A dnaN plasmid shuffle strain for rapid in vivo analysis of mutant Escherichia coli β clamps provides insight into the role of clamp in umuDC-mediated cold sensitivity

Abstract

The E. coli umuDC gene products participate in two temporally distinct roles: UmuD2C acts in a DNA damage checkpoint control, while UmuD'2C, also known as DNA polymerase V (Pol V), catalyzes replication past DNA lesions via a process termed translesion DNA synthesis. These different roles of the umuDC gene products are managed in part by the dnaN-encoded β sliding clamp protein. Co-overexpression of the β clamp and Pol V severely blocked E. coli growth at 30°C. We previously used a genetic assay that was independent of the ability of β clamp to support E. coli viability to isolate 8 mutant clamp proteins (βQ61K, βS107L, βD150N, βG157S, βV170M, βE202K, βM204K and βP363S) that failed to block growth at 30°C when co-overexpressed with Pol V. It was unknown whether these mutant clamps were capable of supporting E. coli viability and normal umuDC functions in vivo. The goals of this study were to answer these questions. To this end, we developed a novel dnaN plasmid shuffle assay. Using this assay, βD150N and βP363S were unable to support E. coli viability. The remaining 6 mutant clamps, each of which supported viability, were indistinguishable from β+ with respect to umuDC functions in vivo. In light of these findings, we analyzed phenotypes of strains overexpressing either β clamp or Pol V alone. The strain overexpressing β+, but not those expressing mutant β clamps, displayed slowed growth irrespective of the incubation temperature. Moreover, growth of the Pol V-expressing strain was modestly slowed at 30°, but not 42°C. Taken together, these results suggest the mutant clamps were identified due to their inability to slow growth rather than an inability to interact with Pol V. They further suggest that cold sensitivity is due, at least in part, to the combination of their individual effects on growth at 30°C.

Figure 1. Summary of the positions of β clamp mutations.

Shown are (A) front and (B) side views of the β clamp on DNA (PDB: 3BEP). Amino acid positions bearing substitutions that failed to confer cold sensitive growth when co-overexpressed with Pol V are represented as red sticks in the green clamp protomer. The two residues mutated in the dnaN159(Ts) allele (β159; G66→E and G174→A) are indicated as red spacefill in the blue clamp protomer. Loops 1–3 of clamp are higlighted in orange in the blue clamp protomer; loops 1 and 2 contacted DNA in the crystal , . The grey ovals represent the approximate location of the hydrophobic cleft present in each clamp protomer that contacts the CBM located in most, if not all clamp partners. This image was generated using PyMOL v1.5.0.2.

Figure 2. Design of the dnaN^–1FS allele and its use in the plasmid shuffle assay.

(A) Genomic structure of the dnaA-dnaN-recF operon. Genes in grey are essential for cell viability, while those in white are non-essential. Blackened triangles represent approximate positions of confirmed promoters, based on EcoGene 3.0 (http://www.ecogene.org). Gross structure of the dnaA–dnaN^–1FS–tet–recF cassette is depicted below. ΔXhoI represents the approximate location of the –1 frameshift mutation present within the dnaN ^–1FS allele. The dnaN ^–1FS allele is predicted to express a protein of 134 residues: the N-terminal 49 residues are identical to the wild-type β clamp protein (white), while the C-terminal 85 residues are distinct and result from the −1 frameshift mutation (light grey). The majority of the dnaN^–1FS allele is not translated (black), due to the premature stop codon at position 135 resulting from the altered reading frame. Relative positions of oligonucleotide primer pairs (see Table 1) used for diagnostic PCR amplification or nucleotide sequence analysis are shown. Expected sizes (in bp) for products of PCR amplified fragments using the noted primer pairs are indicated. (B) The MS201 strain contains dnaN^–1FS allele on its chromosome, and bears the Amp^R plasmid pAMPdnaN⁺, which expresses physiological levels of wild type β clamp that supports viability. After transforming strain MS201 to Cam^R with pACM/pACM-derivatives containing the indicated dnaN allele, representative pAMPdnaN⁺ and pACM (or pACM derivative) double transformants are passaged for ∼100 generations before patching onto LB-Amp and LB-Cam plates to score for pAMPdnaN⁺ retention (i.e., Amp^R). If the mutant clamp expressed from the pACM plasmid can support viability, pAMPdnaN⁺ is lost, and cells display an Amp^S Cam^R phenotype. If the mutant clamp expressed from pACM cannot support viability, the wild type clamp-expressing plasmid pAMPdnaN⁺ is retained, and cells display an Amp^R Cam^R phenotype. As controls for strains that readily lost pAMPdnaN⁺, we verified the nucleotide sequence of the plasmid encoded dnaN allele, as well as the structure of the chromosomal dnaN^–1FS allele (see Materials and Methods).

