Inhibiting translation elongation can aid genome duplication in Escherichia coli

Abstract

Conflicts between replication and transcription challenge chromosome duplication. Escherichia coli replisome movement along transcribed DNA is promoted by Rep and UvrD accessory helicases with Δrep ΔuvrD cells being inviable under rapid growth conditions. We have discovered that mutations in a tRNA gene, aspT, in an aminoacyl tRNA synthetase, AspRS, and in a translation factor needed for efficient proline-proline bond formation, EF-P, suppress Δrep ΔuvrD lethality. Thus replication-transcription conflicts can be alleviated by the partial sacrifice of a mechanism that reduces replicative barriers, namely translating ribosomes that reduce RNA polymerase backtracking. Suppression depends on RelA-directed synthesis of (p)ppGpp, a signalling molecule that reduces replication-transcription conflicts, with RelA activation requiring ribosomal pausing. Levels of (p)ppGpp in these suppressors also correlate inversely with the need for Rho activity, an RNA translocase that can bind to emerging transcripts and displace transcription complexes. These data illustrate the fine balance between different mechanisms in facilitating gene expression and genome duplication and demonstrate that accessory helicases are a major determinant of this balance. This balance is also critical for other aspects of bacterial survival: the mutations identified here increase persistence indicating that similar mutations could arise in naturally occurring bacterial populations facing antibiotic challenge.

Figure 1.

A mutation in an aspartyl tRNA gene suppresses the need for accessory replicative helicases and for anti-backtracking factors. (A) Retention or loss of pRC7uvrD (pAM407) from strains without or with Δrep ΔuvrD deletions as judged by blue/white colony color on LB plates containing X-gal and IPTG. Fractions of white colonies are indicated below each image with actual numbers of white versus total colonies counted in parentheses. (B) DNA content of the indicated strains as monitored by flow cytometry under run out conditions in LB. The number of chromosome equivalents per cell is shown below. (C) The viability of greA⁺ greB⁺ versus ΔgreA ΔgreB cells without and with aspT[t8c] as monitored by serial dilutions of liquid cultures grown at 30°C and plated subsequently on LB agar at 30°C and 42°C.

Figure 2.

The impact of aspT[t8c] on growth and its relevance to Δrep ΔuvrD inviability. (A) Growth of (i) aspT⁺efp⁺ (TB28), (ii) aspT[t8c] efp⁺ (KM231) and (iii) aspT⁺ Δefp (MH299) in LB at 37°C as monitored by absorbance at 600 nm. (B) Assessment of the ability of pRC7uvrD (pAM407) to be lost from Δrep ΔuvrD cells by reducing growth rates via culturing at 37°C and 25°C for the indicated times on defined rich medium containing glycerol as a carbon source.

Figure 3.

A mutation in aspartyl tRNA synthetase suppresses Δrep ΔuvrD lethality. (A) The indicated strains were grown overnight in high salt medium at 30°C and then serial dilutions plated onto low and high salt plates containing 100 μg ml⁻¹ ampicillin and incubated at either 30°C or 42°C. (B) Suppression of Δrep ΔuvrD lethality by aspS^P555S on rich medium was analysed by monitoring retention or loss of pRC7uvrD (pAM407) from the indicated strains at 30°C on either low or high salt LB medium.

Figure 4.

Ribosomal pausing decreases the requirement for accessory helicase activity. Retention or loss of pRC7rep (pAM403) was monitored at 37°C on LB X-gal IPTG plates for the indicated strains.

Figure 5.

(p)ppGpp synthesis is needed for suppression of Δrep ΔuvrD lethality by aspT[t8c] and Δefp. (A) Flow cytometric detection of in vivo levels of RpoS-mCherry fluorescence in the indicated strains. All strains contain the rpoS-mCherry fusion with the exception of (i) MG1655. (B) Survival after challenge with ciprofloxacin. The strains are (i) TB28, (ii) KM241, (iii) N5777, (iv) KM231 and (v) MH372. (C) Retention and loss of pRC7uvrD (pAM407) on LB X-gal IPTG agar in relA⁺ and ΔrelA strains.

Figure 6.

Δefp but not aspT[t8c] can bypass the need for wild type Rho activity. (A) and (B) The indicated strains were grown in liquid culture in the absence of bicyclomycin and their ability to continue to divide with reduced Rho activity was assessed after serial dilution onto plates without and with 25 μg ml⁻¹ bicyclomycin. (C) Loss of pRC7rep (pAM403) on LB X-gal IPTG agar in rho⁺ (i, iii, v) and rho[A243E] (ii, iv, vi) strains.

Figure 7.

The ability of Δefp to bypass the requirement for wild type Rho activity is dependent on (p)ppGpp synthesis. (A) and (B) Strains were grown in the absence of bicyclomycin and then serial dilutions were plated onto LB agar without and with 25 μg ml⁻¹ bicyclomycin.