Distribution of recombination events during gene exchange <i>in vitro</i>, mediated by purified DNA or by cell-cell interactions.

<p>Recombination events mapped throughout the genomes of 22 recombinants from each cross format were sorted by length of recombinant segment (in 2-kbp increments) and aggregated to display average values per strain. <b>A.</b> Number of recombination events in each size class. <b>B</b>. Total amount of DNA transferred due to events in each size class. <b>C.</b> Cumulative amount of DNA incorporated due to events in all size classes up to the indicated segment length. <b>DNA</b>, transformation after treatment with CSP in CDM (CP2204 recipient, CP2215 donor); <b>Filter</b>, cell assemblage treated with CSP in CDM (CP2204 recipient, CP2215 donor); <b>Biofilm</b>, co-culture in CDM (R36A recipient, CP2215 donor).</p

Recombination events in saturating DNA transformation.

<p>Circos plot visualizes the whole <i>S</i>. <i>pneumoniae</i> CP2215 genome with bp coordinates in 50-kb intervals labelled outside of the ring. Red lines in the first inner ring represent SNPs between strain CP2215 (donor strain) and CP2204 (recipient strain for saturating DNA experiments). Subsequent inner rings represent recombinants from the saturating DNA experiments. Lines on the inner rings represent SNPs transferred from donor to the recombinant. As expected, each recombinant strain carries one recombination event for each selected marker (Nov^R near 770 kbp and/or Spc^R near 212 kbp), plus some additional events nearby due to linkage, plus some events much farther away due to congression. Positions marked as S, N indicate positions of the Nov^R and Spc^R selective markers.</p

Schematic description of experimental workflow and data analysis representing the process of analysis for the entire project.

<p>Recipient and donor strains were known to be ~16000 SNPs apart and three experimental set-ups were conceived to test the effect of environment on recombination; saturating DNA, filter assemblage and biofilm co-culture. Recombinants were then recovered by antibiotic selection and sequenced. Final recombination events were determined by a mapping algorithm that identified variant regions (of the ~16000) which were matching between recombinant and donor but not recipient and therefore inferred to have been transferred.</p

Evolution via recombination: Cell-to-cell contact facilitates larger recombination events in <i>Streptococcus pneumoniae</i>

<div><p>Homologous recombination in the genetic transformation model organism <i>Streptococcus pneumoniae</i> is thought to be important in the adaptation and evolution of this pathogen. While competent pneumococci are able to scavenge DNA added to laboratory cultures, large-scale transfers of multiple kb are rare under these conditions. We used whole genome sequencing (WGS) to map transfers in recombinants arising from contact of competent cells with non-competent ‘target’ cells, using strains with known genomes, distinguished by a total of ~16,000 SNPs. Experiments designed to explore the effect of environment on large scale recombination events used saturating purified donor DNA, short-term cell assemblages on Millipore filters, and mature biofilm mixed cultures. WGS of 22 recombinants for each environment mapped all SNPs that were identical between the recombinant and the donor but not the recipient. The mean recombination event size was found to be significantly larger in cell-to-cell contact cultures (4051 bp in filter assemblage and 3938 bp in biofilm co-culture <i>versus</i> 1815 bp with saturating DNA). Up to 5.8% of the genome was transferred, through 20 recombination events, to a single recipient, with the largest single event incorporating 29,971 bp. We also found that some recombination events are clustered, that these clusters are more likely to occur in cell-to-cell contact environments, and that they cause significantly increased linkage of genes as far apart as 60,000 bp. We conclude that pneumococcal evolution through homologous recombination is more likely to occur on a larger scale in environments that permit cell-to-cell contact.</p></div

Recombination events in filter assemblage.

<p>Circos plot visualizes the whole <i>S</i>. <i>pneumoniae</i> CP2215 genome with bp coordinates in 50-kb intervals labelled outside of the ring. Red lines in the first inner ring represent SNPs between strain CP2215 (donor strain) and CP2204 (recipient strain for filter assemblage experiments). Subsequent inner rings represent recombinants from the filter assemblage experiments. Lines on the inner rings represent SNPs transferred from donor to the recombinant. As expected, each recombinant strain carries one recombination event for each selected marker (Nov^R near 770 kbp and/or Spc^R near 212 kbp), plus some additional events nearby due to linkage, plus some events much farther away due to congression. Positions marked as S, N indicate positions of the Nov^R and Spc^R selective markers.</p

Recombination patterns reported for <i>S</i>. <i>pneumoniae</i> in nature and <i>in vitro</i>.

<p>Frequencies of recombination events as a function of segment size, plotted for six retrospective global analyses of exchange events in nature and one global analysis of products of genetic transformation <i>in vitro</i>. Recombinant segments were aggregated in 2-kbp increments. Panels display both the number of events in each size class (<b>blue</b>) and total mass of DNA (kbp) incorporated during those events (<b>orange</b>). Data extracted from cited publications. <b>A.</b> 173 strains of the PMEN4 lineage: 451 events total. [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007410#pgen.1007410.ref012" target="_blank">12</a>]. <b>B.</b> 189 strains of the PMEN2 lineage: 616 events total [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007410#pgen.1007410.ref055" target="_blank">55</a>]. <b>C.</b> Two vaccine escape strains: 27 events total [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007410#pgen.1007410.ref010" target="_blank">10</a>]. <b>D.</b> Two ST13 strains from one patient: 23 events total [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007410#pgen.1007410.ref011" target="_blank">11</a>]. <b>E.</b> 241 strains of the PMEN1 lineage: ~800 events total [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007410#pgen.1007410.ref056" target="_blank">56</a>]. <b>F.</b> 426 strains receiving DNA from PMEN1 lineage: ~24 events total [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007410#pgen.1007410.ref004" target="_blank">4</a>]. <b>G.</b> 84 strains of PMEN1 strain 23-F-R transformed by Kan^R DNA from strain TIGR4: 107 RSS events [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007410#pgen.1007410.ref009" target="_blank">9</a>].</p

Summary of recombination events recovered from 66 recombinants in three experimental environments^a.

