Genome plasticity is governed by double strand break DNA repair in Streptomyces

Abstract

The linear chromosome of the bacterium Streptomyces exhibits a remarkable genetic organization with grossly a central conserved region flanked by variable chromosomal arms. The terminal diversity co-locates with an intense DNA plasticity including the occurrence of large deletions associated to circularization and chromosomal arm exchange. These observations prompted us to assess the role of double strand break (DSB) repair in chromosome plasticity following. For that purpose, DSBs were induced along the chromosome using the meganuclease I-SceI. DSB repair in the central region of the chromosome was mutagenic at the healing site but kept intact the whole genome structure. In contrast, DSB repair in the chromosomal arms was mostly associated to the loss of the targeted chromosomal arm and extensive deletions beyond the cleavage sites. While homologous recombination occurring between copies of DNA sequences accounted for the most part of the chromosome rescue events, Non Homologous End Joining was involved in mutagenic repair as well as in huge genome rearrangements (i.e. circularization). Further, NHEJ repair was concomitant with the integration of genetic material at the healing site. We postulate that DSB repair drives genome plasticity and evolution in Streptomyces and that NHEJ may foster horizontal transfer in the environment.

Figure 1

I-SceI DSB induction and chromosomal survey device. (A) I-SceI mediated DSB targeting in S. ambofaciens chromosome. The target sequences for I-SceI meganuclease are carried by the IKI cassette designed with neo gene, conferring resistance to kanamycine, flanked by two convergent I-SceI restriction sites (dark triangle). Red and blue boxes represent left and right replichores of the chromosome. Grey arrows represent the terminal inverted repeats at each chromosome extremity. The IKI cassette was integrated either in the core genome (IKI-C, position 4.94 Mb), in the left chromosomal arm (IKI-L, position 0.32 Mb) or in the right chromosomal arm (IKI-R, position 7.91 Mb). The cassette was inserted at IKI-L or IKI-R loci through homologous recombination. The IKI-C locus corresponds to the attachment site of phage φBT1 (attB φBT1) where pMS82 conjugative plasmid carrying IKI cassette (therefore named pMS82-IKI) was integrated. Meganuclease encoding I-SceI gene was carried by pDYN6902 conjugative plasmid and integrated in the chromosome at the attachment site of phage φC31 (attB φC31). Scissors symbolize the DSB (I-SceI restriction sites) triggered by the meganuclease. (B) Distribution of the loci used for chromosome mapping after DSB induction. T stands for loci included in the TIRs. L, C and R for left, central and right loci, respectively. The data analysis is schematically represented on Fig. 4.

Figure 2

Kanamycin sensitive (Kan^S) clones appearance after DSB generation at IKI-C. Proportion of Kan^S clones obtained in each independent lineage (symbolized by a flake) of WT, ∆kuA and ∆ligCD contexts. For each of the 50 lineages, an average of 230 clones was analyzed by replica-plating with and without kanamycin.

Figure 3

DNA repair features after DSB generation at IKI-C. (A) IKI-C locus: the left panel shows the 30 kb region surrounding the insertion site of pMS82-IKI at IKI-C locus. Scale is in kilobases. Locus_tags of the CDS delimiting the region are indicated according to Thibessard et al.. The right panel focuses on the IKI cassette structure and sequence. Since the I-SceI recognition site is a non palindromic sequence, the resulting restricted ends are distinguished by two different colors (orange and purple). The neo gene is colored in yellow while hygromycin resistance gene (hyg^R) is colored in cyan. All DNA repair event examples are represented at these two different scales (see B). (B) Examples of deletion events occurring at IKI-C at the kilobase (left panel) and nucleotide (right panel) scales. Grey boxes represent the region flanking IKI-C cassette. Deleted regions are symbolized by dotted lines. The ∆ symbol indicates the number of deleted nucleotides at each restricted end. When the ∆ symbol is followed by neo, it indicates a complete deletion of the kanamycin resistance gene. (C) DNA integration cases. Nucleotides added by fill-in synthesis are colored in grey. Nucleotides added by non-template addition are colored in green. DNA fragments integrated during repair are boxed in green. Microhomologies present at the repair scar are underlined.

