Beta-galactosidase activity in <i>M. smegmatis</i> expressing <i>lacZ</i> under the control of the promoter regions of the <i>M. tuberculosis</i> H37Rv <i>lipF</i>, <i>pks2</i>, <i>msl3</i> and <i>fadD21</i> genes.

<p>The <i>lipF</i>, <i>pks2</i>, <i>msl3</i> and <i>fadD21</i> promoter-<i>lacZ</i> fusions carried by the pJEM15 plasmid were introduced into <i>M. smegmatis</i>. The positive control is <i>M. smegmatis</i> transformed with pJEM31(PAN) containing <i>lacZ</i> under the control of a mobile genetic element normalized against <i>M. smegmatis</i> transformed with pJEM15 empty vector. The results shown are the mean of two independent experiments.</p

DNase I footprinting assay of the <i>pks2</i> and <i>msl3</i> promoter regions with PhoP-P.

<p>Radiolabeled PCR fragments corresponding to (A) pks2 (−136 to +62), (B) msl3 (−312 to −79), (C) fadD21(−194 to +7) and (D) lipF (−639 to −450) were used as DNA targets. Various amounts of PhoP-P were used with pks2 and msl3 (lane 1: 0; lane 2: 1.8 pmol; lane 3: 3.6 pmol; lane 4: 7.2 pmol and lane 5: 14.4 pmol) and were incubated with 0.2 pmol of DNA before DNase 1 digestion. For fadD21 and lipF different amount of PhoP-P were used ((lane 1: 0; lane 2: 1.8 pmol; lane 3: 18 pmol; lane 4: 180 pmol and lane 5: 900 pmol) and were incubated with 1 pmol of DNA before DNase I digestion. Lane 6: A+G Maxam and Gilbert reaction. Protected regions are indicated by a line with colored regions. The blue, orange and red segments correspond to the DR1, DR2 and DR3 sites, respectively.</p

Identification of the DR1–3 sites on the DNA promoters recognized byPhoP.

<p>For each DNA sequence shown to bind PhoP-P, sequences with a complete or partial match to the DR1, DR2 and DR3 sites were identified (underlined). The degree of conservation of the consensus sequence was calculated and is shown as the ratio of matching nucleotides to the consensus: % identity.</p

Electrophoretic mobility assays with the lipF, pks2, msl3 and fadD21 promoter fragments and PhoP-P.

<p>2A. The large DNA fragments initially selected — lipFa (222 bp), pks2 (148 bp), msl3a (235 bp), and fadD21 (210 bp) — were incubated with PhoP-P in the presence of poly dI-dC at 10 µg/ml and run on a native polyacrylamide gel (8%), in 0.5× TBE buffer. Complete binding was assessed on the basis of the amount of shifted material for each fragment in the presence of 4 µM of PhoP-P (protein/DNA ratio: 100/1), except for lipFa, for which 8 µM PhoP-P was required (protein/DNA ratio: 200/1). 2B. Smaller DNA fragments — lipFa1 (68 bp), pks2a (59 bp), pks2b (40 bp), pks2d (104 bp), msl3a1 (45 bp), msl3a2 (83 pb), fadD21a (55 bp) and fadD21b (79 bp) — were designed and incubated with PhoP-P in the presence of poly dI-dC at 10 µg/ml. Protect40 (40 bp) was tested in all mobility shift assays, as a positive control <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042876#pone.0042876-Gupta1" target="_blank">[26]</a>. 2C. Protect40 (40 bp) fragments with an altered sequence were used: Pho1 (DR1 altered sequence), Pho2 (DR2 altered sequence) and Pho3 (DR1 and DR2 altered sequences). Wild-type Protect40 (40 bp) was tested as a positive control <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042876#pone.0042876-Gupta1" target="_blank">[26]</a>.</p

List of DNA fragments used in this study.

b<p>Fragments used for electrophoretic mobility shift assays.</p

List of primers used in this study.

a<p>Fragments used for transcriptional fusions.</p>b<p>Fragments used for electrophoretic mobility shift assays.</p>c<p>Fragments used for DNase protection assay.</p

Schematic diagram of the DNA sequences tested in electrophoretic mobility assays.