<p>Summary of recombination events recovered from 66 recombinants in three experimental environments<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007410#t001fn001" target="_blank">^a</a>.</p

Expanded linkage during gene transfer in direct cell-cell interactions.

<p>Probability of inclusion in a recombinant segment is displayed as a function of genome position, within 40 kbp of the selected marker, <i>nov-1</i> (bp 777,151). Co-transfer rates for Nov^R transformants recovered from DNA transformation (<b>DNA</b>), short-term mixed strain assemblage (<b>Filter</b>), or biofilm co-culture (<b>Biofilm</b>), totalling 21, 22, and 14 strains, respectively. Background congression rates were 0.7%, 1.9%, and 2.2% respectively.</p

Image_2_Microbial DNA extraction of high-host content and low biomass samples: Optimized protocol for nasopharynx metagenomic studies.TIFF

IntroductionLow microbial biomass and high human DNA content in nasopharyngeal aspirate samples hinder comprehensive characterization of microbiota and resistome. We obtained samples from premature infants, a group with increased risk of developing respiratory disorders and infections, and consequently frequent exposure to antibiotics. Our aim was to devise an optimal protocol for handling nasopharyngeal aspirate samples from premature infants, focusing on host DNA depletion and microbiome and resistome characterization.MethodsThree depletion and three DNA extraction protocols were compared, using RT-PCR and whole metagenome sequencing to determine the efficiency of human DNA removal, taxonomic profiling and assignment of antibiotic resistance genes. Protocols were tested using mock communities, as well as pooled and individual patient samples.ResultsThe only extraction protocol to retrieve the expected DNA yield from mock community samples was based on a lytic method to improve Gram positive recovery (MasterPure™). Host DNA content in non-depleted aliquots from pooled patient samples was 99%. Only samples depleted with MolYsis™ showed satisfactory, but varied reduction in host DNA content, in both pooled and individual patient samples, allowing for microbiome and resistome characterisation (host DNA content from 15% to 98%). Other depletion protocols either retrieved too low total DNA yields, preventing further analysis, or failed to reduce host DNA content. By using Mol_MasterPure protocol on aliquots from pooled patient samples, we increased the number of bacterial reads by 7.6 to 1,725.8-fold compared to non-depleted reference samples. PCR results were indicative of achieved microbial enrichment. Individual patient samples processed with Mol_MasterPure protocol varied greatly in total DNA yield, host DNA content (from 40% to 98%), species and antibiotic resistance gene richness.DiscussionDespite high human DNA and low microbial biomass content in nasopharynx aspirates of preterm infants, we were able to reduce host DNA content to levels compatible with downstream shotgun metagenomic analysis, including bacterial species identification and coverage of antibiotic resistance genes. Whole metagenomic sequencing of microbes colonizing the nasopharynx may contribute to explaining the possible role of airway microbiota in respiratory conditions and reveal carriage of antibiotic resistance genes.</p

Image_3_Microbial DNA extraction of high-host content and low biomass samples: Optimized protocol for nasopharynx metagenomic studies.TIFF

IntroductionLow microbial biomass and high human DNA content in nasopharyngeal aspirate samples hinder comprehensive characterization of microbiota and resistome. We obtained samples from premature infants, a group with increased risk of developing respiratory disorders and infections, and consequently frequent exposure to antibiotics. Our aim was to devise an optimal protocol for handling nasopharyngeal aspirate samples from premature infants, focusing on host DNA depletion and microbiome and resistome characterization.MethodsThree depletion and three DNA extraction protocols were compared, using RT-PCR and whole metagenome sequencing to determine the efficiency of human DNA removal, taxonomic profiling and assignment of antibiotic resistance genes. Protocols were tested using mock communities, as well as pooled and individual patient samples.ResultsThe only extraction protocol to retrieve the expected DNA yield from mock community samples was based on a lytic method to improve Gram positive recovery (MasterPure™). Host DNA content in non-depleted aliquots from pooled patient samples was 99%. Only samples depleted with MolYsis™ showed satisfactory, but varied reduction in host DNA content, in both pooled and individual patient samples, allowing for microbiome and resistome characterisation (host DNA content from 15% to 98%). Other depletion protocols either retrieved too low total DNA yields, preventing further analysis, or failed to reduce host DNA content. By using Mol_MasterPure protocol on aliquots from pooled patient samples, we increased the number of bacterial reads by 7.6 to 1,725.8-fold compared to non-depleted reference samples. PCR results were indicative of achieved microbial enrichment. Individual patient samples processed with Mol_MasterPure protocol varied greatly in total DNA yield, host DNA content (from 40% to 98%), species and antibiotic resistance gene richness.DiscussionDespite high human DNA and low microbial biomass content in nasopharynx aspirates of preterm infants, we were able to reduce host DNA content to levels compatible with downstream shotgun metagenomic analysis, including bacterial species identification and coverage of antibiotic resistance genes. Whole metagenomic sequencing of microbes colonizing the nasopharynx may contribute to explaining the possible role of airway microbiota in respiratory conditions and reveal carriage of antibiotic resistance genes.</p