔdinB883 is assumed to be completely dependent upon the presence of UmuDC protein; since the ΔdinB883 mutation did not sensitize the ΔumuDC strain to UV radiation killing throughout log phase and early stationary phase growth. The DNA damage checkpoint activity of UmuDC was clearly affected by ΔdinB883 as shown by testing a umuC104 ΔdinB883 double-mutant. The sensitivities of the ΔumuDC strain and the ΔdinB883 ΔumuDC double-mutant strain were significantly greater than for the ΔdinB883 strain, suggesting that the ΔdinB883 allele only partially suppresses UmuDC activity. The ΔdinB883 mutation partially sensitized (fivefold) uvrA and uvrB strains to UV radiation, but did not sensitize a ΔrecA strain. A comparison of the DNA sequences of the ΔdinB883 allele with the sequences of the Δ(dinB-yafN)882(::kan) and ΔdinB749 alleles, which do not sensitize cells to UV radiation, revealed ΔdinB883 is likely a "gain-of-function" mutation. The ΔdinB883 allele encodes the first 54 amino acids of wild-type DinB followed by 29 predicted residues resulting from the continuation of the dinB reading frame into an adjacent insertion fragment. The resulting polypeptide is proposed to interfere directly or indirectly with UmuDC function(s) involved in protecting cells against the lethal effects of radiation.

Keywords: Checkpoint; DinB; Escherichia coli; TLS; UV radiation sensitivity; UmuDC.

Fig 1

The ΔdinB883 allele reduces cell survival after exposure to radiation. (A) Survival data are shown for logarithmic phase E. coli wild-type (SR2227, ●) and ΔdinB883 (SR4109, ○) strains after UV radiation exposure in buffer. (B) Same format as in A, except strains were irradiated with 160 kV X-rays, aerobically. Data are means ± SD from triplicate experiments.

Fig. 2

The ΔdinB883 allele does not sensitize a ΔumuDC strain to UV radiation. Growth and survival data are shown for E. coli wild-type (SR2227), ΔdinB883 (SR4109), ΔumuDC (SR4128), and ΔdinB883 ΔumuDC (SR4129) strains incubated overnight, diluted (1:50) into fresh LB broth supplemented with 0.1% glucose and incubated at 37ºC with aeration. Samples were taken at times indicated to determine values for OD₅₅₀ and survival following UV radiation exposure at 25 J m^-2. Cells were irradiated on tryptone plates. Survival data are means ± SD from triplicate experiments. Since OD₅₅₀ values were nearly identical for all four strains, only average values are shown.

Fig. 3

The ΔdinB883 allele sensitizes uvrA and uvrB strains to UV radiation. Survival data are shown for logarithmic phase E. coli uvrA (SR4172, ●), uvrA ΔdinB883 (SR4173, ○), uvrB (SR4175, ▼) and uvrB ΔdinB883 (SR4177, Δ) strains UV-irradiated in buffer. Data are means ± SD from triplicate experiments. The survival curves for ΔdinB883 strains are significantly different from those for paired dinB+ strains over the range of 9-15 J m^-2 (P<0.05).

Fig. 4

The ΔdinB883 allele does not sensitize a ΔrecA strain to UV radiation. Survival data are shown for logarithmic phase E. coli ΔrecA (SR4178, ●) and ΔrecA ΔdinB883 (SR4179, ○) strains UV-irradiated in buffer. Data are means ± SD from triplicate experiments. The two survival curves shown are not significantly different (P>0.05).

Fig. 5

The ΔdinB883 allele sensitizes an umuC104 strain to UV radiation. Survival data are shown for logarithmic phase E. coli umuC104 lexA51 (SR4385, ●) and umuC104 lexA51 ΔdinB883 (SR4391, ○) strains UV-irradiated in buffer. Data are means ± SD from triplicate experiments.

Fig. 6

The ΔdinB883 allele uniquely has the ability to sensitize wild-type cells to UV radiation at 25 J m^-2. Survival data is shown for E. coli strains; dinB⁺ (SMR4562 and SR2227), ΔdinB882 (SMR6111), ΔdinB749 (JW0221-1) and ΔdinB883 (SR4109). Cells were prepared and UV-irradiated to determine survival as in Fig. 2. Data are means ± SD from 5-10 experiments per strain. Strain SR4109 is significantly more sensitive than all other strains (P<0.05).

Fig. 7

DNA and amino acid (AA) sequences for wild-type (dinB⁺) and ΔdinB mutant strains. DNA and predicted fusion polypeptide sequences are shown for dinB⁺ (wild-type, SR2227, not UV radiation sensitive), ΔdinB883 (SR4109, significantly UV radiation sensitive), and ΔdinB882 (SMR6111, not UV radiation sensitive) strains. The nucleotide sequences are identical for the three strains for the first 53 codons. Codon 54 differs in the ΔdinB883 and ΔdinB882 strains from the dinB⁺ DNA sequence but the AA remains the same. After codon 54 the dinB⁺ DNA sequence and resulting AAs differs from those of ΔdinB883 and ΔdinB882. From codon positions 55 to 61, the AA sequences are identical for ΔdinB883 and ΔdinB882 as are the DNA sequences except for codon 61. After codon 61 the DNA and AA sequences both differ in the two ΔdinB strains. The nucleotides of the kan^r insertion fragment fused to ΔdinB sequence begin opposite AA position 54 (3^rd position), but the AA sequence is not affected until AA position 55.