Influence of repeat composition on mycoplasma gene expression.

<p>Influence of repeat composition on mycoplasma gene expression.</p

Repetitive Elements in <i>Mycoplasma hyopneumoniae</i> Transcriptional Regulation

<div><p>Transcriptional regulation, a multiple-step process, is still poorly understood in the important pig pathogen <i>Mycoplasma hyopneumoniae</i>. Basic motifs like promoters and terminators have already been described, but no other cis-regulatory elements have been found. DNA repeat sequences have been shown to be an interesting potential source of cis-regulatory elements. In this work, a genome-wide search for tandem and palindromic repetitive elements was performed in the intergenic regions of all coding sequences from <i>M</i>. <i>hyopneumoniae</i> strain 7448. Computational analysis demonstrated the presence of 144 tandem repeats and 1,171 palindromic elements. The DNA repeat sequences were distributed within the 5’ upstream regions of 86% of transcriptional units of <i>M</i>. <i>hyopneumoniae</i> strain 7448. Comparative analysis between distinct repetitive sequences found in related mycoplasma genomes demonstrated different percentages of conservation among pathogenic and nonpathogenic strains. qPCR assays revealed differential expression among genes showing variable numbers of repetitive elements. In addition, repeats found in 206 genes already described to be differentially regulated under different culture conditions of <i>M</i>. <i>hyopneumoniae</i> strain 232 showed almost 80% conservation in relation to <i>M</i>. <i>hyopneumoniae</i> strain 7448 repeats. Altogether, these findings suggest a potential regulatory role of tandem and palindromic DNA repeats in the <i>M</i>. <i>hyopneumoniae</i> transcriptional profile.</p></div

Pipeline of repeat search strategy.

<p>Up to 500 bp of 5’ intergenic regions (IR) of all <i>M</i>. <i>hyopneumoniae</i> strain 7448 (MHP_7448) CDSs were used for <i>in silico</i> prediction of tandem and palindromic DNA repeats.</p

Conservation of repetitive elements in <i>Mycoplasma</i> genomes.

<p>Graphics represent percent of relative conservation (%) of conserved (A), nonconserved (B) and absent (C) SSR, SSRM, and DR repeats found in <i>M</i>. <i>hyopneumoniae</i> strain 7448 intergenic regions in relation to strains 7422, J and <i>M</i>. <i>flocculare</i> (MFL) orthologues. A pathogenic versus nonpathogenic comparison was done and defined as P-nP. Repeats conserved between <i>M</i>. <i>hyopneumoniae</i> strain 7448 and <i>M</i>. <i>hyopneumoniae</i> strain 7422 were investigated in relation to MHP_J (P-nP (sp)) and MFL (P-nP(gn)). (D) Graphic represents the average of all tandem repeats found in each situation tested.</p

Repeat distribution in the intergenic regions of <i>M</i>. <i>hyopneumoniae</i> strain 7448.

<p>(A) All intergenic regions of the genes were analysed in relation to the presence of tandem repeats, palindromic sequences or putative promoter motifs. (B) Tandem repeats and palindromic sequences were distributed in SSR, SSRM, DR, PAL and PALG throughout genes intergenic regions. The same was done for the 5’ upstream region of the first gene of each transcription unit described in genome (C) and (D).</p

Pipeline of tandem repeat comparison analysis among mycoplasma genomes.

<p>An example of nonconserved SSR between <i>M</i>. <i>hyopneumoniae</i> strain 7448 (MHP_7448), and <i>M</i>. <i>hyopneumoniae</i> strain J (MHP_J) can be observed above. A (TAT)₉ repeat found in the MHP_7448 gene intergenic region was classified as nonconserved, as a (TAT)₁₁ repeat was localized in the respective orthologous intergenic region in MHP_J. Abbreviations: <i>M</i>. <i>hyopneumoniae</i> strain 7422 (MHP_7422); <i>M</i>. <i>flocculare</i> (MFL). *First CDS of the transcription unit.</p

Pipeline of repeat conservation in putative adhesin-coding genes and differentially expressed genes from strains of <i>M</i>. <i>hyopneumoniae</i>.

<p>(A) Comparison analysis of adhesins, with an example of relative conservation between MHP_7448 genes that have 5 PALG repeats and the respective orthologues in MHP_J that have only 3. The percentage (%) conservation between MHP_7448 and MHP_J repeats was obtained by dividing the number of repeat elements. (B) Repeats present in differentially expressed (DE) genes found in <i>M</i>. <i>hyopneumoniae</i> strain 232 (MHP_232) were compared with those found in MHP_7448 in the same way as in (A). Abbreviations as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0168626#pone.0168626.g002" target="_blank">Fig 2</a>.</p

Workflow of samples preparation for metagenomic sequencing.

<p>The figure shows the samples collection; bacteria cell selection; bacteria DNA extraction and shotgun metagenomic sequencing. M01: tracheal and lungs lavage pool from 20 pneumonic lungs. M02: tracheal and lungs lavage pool from 20 lungs without both swine enzootic pneumonia and infection macroscopic signs.</p

Microbiome overview in swine lungs

<div><p><i>Mycoplasma hyopneumoniae</i> is the etiologic agent of swine enzootic pneumonia. However other mycoplasma species and secondary bacteria are found as inhabitants of the swine respiratory tract, which can be also related to disease. In the present study we have performed a total DNA metagenomic analysis from the lungs of pigs kept in a field condition, with suggestive signals of enzootic pneumonia and without any infection signals to evaluate the bacteria variability of the lungs microbiota. Libraries from metagenomic DNA were prepared and sequenced using total DNA shotgun metagenomic pyrosequencing. The metagenomic distribution showed a great abundance of bacteria. The most common microbial families identified from pneumonic swine’s lungs were <i>Mycoplasmataceae</i>, <i>Flavobacteriaceae</i> and <i>Pasteurellaceae</i>, whereas in the carrier swine’s lungs the most common families were <i>Mycoplasmataceae</i>, <i>Bradyrhizobiaceae</i> and <i>Flavobacteriaceae</i>. Analysis of community composition in both samples confirmed the high prevalence of <i>M</i>. <i>hyopneumoniae</i>. Moreover, the carrier lungs had more diverse family population, which should be related to the lungs normal flora. In summary, we provide a wide view of the bacterial population from lungs with signals of enzootic pneumonia and lungs without signals of enzootic pneumonia in a field situation. These bacteria patterns provide information that may be important for the establishment of disease control measures and to give insights for further studies.</p></div

p-values of the ratio of mapped reads between samples.

<p>The x-axis shows the most prevalent bacteria species reported. The y-axis shows the p-values from normalized ratio of mapped reads. The red line represents the significant threshold set at an α = 0.10.</p