A genome-wide genetic interaction platform for MRSA reveals connections between cell division and the cell envelope

The bacterial cell cycle is an ensemble of integrated pathways that coordinates cell growth and division. Although these pathways contain a suite of promising antimicrobial targets, their functional organization and interrelationships have only been sparsely mapped. Here, we established a dual-CRISPRi platform for systematic genetic interaction profiling in methicillin-resistant Staphylococcus aureus (MRSA), a major drug-resistant pathogen. Our platform achieved genome-wide coverage across a selected set of 51 cell cycle genes, surveying 115,497 gene-pairs and uncovering hundreds of genetic interactions, both positive (suppressors) and negative (synthetic sick/lethal). These interactions identified two major connections between cell division and the cell envelope. First, we identified a suppressive relationship between a defined set of cell division genes, including components of the major septal peptidoglycan biosynthesis complex PBP1-FtsWL-DivIBC, and fatty acid or phospholipid biosynthesis. Simultaneous inhibition of these lipid biosynthesis pathways suppressed the fitness and morphological defects caused by knocking down expression of cell division genes, revealing that toxic membrane accumulation contributes to the lethality of disrupting cell division. Second, we uncovered a synthetic lethal relationship between a distinct set of cell division genes and the conserved dltXABCD operon, linking cell division to the modification of teichoic acid surface polymers. Our results establish a versatile platform for interrogating selected gene sets in MRSA, providing a map of cell cycle interactions, and uncovering links between cell division and cell envelope pathways.