Mutations Affecting Potassium Import Restore the Viability of the Escherichia coli DNA Polymerase III holD Mutant

International audienceMutants lacking the ψ (HolD) subunit of the Escherichia coli DNA Polymerase III holoenzyme (Pol III HE) have poor viability, but a residual growth allows the isolation of spontaneous suppressor mutations that restore ΔholD mutant viability. Here we describe the isolation and characterization of two suppressor mutations in the trkA and trkE genes, involved in the main E. coli potassium import system. Viability of ΔholD trk mutants is abolished on media with low or high K+ concentrations, where alternative K+ import systems are activated, and is restored on low K+ concentrations by the inactivation of the alternative Kdp system. These findings show that the ΔholD mutant is rescued by a decrease in K+ import. The effect of trk inactivation is additive with the previously identified ΔholD suppressor mutation lexAind that blocks the SOS response indicating an SOS-independent mechanism of suppression. Accordingly, although lagging-strand synthesis is still perturbed in holD trkA mutants, the trkA mutation allows HolD-less Pol III HE to resist increased levels of the SOS-induced bypass polymerase DinB. trk inactivation is also partially additive with an ssb gene duplication, proposed to stabilize HolD-less Pol III HE by a modification of the single-stranded DNA binding protein (SSB) binding mode. We propose that lowering the intracellular K+ concentration stabilizes HolD-less Pol III HE on DNA by increasing electrostatic interactions between Pol III HE subunits, or between Pol III and DNA, directly or through a modification of the SSB binding mode; these three modes of action are not exclusive and could be additive. To our knowledge, the holD mutant provides the first example of an essential protein-DNA interaction that strongly depends on K+ import in vivo

K⁺ uptake systems in <i>E</i>. <i>coli</i>.

<p>(Adapted from [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006114#pgen.1006114.ref021" target="_blank">21</a>] and [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006114#pgen.1006114.ref028" target="_blank">28</a>]).</p

<i>trkA</i> inactivation does not rescue the Δ<i>holD</i> mutant by affecting SOS induction or SSB binding.

<p>Isolated colonies obtained on M9 as described in the legend of <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006114#pgen.1006114.g002" target="_blank">Fig 2</a> were suspended in MK0 salts and 5 μl drops of serial dilutions were plated on M9 and LB (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006114#pgen.1006114.s005" target="_blank">S5 Fig</a> for growth on MK1, MK22, MK115). LB plates were incubated overnight at 37°C and 42°C, and for two days at 30°C. M9 casamino acids plates were incubated for two days at all temperatures. The strains used here to generate plasmid-less colonies are: <i>holD</i> Δ<i>trkA</i>::cm, JJC6898; <i>holD lexAind</i>, JJC6420; <i>holD lexAind</i> Δ<i>trkA</i>::cm, JJC7008; <i>holD recF</i>, JJC7058; <i>holD</i> Δ<i>trkA</i>::cm <i>recF</i>, JJC7063; <i>holD argE</i>::<i>ssb</i>, JJC6394; <i>holD argE</i>::<i>ssb</i> Δ<i>trkA</i>::cm, JJC7000.</p

<i>trkA</i> gene inactivation decreases <i>E</i>. <i>coli</i> intracellular K⁺ concentration.

<p><i>trkA</i> gene inactivation decreases <i>E</i>. <i>coli</i> intracellular K⁺ concentration.</p

The <i>holD trkA</i> mutant is resistant to an increased level of DinB, but less than wild-type.

<p>Wild-type, (JJC6534), <i>lexADef</i> (JJC6904), <i>holD</i> Δ<i>trkA</i>::cm (JJC6927) and <i>holD</i> Δ<i>trkA</i>::<i>cm lexAind</i> (JJC7004) strains were transformed with plasmids pGB2 (plain bars), pGB-DinB (dotted bars) and pGB-DinBΔC5 (hatched bars). Transformants were selected on M9 spc plates. For each experiment, the number of pGB-DinB and pGB-DinBΔC5 transformants was normalized to the number of pGB2 transformants and the averages of three independent experiments are shown. Colonies obtained with pGB-DinB in the <i>holD trkA lexAind</i> mutant were smaller than pGB2 and pGB-DinBΔC5 colonies in the same strain, and than pGB-DinB colonies in wild-type and LexADef strains.</p

Viability of the <i>holD trkA</i> mutant in liquid medium.

