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, 9 (9), e1003819

Natural Genetic Transformation Generates a Population of Merodiploids in Streptococcus Pneumoniae

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Natural Genetic Transformation Generates a Population of Merodiploids in Streptococcus Pneumoniae

Calum Johnston et al. PLoS Genet.

Abstract

Partial duplication of genetic material is prevalent in eukaryotes and provides potential for evolution of new traits. Prokaryotes, which are generally haploid in nature, can evolve new genes by partial chromosome duplication, known as merodiploidy. Little is known about merodiploid formation during genetic exchange processes, although merodiploids have been serendipitously observed in early studies of bacterial transformation. Natural bacterial transformation involves internalization of exogenous donor DNA and its subsequent integration into the recipient genome by homology. It contributes to the remarkable plasticity of the human pathogen Streptococcus pneumoniae through intra and interspecies genetic exchange. We report that lethal cassette transformation produced merodiploids possessing both intact and cassette-inactivated copies of the essential target gene, bordered by repeats (R) corresponding to incomplete copies of IS861. We show that merodiploidy is transiently stimulated by transformation, and only requires uptake of a ~3-kb DNA fragment partly repeated in the chromosome. We propose and validate a model for merodiploid formation, providing evidence that tandem-duplication (TD) formation involves unequal crossing-over resulting from alternative pairing and interchromatid integration of R. This unequal crossing-over produces a chromosome dimer, resolution of which generates a chromosome with the TD and an abortive chromosome lacking the duplicated region. We document occurrence of TDs ranging from ~100 to ~900 kb in size at various chromosomal locations, including by self-transformation (transformation with recipient chromosomal DNA). We show that self-transformation produces a population containing many different merodiploid cells. Merodiploidy provides opportunities for evolution of new genetic traits via alteration of duplicated genes, unrestricted by functional selective pressure. Transient stimulation of a varied population of merodiploids by transformation, which can be triggered by stresses such as antibiotic treatment in S. pneumoniae, reinforces the plasticity potential of this bacterium and transformable species generally.

Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. R2597 harbors a duplication of the codY chromosomal region.
(A) Increased sequence coverage of a 107.4 kb region (green) containing codY suggests duplication. (B) Scale map of the duplication limits, bordered by IS861 sequences. (C) Coverage of the right hand limit of the duplication shows around 1/3 of the IS861 R is duplicated. (D) Coverage of codY and trim sequences shows under-representation of trim.
Figure 2
Figure 2. The R2597 duplication is a TD in the chromosome.
(A) Predicted R2597 chromosome structure. A–E, duplicated regions; X/Z, flanks; Y, codY; R1/R2 flanking IS861; R2/1, predicted hybrid IS861. PCR detected the predicted junction in R2597. Green rectangle, TD junction; black rectangle, sequence represented in Figure 1B. (B) Representation of the R2/1 hybrid at the TD junction in R2597 identified by sequencing. Mismatches are represented by vertical black lines, extending either above (R1) or below (R2) the diagram. Horizontal green line represents region within which R1 and R2 recombined. Green arrows represent limits of recombination between R1 and R2. (C) PFGE analysis of R2597. Open arrows, restriction fragments predicted in Table S1. Red arrowhead, merodiploid-specific fragment revealed by hybridization. Although the lanes shown were part of the same initial gel/membrane, lanes present between them have been removed to simplify comparison between lanes.
Figure 3
Figure 3. Self-transformation generates merodiploids.
(A) Increased survival of lethal transformants after transformation with codY::trim (grey bars) or codY::spc (black bars) cassettes in presence of chromosomal DNA from R800 or D39. (B) Maps of codY+ and codY::trim loci, and detection of codY- (red, scodY1-scodY2 primers) and trim-specific (blue, strim1/strim2 primers) sequences by PCR in TrimR clones recovered from transformation with either codY::trim PCR alone (#1–10), or codY::trim PCR in presence of R800 chromosomal DNA (#11–20). (C) Detection of TD junction by PCR with CJ242/merod-b primers in clones #1–5 and #11–15 from Figure 3B. 9/10 of these clones possessed the TD junction. The one clone without the TD junction (clone #1, R3022) was found to possess an additive insertion of the codY::trim cassette in close proximity to the codY locus, creating a very small TD (See Figure S4B–E). (D) PFGE analysis of chromosomal DNA from clone #11 (R3023), digested by SmaI and hybridized with codY-specific probe. Open red arrow, expected wild-type codY ethidium bromide stained band; closed red arrowhead, R3023 codY fragment, expected for codY +/codY::trim merodiploid; open red arrowhead, wild-type codY band weakly present in R3023. Although the lanes shown were part of the same initial gel/membrane, lanes present between them have been removed to simplify comparison between lanes. (E) Representation of the R2/1 hybrid at the TD junction in R3023 identified by sequencing, layout as in Figure 2B.
Figure 4
Figure 4. R-NRf donor fragments promote merodiploid formation, allowing survival of otherwise lethal codY::trim transformants.
(A) Co-transformation of 100 ng mL−1 codY::trim with R1-A (red), R2-Z (orange), R1-A and R2-Z (green) or comparable non-R-NRf fragments (AB, grey). (B) R TD junction sequence of three independent clones. Layout as in Figure 2B. (C) Donor fragments with only repeats do not induce merodiploidy. DNA concentrations: 100 ng mL−1 for codY::trim cassette with 100 ng mL−1 of R1 or R2 PCR fragment; 50 ng mL−1 R1-A and 50 ng mL−1 R2-Z as positive control. PCR primers used: R1, merod-a1/CJ241; R2, merod-a2/CJ243. (D) Diagrammatic representation of the codY chromosomal region with location of primers used to produce PCR fragments assayed in transformation in combination with the codY::trim cassette (panel E). (E) Cooperativity requires two R-NRf fragments with same polarity. DNA concentrations: 100 ng mL−1 for codY::trim cassette; 50 ng mL−1 of each R-NRf fragment in each test, giving a total of 100 ng mL−1 fragment per test.
Figure 5
Figure 5. Model of merodiploid formation during pneumococcal transformation.
(A) Alternative pairing between R-NRf sequences triggers unequal crossing over. Letters as in Figure 1D. (B) Spontaneous restoration of complementary strand integrity. (C) Restoration of complementary strand integrity stimulated by R2-Z donor fragment. (D) Resolution of chromosome dimer to produce one tandem-duplicated chromosome and one abortive chromosome. oriC, origin of replication; terC, terminus of replication; Δ, deletion; †, abortive chromosome. (E) Predicted abortive chromosome structure. Layout as in Figure 2A. Red square; abortive chromosome junction. Detection of abortive chromosome junction by primers in Figure 3C. Diagram of R1/2 sequence at abortive chromosome junction amplified after R1-A transformation, determined by sequencing of PCR fragments. Vertical lines, R1/R2 mismatches; red horizontal line, mixed sequence .(F) Saturation of the Hex system establishes frequent alternative pairing of R-NRf fragment. Transformation efficiencies of RifR and NovR point mutations (carried by R304 chromosomal DNA) compared in hex (R246) or hex+ (R800) recipients in the presence or absence of R-NRf PCR fragments. Transformation efficiencies normalized to SmR (rpsL41) point mutation, resistant to Hex. hex recipient, shades of gray; hex + recipient, shades of blue.
Figure 6
Figure 6. Transformation does not select pre-existing structures but creates de novo merodiploids.
(A) Detection of tandem-duplication junction by PCR on cultures at different time points after transformation with R-NRf fragments alone. (B) Detection of abortive chromosome junction on same transformed cultures as in panel A. (C) Detection of tandem-duplication and abortive chromosome junctions by PCR on cultures at different time points after self-transformation. (D) Integration of donor R1*-A fragment during merodiploid formation. Right panel shows BamHI restriction of TD junction PCRs recovered from TrimR clones transformed with codY::trim and R1*-A PCR fragments. Undigested PCR shown in green, products of BamHI digestion shown in orange. Left panel shows BamHI digestion of TD junction PCR amplified from population transformed with R1-A or R1*-A. −, undigested PCR fragment; +, BamHI-digested PCR fragment.
Figure 7
Figure 7. Transformation-triggered merodiploidy is a general process.
(A) Chromosomal location of generated TDs. Size, identity and homology of repeat sequences noted. (B) PCR detection of TD junctions in the transformed population 40 min after uptake of appropriate R-NRf fragments (R1-A+R2-Z; R3-A+R4-Z; R5-A+R6-Z). (C) PCR detection of TD junctions in the transformed population 40 min after self-transformation. PCRs carried out on same culture of transformed cells. Control PCRs without transforming DNA on the same cells as in Figure 5B. (D) Detection of TD #4 junction by PCR with primers in Figure S5G, PCR carried out on population transformed with R4-Z fragment. (E) Schematic representation of proposed evolutionary potential of merodiploidy triggered by transformation. Stresses such as antibiotics can induce competence in S. pneumoniae and other species . NC, non-competent; C, competent; TD, tandem-duplication where numbers represent different merodiploids. Small arrows indicate subsequent cell divisions. Crossed-out TD indicates loss of duplication due to intrinsic instability.

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Grant support

This work was supported in part by the European Community's Seventh Framework Programme FP7/2007-2013 under grant agreement number HEALTH-F3-2009-222983 (Pneumopath project). SC was the recipient of a PhD thesis fellowship from the Ministère de la Recherche 2007-2009) and from the Fondation pour la Recherche Médicale (2010). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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