Figure 4

DNA rearrangements occurring in survivors after DSB induction at IKI-L and IKI-R. The chromosome of survivors were mapped using a set of targets for PCR amplication distributed along the chromosome (Fig. 1B). The scale is given in Mb. Grey arrows represent the terminal inverted repeats at each chromosome extremity. The DSB sites (IKI-L or IKI-R) are represented by the vertical white dotted lines. The black boxes represent the deleted areas. The occurrence of each deletion pattern (among 20 strains analyzed per chromosomal arm) and the strains chosen for whole genome sequencing are mentioned on the right.

Figure 5

Chromosomal arm replacement by homologous recombination after DSB induction at IKI-L. (A) Analysis of the coverage of sequencing reads along the chromosome of a DSB survivor at IKI-L (WT17). Genome sequencing reads of strain WT17 plotted along the S. ambofaciens ATCC 23877 genome as a reference are shown as a pink area. Yellow boxes symbolize HR1-R and HR1-L (two duplicated copies of a sigma factor encoding gene: hasL, SAM23877_0517, and hasR, SAM23877_6930). (B) Hybrid read resulting from recombination between HR1-L and HR1-R sequences. HR1-L and HR1-R sequences were aligned with one typical hybrid_read found in WT17 sequencing data. The alignment shown covers the 3’ end of the hasL/R sequences (from position 600 to 846). Polymorphism is symbolized with a colored background. The stop codon is boxed. (C) WT17 chromosome structure. The hybrid HR1 sequence is labelled HR1-L/R. The I-SceI cut at IKI-L resulted in the loss of the left chromosomal arm and in a homologous recombination event between HR1-R and HR1-L leading to the duplication of the right chromosomal arm over 825 kb.

Figure 6

Chromosomal circularization after DSB induction at IKI-L. HR2-L and HR2-R are two homologous regions sharing over 99% nucleotide identity along 1,672 bp and are symbolized as salmon pink boxes. The hybrid HR2 sequence is labelled HR2-L/R. HR3 is a region including two identical transposase encoding genes (SAM23877_7417 and SAM23877_7422) in opposite direction. Similarly, HR4 is a region including two identical transposase encoding genes (SAM23877_7326 and SAM23877_7404) in opposite direction. HR3 and HR4 are symbolized as purple boxes. Illegitimate recombination scars are represented by black wave lines. Green boxes represent inserted DNA stretches. Amplified sequences are marked out by light blue boxes between brackets. In some cases, circularization occurred by simple event of fusion between sequences present on both arms with (a) or without (b) DNA insertion. The other cases are typified by a common feature, which consists in the deletion of the left arm until the HR2-L locus and its recombination with its homologous region on the right arm (HR2-R), leading to the formation of a hybrid HR2-R/L sequence. This arm replacement is concomitant with circularization through different molecular event: HR involving HR3 or HR4 (c), IR (d) sometimes accompanied by DNA amplification (e) or DNA insertion (f). For case e, the read coverage level of the amplified region prevents the identification of the fusion sequence and thus the nature of the recombination event. For case (g), the circularization may have occurred through recombination between two short homologous sequences (41 bp) in opposite direction.

Figure 7

Fates of the chromosome structure following DSB and arm replacement. Red and blue boxes represent left and right replichores of the chromosome respectively. Grey arrows and the grey oval represent the terminal inverted repeats and the terminal proteins attached to them. HR2-L and HR2-R are symbolized as salmon pink boxes. The hybrid HR1 and HR2 sequences are labelled HR1-L/R and HR2-L/R respectively. HR3 and HR4 are symbolized as purple boxes. The AUD90 locus, susceptible to DNA amplification is marked out by light blue boxes between brackets. The tap-tpg locus encoding the terminal associated protein and the terminal protein respectively is represented by a black star. The yellow PacMan symbolizes the accessibility of the chromosome end to nuclease activities. After DSB induction at IKI-L, DNA resection progresses towards the inner parts of the chromosome concomitantly with the loss of the distal part of the arm. In case (1), DNA resection does not reach the tap-tpg locus, and arm replacement occurs by HR between HR1-R and HR1-L. Since tap-tpg locus is preserved, the linear configuration of the rearranged chromosome is stable. In case (2), DNA resection gets past tap-tpg locus and arm replacement is initiated by HR between HR2-R and HR2-L. The tap-tpg being deleted, this chromosome is unstable and irremediably leads to stabilization through circularization by different ways involving either HR3, or HR4 or an amplified sequence as previously shown.