Figure 3. Respective abilities of mutant β clamp proteins to support DNA damage-induced mutagenesis.

Frequencies of (A) UV- or (B) MMS-induced mutagenesis were measured as described in Material and Methods using strains RW118 (WT; dnaN⁺ umuD⁺C⁺), RW120 (ΔumuD; dnaN⁺ ΔumuDC595::cat), or the umuD⁺C⁺ plasmid shuffle strains MS202 (β⁺), MS203 (β^Q61K), MS204 (β^S107L), MS205 (β^G157S), MS206 (β^V170M), MS207 (β^E202K) and MS208 (β^M204K), as indicated. Results represent the average of 5 independent determinations. Error bars represent one standard deviation. P-values ≤0.05 are indicated, and were calculated using the Student's t-test.

Figure 4. Ability of the mutant clamp strains to survive UV irradiation.

(A) UV sensitivity of isogenic dnaN⁺ umuD⁺C⁺ (RW118) and dnaN⁺ ΔumuDC (RW120) strains was measured as described in Materials and Methods. (B) UV sensitivity of the umuD⁺C⁺ plasmid shuffle strains MS202 (β⁺), MS203 (β^Q61K), MS204 (β^S107L), MS205 (β^G157S), MS206 (β^V170M), MS207 (β^E202K) and MS208 (β^M204K) was measured similarly. These experiments were performed in triplicate; results from one representative experiments are shown.

Figure 5. Effect of overexpression of β clamp on growth of AB1157.

Representative images of LB agar plates showing pBR322 (control) or pJRC210 (β⁺) transformants of strain AB1157 following incubation at (A) 30° or (B) 42°C, as noted. (C) Diameters of representative CFUs shown in panels A and B were measured as described in Materials and Methods. The asterisk (*) indicates strains whose average colony diameter was below the measurement limit of 0.2 mm. Error bars represent one standard deviation. P-values ≤0.05 are indicated, and were calculated using the Student's t-test. (D) Shown are representative images of M9 agar plates of pBR322 (control) or pJRC210 (β⁺) transformants following incubation for 16 hrs at either 30° or 42°C. Colony diameters in panel D were not measured due to the small size of the pJRC210 transformants (i.e., diameters were <0.2 mm). Each transformation experiment was performed at least 3 independent times; results from one representative experiment are shown.

Figure 6. Effect of overexpression of the different umuDC gene products on growth of AB1157.

Average colony diameters of pGB2 (control), pGY9739 (UmuD₂C) or pGY9738 (UmuD'₂C) transformants of strain AB1157 following growth at either 30°C or 42°C, as noted, are shown. No colonies were observed for the AB1157 pGY9739 transformant. Experiments were performed at least twice. Error bars represent one standard deviation. P-values ≤0.05 are indicated, and were calculated using the Student's t-test.

Figure 7. Effect of overexpression of different mutant clamps on growth of AB1157.

(A) Shown are representative images of LB agar plates of AB1157 transformants following 18 hrs of growth at 30°C using the indicated plasmids. (B) Colonies were measured as described in Materials and Methods, and their respective sizes are represented relative to that observed for the AB1157(pBR322) control strain, which was set equal to 1.0. The asterisk (*) indicates strains whose average colony diameter was below the measurement limit of 0.2 mm. Experiments were performed at least twice. Error bars represent one standard deviation. P-values ≤0.05 are indicated, and were calculated using the Student's t-test.

Figure 8. Mutant clamps confer resistance to HU.

HU sensitivity was measured as described in Material and Methods using plasmid shuffle strains MS202 (β⁺), MS203 (β^Q61K), MS204 (β^S107L), MS205 (β^G157S), MS206 (β^V170M), MS207 (β^E202K) and MS208 (β^M204K). This experiment was performed at least twice; results from one representative experiment are shown.