<p>Overnight cultures of wild-type (JJC1392), <i>trkA</i> (JJC6800) and <i>holD trkA</i> [pAM-holD] (JJC6898) strains were grown at 30°C in LB, M9, MK1, MK115 medium, diluted, and further grown at 37° in the same medium for 7 hours, as described in the Materials and Methods. (A) Representative growth curves of <i>holD trkA</i> (JJC6898) in LB (full line, diamond), M9 (full line, square), MK1 (dashed line, circles), MK115 (dashed line, triangles). (B) The doubling time expressed in minutes was calculated from the exponential part of the growth curve, averages of three independent determinations are shown with standard deviations. No generation time could be calculated for the <i>holD trkA</i> mutant grown in MK115 and in MK1, as growth at 37°C stopped after 2 to 3 generations (NA = not applicable).</p

Viability of Δ<i>holD trkA</i> and Δ<i>holD trkE</i> mutants is abolished by a high K⁺ concentration, and in a Kdp-dependent way at a low K⁺ concentration.

<p>In a first step, isolated colonies were obtained by plating appropriate dilutions of wild-type, Δ<i>trkA</i>, <i>holD trkE sup</i> or Δ<i>trkA</i> Δ<i>kdp</i> overnight cultures on M9, or appropriate dilutions of pAM-holD containing cultures grown for 8 hours in the absence of IPTG at 30°C, and incubating plates for 3 days at 30°C. These isolated colonies were suspended in 1 ml MK0 salt medium. Serial dilutions were made and 5 μl drops of each dilution were plated on MK plates containing different K⁺ concentrations as indicated (MK1 1 mM, MK22 22 mM, MK115 115 mM; see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006114#pgen.1006114.s002" target="_blank">S2 Fig</a> for growth on MK0.2, M9, MMA, and LB). Pictures of wild-type and Δ<i>trkA</i> strains at 37°C were taken after 24 h incubation; all other plates were incubated for two days at the indicated temperatures. Wild-type, JJC1392; Δ<i>trkA</i>::cm, JJC6800; <i>holD trkE sup</i>, JJC6389 (the original suppressed clone); Δ<i>trkA</i>⁸⁴ Δ<i>kdp</i>::cm, JJC7030. The strains used to generate plasmid-less colonies are: <i>holD</i>, JJC6869; <i>holD</i> Δ<i>trkA</i>::cm, JJC6898; <i>holD</i> Δ<i>trkA</i>⁸⁴, JJC6969; <i>holD</i> Δ<i>kdp</i>::cm JJC6750; <i>holD</i> Δ<i>trkA</i>⁸⁴ Δ<i>kdp</i>::cm, JJC6819; <i>holD</i> Δ<i>trkE</i>::<i>kan</i>, JJC7173; <i>holD</i> Δ<i>trkE</i>::kan Δ<i>kdp</i>::cm, JJC7223.</p

<i>trkA</i> inactivation restores Δ<i>holC</i> and Δ<i>holC</i> Δ<i>holD</i> viability.

<p>Isolated colonies obtained on M9 as described in the legend of <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006114#pgen.1006114.g002" target="_blank">Fig 2</a> were suspended in MK0 salts and 5 μl drops of serial dilutions were plated on the indicated medium (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006114#pgen.1006114.s004" target="_blank">S4 Fig</a> for growth on MK1, MK22 and MK115). LB plates were incubated overnight at 37°C and 42°C, and for two days at 30°C. M9 casamino acids plates were incubated for two days at all temperatures. The strains used to generate plasmid-less colonies are: <i>holC</i>, JJC6748; <i>holC</i> Δ<i>trkA</i>⁸⁴, JJC6827; <i>holC holD</i>, JJC6774<i>; holC holD</i> Δ<i>trkA</i>⁸⁴, JJC6828.</p

The <i>holD trkA</i> mutant is sensitive to RNaseH overexpression.

<p>The <i>holD trkA</i> mutant is sensitive to RNaseH overexpression.</p