Distinguishing Characteristics of Hyperrecombinogenic RecA Protein from Pseudomonas aeruginosa Acting in Escherichia coli

In Escherichia coli, a relatively low frequency of recombination exchanges (FRE) is predetermined by the activity of RecA protein, as modulated by a complex regulatory program involving both autoregulation and other factors. The RecA protein of Pseudomonas aeruginosa (RecA(Pa)) exhibits a more robust recombinase activity than its E. coli counterpart (RecA(Ec)). Low-level expression of RecA(Pa) in E. coli cells results in hyperrecombination (an increase of FRE) even in the presence of RecA(Ec). This genetic effect is supported by the biochemical finding that the RecA(Pa) protein is more efficient in filament formation than RecA K72R, a mutant protein with RecA(Ec)-like DNA-binding ability. Expression of RecA(Pa) also partially suppresses the effects of recF, recO, and recR mutations. In concordance with the latter, RecA(Pa) filaments initiate recombination equally from both the 5′ and 3′ ends. Besides, these filaments exhibit more resistance to disassembly from the 5′ ends that makes the ends potentially appropriate for initiation of strand exchange. These comparative genetic and biochemical characteristics reveal that multiple levels are used by bacteria for a programmed regulation of their recombination activities

Two RecA Protein Types That Mediate Different Modes of Hyperrecombination▿

RecAX53 is a chimeric variant of the Escherichia coli RecA protein (RecAEc) that contains a part of the central domain of Pseudomonas aeruginosa RecA (RecAPa), encompassing a region that differs from RecAEc at 12 amino acid positions. Like RecAPa, this chimera exhibits hyperrecombination activity in E. coli cells, increasing the frequency of recombination exchanges per DNA unit length (FRE). RecAX53 confers the largest increase in FRE observed to date. The contrasting properties of RecAX53 and RecAPa are manifested by in vivo differences in the dependence of the FRE value on the integrity of the mutS gene and thus in the ratio of conversion and crossover events observed among their hyperrecombination products. In strains expressing the RecAPa or RecAEc protein, crossovers are the main mode of hyperrecombination. In contrast, conversions are the primary result of reactions promoted by RecAX53. The biochemical activities of RecAX53 and its ancestors, RecAEc and RecAPa, have been compared. Whereas RecAPa generates a RecA presynaptic complex (PC) that is more stable than that of RecAEc, RecAX53 produces a more dynamic PC (relative to both RecAEc and RecAPa). The properties of RecAX53 result in a more rapid initiation of the three-strand exchange reaction but an inability to complete the four-strand transfer. This indicates that RecAX53 can form heteroduplexes rapidly but is unable to convert them into crossover configurations. A more dynamic RecA activity thus translates into an increase in conversion events relative to crossovers

Structure of RecX protein complex with the presynaptic RecA filament: Molecular dynamics simulations and small angle neutron scattering

Using molecular modeling techniques we have built the full atomic structure and performed molecular dynamics simulations for the complexes formed by Escherichia coli RecX protein with a single-stranded oligonucleotide and with RecA presynaptic filament. Based on the modeling and SANS experimental data a sandwich-like filament structure formed two chains of RecX monomers bound to the opposite sides of the single stranded DNA is proposed for RecX::ssDNA complex. The model for RecX::RecA::ssDNA include RecX binding into the grove of RecA::ssDNA filament that occurs mainly via Coulomb interactions between RecX and ssDNA. Formation of RecX::RecA::ssDNA filaments in solution was confirmed by SANS measurements which were in agreement with the spectra computed from the molecular dynamics simulations

Effects of RecX proteins on the growth curves of cells expressing wild type RecA protein or RecA D112R protein.

<p>Cells containing pRecA Ec or pRecA [D112R] were grown in LB with Ampicillin 100mkg/ml. Where indicated, the cells contained plasmid pEAW847, expressing the RecX and RecA proteins from <i>E</i>.<i>coli</i>, pEAW858 –RecX and RecA D112R proteins from <i>E</i>. <i>coli</i>, pEAW959 –RecA from <i>E</i>.<i>coli</i> and RecX_NG, or pEAW958 –RecA D112R and RecX_NG.</p

Effect of RecX protein expression on measured FRE levels in strains expressing RecA or RecA D112R proteins.

<p>The strain used is JC10289 (AB1157 ΔrecA) engineered with a deletion of the araBAD operon as described in Materials and Methods. Plasmids were constructed to express both the indicated RecA (from the <i>tac</i> promoter, uninduced) and the indicated RecX (from the <i>ara</i> promoter, with arabinose added as an inducer as indicated) proteins.</p

Growth disadvantage conferred by RecA D112R.

<p>(A) Growth curves for <i>E</i>. <i>coli</i> cells expressing either the wild type RecA protein (red circles) from plasmid pRecA or the RecA D112R variant (black squares) from plasmid pRecA[D112R]. (B) Cell growth competition assays. Assays were carried out as described in Methods. The top trial (red circles) shows a competition between two wild type cultures, one of which carries the Ara⁻mutation. The lower one shows a competition between wild type cells and cells expressing RecA D112R at the normal chromosomal <i>recA</i> locus. The <i>ara</i>^<i>−</i>mutation conferring a red color to colonies is present in the cells expressing wild type RecA in this experiment. Colony counts revealing the % of cells expressing the mutant RecA protein are plotted as a function of the daily growth cycle of the experiment. (C) Resistance to ultraviolet light. Experiments were carried out as described in Materials and Methods. The strains compared are MG1655 and EAW166 (RecA D112R expressed on the chromosome at the normal <i>recA</i> locus).</p

RecA protein expression in cells that have lost the hyperrec phenotype.

<p>Intracellular RecA protein was measured by Western blotting as described in Materials and Methods. Each lane reflects the RecA protein content of 5 X 10⁷ cells. Lane 1 is a marker. Lanes 2 and 3 represent expression of either EcRecA (lane 2) or RecA D112R (lane 3) from their respective plasmids controlled by the <i>tac</i> promoter in the absence of induction. Lane 4 shows an empty vector control. Lanes 5–8 show results from de-evolved strains 7, 8, 12, and 13 respectively as labeled.</p

Loss of the hyperrec phenotype in strains expressing RecA D112R protein.

<p>The second column documents ΔFRE, the relative increase in the frequency of genetic exchange per chromosomal DNA unit length (ΔFRE is a ratio of the FRE observed with the indicated strain divided by FRE for comparable cells expressing the wild type RecA protein), after continuous culture for 70 cell generations. The third column shows results obtained after the plasmids from the cultured strains were isolated and used to again transform strain JC10289. Four of the plasmids (from cultures 8, 11, 12, and 13) still produce an elevated ΔFRE. In the final column, the strains were cured of their resident plasmid, and re-transformed with pRecA D112R. The lower ΔFRE scores in some of these strains reflect the effects of chromosomal mutations.</p

Loss of hyperrec phenotype in cells expressing RecA D112R upon continuous culture.

<p>Multiple cultures of strain JС10289, expressing RecA D112R protein from plasmid pRecA[D112R] were grown as described in Materials and Methods for 70 generations. ΔFRE was determined for each culture at the indicated times, and is the ratio of the FRE value measured for JC10289 with pRecA[D112R] to the FRE value for JC10289 expressing wild type RecA protein.</p