Genome-wide mapping of genetic suppression events in Streptococcus pneumoniae

Historically, suppressor analysis has been a powerful tool for uncovering the functions of essential genes. In this study, we report Sup-seq, a transposon-sequencing technique with random DNA barcodes that enables genome-wide detection of genetic suppression. When applied to seven essential genes in Streptococcus pneumoniae, Sup-seq revealed 53 suppressors across various cellular pathways. These suppressors function through diverse mechanisms, such as gene inactivation, overexpression, or downregulation, depending on the locations and orientations of the transposon insertions. Our proof-of-concept experiments identified a previously uncharacterized transcriptional regulator that controls chaperone and ribosomal protein expression, as well as a mechanism that bypasses the requirement for acetate kinase for growth. Furthermore, we found that phosphatidylglycerol is conditionally essential in pneumococcus, revealing an unexpected link between phospholipid synthesis and potassium transport. These findings demonstrate that Sup-seq is a facile approach for providing mechanistic insights into the functions of essential genes. Additionally, Sup-seq can be adapted to other bacteria, aiding in the identification of new antimicrobial targets. Here the authors present ‘Sup-seq’, a transposon-based sequencing technique that enables genome-wide detection of genetic suppression in bacteria. They use it to identify 53 genetic suppressors of seven essential genes in pneumococcus, revealing new links among metabolism, ribosome assembly, and membrane lipid synthesis.