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

Abstract

The spread of antimicrobial resistance and vaccine escape in the human pathogen Streptococcus pneumoniae can be largely attributed to competence-induced transformation. Here, we studied this process at the single-cell level. We show that within isogenic populations, all cells become naturally competent and bind exogenous DNA. 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 and because a single stranded donor DNA replaces the original allele, transformation efficiency has an upper threshold of approximately 50% of the population. 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.

The acquisition of clinically relevant amoxicillin resistance in Streptococcus pneumoniae requires ordered horizontal gene transfer of four loci

“…Transformation efficiency increased with length of both the overall molecule, and the homologous region (Figure 6A). This is in line with previous studies showing the importance of molecule length and homology in pneumococcal transformation (Kurushima et al, 2020; Morrison and Guild, 1972). We hypothesize that increased length may increase the likelihood of spontaneous contact with the ComGC pilus, while also decreasing the likelihood of homologous region fragmentation by EndA, prior to entry (Morrison and Guild, 1973).…”

“…Transformation efficiency increased with length of both the overall molecule, and the homologous region (Figure 6A). This is in line with previous studies showing the importance of molecule length and homology in pneumococcal transformation (Kurushima et al, 2020;Morrison and Guild, 1972). The efficiencies of the short overlapping fragments were lower than that of the long fragments, although increased slightly when transformed in combination.…”

“…As molecule length affects transformation efficiency (Keller et al, 2019; Kurushima et al, 2020; Lee et al, 1998), an additional experiment was performed, where the amplified fragments were all extended to 6 Kb by the addition of non-homologous DNA amplified from E. coli and ligated to one end using Golden Gate assembly with BsmBI and T4 DNA ligase (NEB, Vazyme). All oligos and the corresponding templates used in the cloning for these experiments are outlined in Table S6, and a comprehensive oligo list can be found in Table S2.…”

Harnessing CRISPR-Cas9 for Genome Editing in Streptococcus pneumoniae D39V

“…Next, an HR template is constructed that consists of the upstream and downstream regions of the deletion target. It has been shown that higher transformation efficiency is observed at higher donor DNA concentrations and with longer homology regions, with a plateau of approximately 1,000 bp of homology ( 34 , 35 ). Therefore, each selected homology arm is designed to be at least 1,000 bp, ensuring efficient homologous recombination.…”

Programming bacteria for multiplexed DNA detection

“…Due to the transformation efficiency and doubling time of the cells, the total experimental time for Cell-Sens to perform detection of a target DNA sequence was approximately one day. To speed up the DNA detection workflow, flow cytometry or fluorescence microscopy could be used to quantify fluorescent cells within hours following transformation before selection 47 . Future work will investigate the generalizability of the DNA sensor to sensing other organisms beyond bacteria, including viruses, fungi or cancer cells 48 .…”