Unbiased homeologous recombination during pneumococcal transformation allows for multiple chromosomal integration events

  1. Jun Kurushima
  2. Nathalie Campo
  3. Renske van Raaphorst
  4. Guillaume Cerckel
  5. Patrice Polard
  6. Jan-Willem Veening

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Abstract

The rapid spread of antimicrobial resistance and vaccine escape in the opportunistic human pathogen Streptococcus pneumoniae can be largely attributed to competence-induced transformation. To better understand the dynamics of competence-induced transformation, we studied this process at the single-cell level. We show that within isogenic populations, all cells become naturally competent and bind exogenous DNA. In addition, we find that transformation is highly efficient and that the chromosomal location of the integration site or whether the transformed gene is encoded on the leading or lagging strand has limited influence on recombination efficiency. Indeed, we have observed multiple recombination events in single recipients in real-time. However, because of saturation of the DNA uptake and integration machinery and because a single stranded donor DNA replaces the original allele, we find that transformation efficiency has an upper threshold of approximately 50% of the population. Counterintuitively, in the presence of multiple transforming DNAs, the fraction of untransformed cells increases to more than 50%. The fixed mechanism of transformation results in a fail-safe strategy for the population as half of the population generally keeps an intact copy of the original genome. Together, this work advances our understanding of pneumococcal genome plasticity.

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  1. eLife

    ###Reviewer #3:

    The properties and mechanism of DNA transformation by Streptococcus pneumoniae have been intensively studied for nearly a century. This elegant and insightful paper develops a powerful new set of quantitative assays based on recombining out stop codons of fluorescent protein fusions to reprise several issues that have largely been addressed by conventional antibiotic resistance selection. This approach leads to new answers for a number of fundamental questions about pneumococcal transformation, thereby re-setting the paradigm in this area. This is an extremely well-written, complete study that answers interesting and important questions about bottlenecks and recombination during transformation of this genetically plastic pathogen.

    This is a rigorous study that represents a substantial amount of work and creative thinking. The results will be of interest to a large audience concerned with genome evolution by transformation in different bacteria, points of limitation, or not, in the different steps in transformation, and mechanisms of recombination. The conclusions of this paper are well supported by extensive, often corroborative data, and provide new insights that go way beyond traditional genetic approaches. Rather complicated assay schemes are presented in highly effective diagrams and descriptions. Some of the new findings include that: all pneumococcal cells become competent and express the competence machinery in response to added competence stimulatory peptide or during natural competence; confirmation of brief non-genetic inheritance of phenotypes during transformation by single-cell tracking of recombination through lineage trees; a ≈50% limitation of transformation through RecA-dependent recombination that is unaffected by mismatch repair or restriction/modification; cell-cycle independence of recombination, regardless of reading strand or distance to the origin of replication; quantitation of direct multiple recombination (up to three was tested); and reduction of transformation recombination by non-homologous DNA.

    Many of these conclusions overturn and/or refine previous results that were obtained by less precise genetic methods. Together, this paper shows that any site or orientation with regard to DNA replication can be transformed in pneumococcal cells, including multiple chromosomal insertions; however, there is an intrinsic limitation to the efficiency of recombination, possibly related to the level of off-marker recombination. This limitation may have implications to pneumococcal evolution.

  2. eLife

    ###Reviewer #2:

    In this work Kurushima et al. use recently developed fluorescent labelling techniques to study natural transformation in the human pathogen Streptococcus pneumoniae. Previously, genetic marker analyses have been used to study the different aspects of this process, but with these new techniques the process can now be studied at the single cell level. The authors used the single cell analysis to identify new transformation bottlenecks and tried to determine why some cells are genetically transformed and others are not. Related experiments have been performed in the past using classic genetics and Kurushima et al. were able to confirm these studies. In that sense, in my opinion, the novelty is limited and no important new molecular insights are provided. They found that the number of cells that are ultimately transformed is plateauing at approximately 50%, despite the fact that most cells bind DNA. This is partially the result of the heteroduplex formed after recombination followed by separation by strand replication, combined with the fact that the DNA binding sites on cells are limited so that there is a competition between DNA markers at saturating DNA concentrations. The authors argue that this mechanism entails a "fail-safe strategy for the population as half of the population generally keeps an intact copy of the original genome". I find this conclusion far-fetched for two reasons.

    Firstly, the DNA recombination event followed by DNA replication will automatically assure that only half the population will inherit the mutation, and to speak of a strategy implies that the organism has specifically evolved this system, but we are dealing here with a well-known and general recombination system found in many organisms that will generally result in a 50/50 distribution. Maybe more importantly, under natural conditions it is highly unlikely that cells encounter saturating levels of tDNA. In their experiments the authors use 3.2 nM DNA for transformation. If my calculation is correct, this would amount to 19xE11 DNA molecules per ml, which seems a bit high when assuming tDNA comes from lysed bacteria. In nature, this number will be much (much) smaller therefore there is no need for the bacterium to come up with a dedicated strategy to assure that not all cells in a population are being transformed.

    Finally, the results are very well presented and the paper makes easy reading.

  3. eLife

    ###Reviewer #1:

    Overall I thought this to be an extremely compelling story, both in terms of general scientific interest and the overall high degree of experimental rigor. Overall, the data provides strong experimental evidence to support the authors conclusions.

    Overall, I found it very interesting the maximal efficiency is capped at 50%, as this makes for a very intriguing evolutionary hedge betting strategy for a naturally competent bacterial pathogen that frequently undergoes both intra and inter-species recombination events. In addition, this study provides a very elegant experimental framework for understanding the finer points of pneumococcal recombination through both clever genetic approaches and rigorous experimental design. The data was presented in a clear, concise manner and the overall manuscript followed a clear and logical progression.

  4. eLife

    ##Preprint Review

    This preprint was reviewed using eLife’s Preprint Review service, which provides public peer reviews of manuscripts posted on bioRxiv for the benefit of the authors, readers, potential readers, and others interested in our assessment of the work. This review applies only to version 2 of the manuscript.

    ###Summary:

    All three reviewers felt that the manuscript was experimentally sound and praised the authors for their use of single cell analysis to tackle the question of why some cells are transformed and others are not in a population of genetically competent Pneumococci. The thoughtful presentation of complicated and extensive data was appreciated by all. Two reviewers were enthusiastic about the study's conclusions regarding bet hedging and the potential for an intrinsic limit on recombination efficiency. The latter reduces the potential for off-marker recombination which, as Reviewer #3 notes, might have implications to pneumococcal evolution. At the same time, Reviewer #2 had some reservations about the significance of the data in light of previous studies of Pneumococcus and other naturally competent organisms. Most importantly, this reviewer questions whether the finding that only a portion of bacteria incorporate exogenous DNA is a particularly novel one and, regardless, whether the saturating DNA concentrations used in the study are representative of a "natural" environment.

  5. Version published to 10.1101/2020.03.15.992354 on bioRxiv