Chromosome segregation dynamics during the cell cycle of Staphylococcus aureus

Research on chromosome organization and cell cycle progression in spherical bacteria, particularly Staphylococcus aureus, remains limited and fragmented. In this study, we established a working model to investigate chromosome dynamics in S. aureus using a Fluorescent Repressor-Operator System (FROS), which enabled precise localization of specific chromosomal loci. This approach revealed that the S. aureus cell cycle and chromosome replication cycle are not synchronized (i.e. they do not initiate simultaneously), with cells exhibiting two segregated origins of replication at the start of the cell cycle. The chromosome has a specific origin-terminus-origin conformation, with origins localizing near the membrane, towards the tip of each hemisphere, or the "cell poles". We further used this system to assess the role of various proteins with a function in S. aureus chromosome biology, focusing on the ParB-parS and SMC-ScpAB systems. Our results demonstrate that ParB binds five parS chromosomal sequences and the resulting complexes are required for specific chromosomal inter-arm alignment, but play a minor role in chromosome segregation. In contrast, the SMC-ScpAB complex plays a key role in S. aureus chromosome biology, contributing to chromosome segregation and spatial organization. Additionally, we systematically assessed and compared the impact of proteins linking chromosome segregation to cell division-Noc, FtsK, SpoIIIE and XerC-on origin and terminus number and positioning. This work provides a comprehensive study of the factors governing chromosome dynamics and organization in S. aureus, contributing to our knowledge on chromosome biology of spherical bacteria.