Direct observation of a crescent-shape chromosome inBacillus subtilis

Abstract

Bacterial chromosomes are folded into tightly regulated threedimensional structures to ensure proper transcription, replication, and segregation of the genomic information. Direct visualization of the chromosomal shape within bacterial cells is hampered by cell wall confinement and the optical diffraction limit. Here, we combine cell-shape manipulation strategies, high resolution fluorescence microscopy techniques, and genetic engineering to visualize the intrinsic shape of the bacterial chromosome in real time in liveBacillus subtiliscells. We show that the chromosome exhibits a crescent shape with a non-uniform DNA density that is increased near the origin of replication (oriC). Additionally, we localized ParB and BsSMC proteins – the key drivers of chromosomal organization – along the contour of the crescent chromosome, showing the highest density nearoriC. Opening of the BsSMC ring complex disrupted the crescent chromosome shape and instead yielded a torus shape. These findings help to understand the threedimensional organization of the chromosome and the main protein complexes that underlie its structure.

DNA supercoiling enhances DNA condensation by ParB proteins

“…Interestingly, previous work showed that brief rifampicin treatment of bacterial cells showed a complete loss of any higher-order organization within the origin region [64][65][66] , which is surprising as ParB proteins should have been unaffected in their ability to locally condense the DNA in the partition complex. This observation is consistent with the hypothesis that supercoiling is a crucial facilitator for the maintenance of the ParB-DNA partition complex, especially considering the small number of ParB proteins (~250-700) in a bacterial cell 23,25 .…”