<p>The DR1, DR2 and DR3 sites are indicated in blue, orange and red, respectively. Binding is indicated by (+), with (−) indicating an absence of binding and (−/+) indicating weak binding. Positions on the DNA regions are shown with respect to the translation start site, in brackets.</p

Initial Characterization of the Pf-Int Recombinase from the Malaria Parasite <em>Plasmodium falciparum</em>

<div><h3>Background</h3><p>Genetic variation is an essential means of evolution and adaptation in many organisms in response to environmental change. Certain DNA alterations can be carried out by site-specific recombinases (SSRs) that fall into two families: the serine and the tyrosine recombinases. SSRs are seldom found in eukaryotes. A gene homologous to a tyrosine site-specific recombinase has been identified in the genome of <em>Plasmodium falciparum</em>. The sequence is highly conserved among five other members of Plasmodia.</p> <h3>Methodology/Principal Findings</h3><p>The predicted open reading frame encodes for a ∼57 kDa protein containing a C-terminal domain including the putative tyrosine recombinase conserved active site residues R-H-R-(H/W)-Y. The N-terminus has the typical alpha-helical bundle and potentially a mixed alpha-beta domain resembling that of λ-Int. Pf-Int mRNA is expressed differentially during the <em>P. falciparum</em> erythrocytic life stages, peaking in the schizont stage. Recombinant Pf-Int and affinity chromatography of DNA from genomic or synthetic origin were used to identify potential DNA targets after sequencing or micro-array hybridization. Interestingly, the sequences captured also included highly variable subtelomeric genes such as <em>var</em>, <em>rif</em>, and <em>stevor</em> sequences. Electrophoretic mobility shift assays with DNA were carried out to verify Pf-Int/DNA binding. Finally, Pf-Int knock-out parasites were created in order to investigate the biological role of Pf-Int.</p> <h3>Conclusions/Significance</h3><p>Our data identify for the first time a malaria parasite gene with structural and functional features of recombinases. Pf-Int may bind to and alter DNA, either in a sequence specific or in a non-specific fashion, and may contribute to programmed or random DNA rearrangements. Pf-Int is the first molecular player identified with a potential role in genome plasticity in this pathogen. Finally, Pf-Int knock-out parasite is viable showing no detectable impact on blood stage development, which is compatible with such function.</p> </div

Characterization of Pf-Int interaction with DNA targets.

<p>10 nM of labeled Selex8-DNA were incubated with increasing amounts of Pf-Int-C192 (lanes 1 to 6) and Pf-Int-C162 (lanes 7 to 12) <b>A</b>) in absence of poly (dI-dC) and <b>B</b>) in the presence of 10 µg/ml of poly (dI-dC).</p

Circular dichroism analysis of the purified recombinant proteins Pf-Int-C162 and Pf-Int-C192.

<p><b>A</b>) CD analysis in far UV. Black dots: experimental CD data. Blue line: modeled CD spectra of Pf-Int-C192. Red line: modeled CD spectra of Pf-Int-C162. The CD spectral analysis showed for both analyzed forms a high degree of α-helical organization and a low percentage of β-sheet (54/60% α-helical and 6/3% β-sheet for Pf-Int-C162/C192). <b>B, C</b>) Stability analysis by thermal denaturation coupled to CD for <b>B</b>) Pf-Int-C162 and <b>C</b>) Pf-Int-C192. Thermal CD transition curves (red lines) for Pf-Int-C162 and Pf-Int-C192 showed a beginning of stability change at approximately 50°C and 40°C, with a completion at 60°C and 50°C. The increase of the dynode signal (blue lines) indicates that the proteins are precipitating at the transition and thus the melting temperatures cannot be precisely determined.</p