The folding of the specific DNA recognition subdomain of the sleeping beauty transposase is temperature-dependent and is required for its binding to the transposon DNA.

The reaction of DNA transposition begins when the transposase enzyme binds to the transposon DNA. Sleeping Beauty is a member of the mariner family of DNA transposons. Although it is an important tool in genetic applications and has been adapted for human gene therapy, its molecular mechanism remains obscure. Here, we show that only the folded conformation of the specific DNA recognition subdomain of the Sleeping Beauty transposase, the PAI subdomain, binds to the transposon DNA. Furthermore, we show that the PAI subdomain is well folded at low temperatures, but the presence of unfolded conformation gradually increases at temperatures above 15°C, suggesting that the choice of temperature may be important for the optimal transposase activity. Overall, the results provide a molecular-level insight into the DNA recognition by the Sleeping Beauty transposase

DNA-binding of folded and unfolded conformations of the PAI subdomain.

<p>(A) 2D [¹H,¹⁵N]-HSQC spectrum of the PAI subdomain collected at the temperature of 5°C in 25 mM aqueous sodium phosphate buffer at pH 5.0 in the presence of 250 mM NaCl. The folded and unfolded conformations exist in slow exchange on the NMR time scale. Thus, two resonances are observed for each residue. Non-overlapping resonances originating from the same residue in both the folded and unfolded conformations are labeled. (B) Cartoon representation of the PAI subdomain (PDB code 2m8e <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112114#pone.0112114-Carpentier1" target="_blank">[7]</a>). The DNA-binding site is colored blue. Side chains of the residues that were used for the analysis of the DNA-binding are labeled and shown as red sticks. (C) Relative intensities of resonances corresponding to the folded and unfolded conformations are plotted as a function of PAI:DNA molar ratio. Relative intensities were calculated by dividing the resonance intensity at a given PAI:DNA molar ratio by the intensity of this resonance in the absence of DNA.</p

2D [¹H,¹⁵N]-HSQC spectra of the PAI subdomain.

<p>The spectra were collected in 25 mM aqueous sodium phosphate buffer at pH 5.0 (top panel) and 7.0 (bottom panel) in the range of temperatures from 5 to 45°C with a 5°C increment.</p

Intrinsic tyrosine fluorescence.

<p>(Left panel) The location of Y46 on the cartoon representation of the PAI subdomain (PDB code 2m8e <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112114#pone.0112114-Carpentier1" target="_blank">[7]</a>) is shown. (Right panel) The integral fluorescence of Y46 at pH 5.0 (squares) and pH 7.0 (circles) is plotted vs. temperature. Shown data is the average of three independent experiments. Error bars in many cases do not exceed the size of the symbol. Solid lines represent best fits of experimental data.</p

Self-diffusion coefficients.

<p>The PAI self-diffusion coefficient at pH 5.0 (squares) and 7.0 (circles) are plotted vs. temperature over the range from 5 to 35°C. Protein samples were prepared in 25 mM sodium phosphate buffer using 100% D₂O. The temperature dependence of the self-diffusion coefficient of BPTI (stars) is shown for comparison. Solid lines represent fits of Arrhenius dependence of the self-diffusion coefficient to experimental data.</p

Urogenital <i>Chlamydia trachomatis</i> multilocus sequence types and genovar distribution in chlamydia infected patients in a multi-ethnic region of Saratov, Russia

<div><p>Background</p><p>This is the first report to characterize the prevalence and genovar distribution of genital chlamydial infections among random heterosexual patients in the multi-ethnic Saratov Region, located in Southeast Russia.</p><p>Methods</p><p>Sixty-one clinical samples (cervical or urethral swabs) collected from a random cohort of 856 patients (7.1%) were <i>C</i>. <i>trachomatis</i> (CT) positive in commercial nucleic acid amplification tests (NAATs) and duplex TaqMan PCRs.</p><p>Results</p><p>Sequence analysis of the VDII region of the <i>ompA</i> gene revealed seven genovars of <i>C</i>. <i>trachomatis</i> in PCR-positive patients. The overall genovars were distributed as E (41.9%), G (21.6%), F (13.5%), K (9.5%), D (6.8%), J (4.1%), and H (2.7%). CT-positive samples were from males (n = 12, 19.7%), females (n = 42, 68.8%), and anonymous (n = 7, 11.5%) patients, with an age range of 19 to 45 years (average 26.4), including 12 different ethnic groups representative of this region. Most patients were infected with a single genovar (82%), while 18% were co-infected with either two or three genovars. The 1156 bp-fragment of the <i>ompA</i> gene was sequenced in 46 samples to determine single nucleotide polymorphisms (SNP) among isolates. SNP-based subtyping and phylogenetic reconstruction revealed the presence of 13 variants of the <i>ompA</i> gene, such as E (E1, E2, E6), G (G1, G2, G3, G5), F1, K, D (D1, Da2), J1, and H2. Differing genovar distribution was identified among urban (E>G>F) and rural (E>K) populations, and in Slavic (E>G>D) and non-Slavic (E>G>K) ethnic groups. Multilocus sequence typing (MLST) determined five sequences types (STs), such as ST4 (56%, 95% confidence interval, CI, 70.0 to 41.3), ST6 (10%, 95% CI 21.8 to 3.3), ST9 (22%, 95% CI 35.9 to 11.5), ST10 (2%, 95% CI 10.7 to 0.05) and ST38 (10%, 95% CI 21.8 to 3.3). Thus, the most common STs were ST4 and ST9.</p><p>Conclusion</p><p><i>C</i>. <i>trachomatis</i> is a significant cause of morbidity among random heterosexual patients with genital chlamydial infections in the Saratov Region. Further studies should extend this investigation by describing trends in a larger population, both inside and outside of the Saratov Region to clarify some aspects for the actual application of <i>C</i>. <i>trachomatis</i> genotype analysis for disease control.</p></div

Clonal grouping of sequence types (STs) of <i>C</i>. <i>trachomatis</i> strains found in our study (marked with gray ovals).

<p>The division into Group I (A) and Group III (B) was based on the EBURST analysis with other STs of these clonal complexes obtained from PubMLST/Chlamydiales database.</p

The <i>ompA</i> and seven housekeeping genes MLST sequence types of Saratov and reference strains of <i>C</i>. <i>trachomatis</i> obtained from the PubMLST database (https://pubmlst.org).

<p>The <i>ompA</i> and seven housekeeping genes MLST sequence types of Saratov and reference strains of <i>C</i>. <i>trachomatis</i> obtained from the PubMLST database (<a href="https://pubmlst.org" target="_blank">https://pubmlst.org</a>).</p

Distribution of genovars in CT-positive patients.

<p><b>(A) According to their gender, place of residence and ethnic origin. (B) According to their age in mono-infected and multiple-infected CT-positive samples</b>. Analysis was done by Fisher’s exact test (two-tailed). Statistically significant differences are indicated by * (<i>p</i><0.05) or by ** (<i>p</i><0.01) or by *** (<i>p</i><0.001). Slavic cohort included Russians, Byelorussians and Ukrainians, Non-Slavic cohort was presented by Caucasians, Jews, Kyrgyzs, Koreans, Moldavians, Germans, Mordovians, and anonymous.</p

Phylogenetic tree demonstrating the relationship of Saratov <i>ompA</i> sequences to references strains from Table 1.

<p>Thick branches, subtending all large clades, represent 100% bootstrap support. The scale at the bottom represents one substitution per site.</p