Precision Genome Engineering in Streptococcus suis Based on a Broad-Host-Range Vector and CRISPR-Cas9 Technology.

Streptococcussuis is an important zoonotic pathogen that causes severe invasive disease in pigs and humans. Current methods for genome engineering of S. suis rely on the insertion of antibiotic resistance markers, which is time-consuming and labor-intensive and does not allow the precise introduction of small genomic mutations. Here we developed a system for CRISPR-based genome editing in S. suis, utilizing linear DNA fragments for homologous recombination (HR) and a plasmid-based negative selection system for bacteria not edited by HR. To enable the use of this system in other bacteria, we engineered a broad-host-range replicon in the CRISPR plasmid. We demonstrated the utility of this system to rapidly introduce multiple gene deletions in successive rounds of genome editing and to make precise nucleotide changes in essential genes. Furthermore, we characterized a mechanism by which S. suis can escape killing by a targeted Cas9-sgRNA complex in the absence of HR. A characteristic of this new mechanism is the presence of very slow-growing colonies in a persister-like state that may allow for DNA repair or the introduction of mutations, alleviating Cas9 pressure. This does not impact the utility of CRISPR-based genome editing because the escape colonies are easily distinguished from genetically edited clones due to their small colony size. Our CRISPR-based editing system is a valuable addition to the genetic toolbox for engineering of S. suis, as it accelerates the process of mutant construction and simplifies the removal of antibiotic markers between successive rounds of genome editing.

Active Human and Porcine Serum Induce Competence for Genetic Transformation in the Emerging Zoonotic Pathogen Streptococcus suis.

The acquisition of novel genetic traits through natural competence is a strategy used by bacteria in microbe-rich environments where microbial competition, antibiotics, and host immune defenses threaten their survival. Here, we show that virulent strains of Streptococcus suis, an important zoonotic agent and porcine pathogen, become competent for genetic transformation with plasmid or linear DNA when cultured in active porcine and human serum. Competence was not induced in active fetal bovine serum, which contains less complement factors and immunoglobulins than adult serum and was strongly reduced in heat-treated or low-molecular weight fractions of active porcine serum. Late competence genes, encoding the uptake machinery for environmental DNA, were upregulated in the active serum. Competence development was independent of the early competence regulatory switch involving XIP and ComR, as well as sigma factor ComX, suggesting the presence of an alternative stress-induced pathway for regulation of the late competence genes required for DNA uptake.