Biology subjects

Gussak, A.

Publications and source records attributed to Gussak, A..

2 recordsLinked to original sources

bioRxiv2020

Complement active human and porcine serum induces natural competence for genetic transformation in the emerging zoonotic pathogen Streptococcus suis

The acquisition of novel genetic traits by natural competence is a strategy used by bacteria in microbe-rich environments including animal or human hosts where microbial competition, antibiotics and host immune defences threaten their survival. We show here that several virulent strains of Streptococcus suis, an important porcine pathogen and zoonotic agent, become naturally competent for genetic transformation with plasmid or genomic DNA when cultured in active porcine and human serum, but not when it is pre-heated for 30 minutes at 56{degrees}C to inactivate complement. Competence is also not induced in active fetal bovine serum, which contains less complement factors and immunoglobulins than adult serum. Late competence genes, encoding the uptake machinery for environmental DNA, were highly upregulated in active serum. Competence development was independent of the early competence regulatory switch suggesting the presence of an alternative stress-induced pathway for regulation of the transformasome, a type 4-like pilus DNA binding and transport apparatus.

microbiology↗

bioRxiv2017

Characterizing a thermostable Cas9 for bacterial genome editing and silencing

CRISPR-Cas9 based genome engineering tools have revolutionized fundamental research and biotechnological exploitation of both eukaryotes and prokaryotes. However, the mesophilic nature of the established Cas9 systems does not allow for applications that require enhanced stability, including engineering at elevated temperatures. Here, we identify and characterize ThermoCas9: an RNA-guided DNA-endonuclease from the thermophilic bacterium Geobacillus thermodenitrificans T12. We show that ThermoCas9 is active in vitro between 20{degrees}C and 70{degrees}C, a temperature range much broader than that of the currently used Cas9 orthologues. Additionally, we demonstrate that ThermoCas9 activity at elevated temperatures is strongly associated with the structure of the employed sgRNA. Subsequently, we develop ThermoCas9-based engineering tools for gene deletion and transcriptional silencing at 55{degrees}C in Bacillus smithii and for gene deletion at 37{degrees}C in Pseudomonas putida. Altogether, our findings provide fundamental insights into a thermophilic CRISPR-Cas family member and establish the first Cas9-based bacterial genome editing and silencing tool with a broad temperature range.

microbiology↗