MukBEF-dependent chromosomal organization in widened Escherichia coli

Abstract

The bacterial chromosome is spatially organized through protein-mediated compaction, supercoiling, and cell-boundary confinement. Structural Maintenance of Chromosomes (SMC) complexes are a major class of chromosome-organizing proteins present throughout all domains of life. Here, we study the role of the Escherichia coli SMC complex MukBEF in chromosome architecture and segregation. Using quantitative live-cell imaging of shape-manipulated cells, we show that MukBEF is crucial to preserve the toroidal topology of the E. coli chromosome and that it is non-uniformly distributed along the chromosome: it prefers locations towards the origin and away from the terminus of replication, and it is unevenly distributed over the origin of replication along the two chromosome arms. Using an ATP hydrolysis-deficient MukB mutant, we find that MukBEF translocation along the chromosome is ATP-dependent, in contrast to its loading onto DNA. MukBEF and MatP are furthermore found to be essential for sister chromosome decatenation. We propose a model that explains how MukBEF, MatP, and their interacting partners organize the chromosome and contribute to sister segregation and recombination. The combination of bacterial cell-shape modification and quantitative fluorescence microscopy paves way to investigating chromosome-organization factors in vivo.

Direct observation of a crescent-shape chromosome inBacillus subtilis

“…In E. coli, for example, direct microscopy observation of chromosomes in expanded-volume cells did resolve the local DNA condensation in the left and right chromosomal arm as well as a lack of structure within the ter domain 16 . The approach furthermore yielded new insight into chromosome replication 27,30 and the role of SMC/MukBEF proteins 15,28 in chromosome organization.…”

“…However, questions remain as Hi-C methods involve extensive cell fixation which can alter the DNA organization 22 , and conclusions are drawn from population averages where the single-cell structure and cell-to-cell variabilities are lost. A promising way to spatially resolve the bacterial chromosome is to use cell-shape manipulation techniques 16,[27][28][29][30] or expansion microscopy (ExM) [31][32][33] . While expansion microscopy, where bacteria are encapsulated in a hydrogel that is stretched, requires cell fixation that precludes live cell imaging 33 , cell-shape manipulation techniques allow for imaging of live cells, while a single chromosome can be maintained via DNA replication halting.…”