Abstract
The bacterial chromosome is under varying levels of mechanical stress due to a high degree of crowding and dynamic protein–DNA interactions experienced within the nucleoid. DNA tension is difficult to measure in cells and its functional significance remains unclear although in vitro experiments have implicated a range of biomechanical phenomena. Using single-molecule tools, we have uncovered a novel protein–DNA interaction that responds to fluctuations in mechanical tension by condensing DNA. We combined tethered particle motion (TPM) and optical tweezers experiments to probe the effects of tension on DNA in the presence of the Hha/H-NS complex. The nucleoid structuring protein H-NS is a key regulator of DNA condensation and gene expression in enterobacteria and its activity in vivo is affected by the accessory factor Hha. We find that tension, induced by optical tweezers, causes the rapid compaction of DNA in the presence of the Hha/H-NS complex, but not in the presence of H-NS alone. Our results imply that H-NS requires Hha to condense bacterial DNA and that this condensation could be triggered by the level of mechanical tension experienced along different regions of the chromosome.
Highlights
IntroductionEpigenetic and transient regulatory mechanisms guide cellular function
Hha and H-NS expression constructs were transformed into the E. coli BL21 (DE3) strain, and the transformants were selected on Luria-Bertani (LB) agar plates supplemented with 100 g/ml ampicillin
We performed a series of single-molecule experiments on the Hha/H-NS complex to determine what effect, if any, Hha may have on the ability of H-NS to alter the properties of the DNA to which it is bound
Summary
Epigenetic and transient regulatory mechanisms guide cellular function. For example, the various topological conformations displayed by the bacterial chromosome. The bare chromosomal DNA of Escherichia coli would have a radius of gyration of ∼5 m, which is significantly larger than the size of the cell (1–2 m). It is no wonder that conformational changes in bacterial DNA, arising from compaction and crowding, are involved in a multitude of cellular processes. It has been suggested that the entropic forces, which such a tightly confined polymer would exhibit, are an important factor in chromosome organization and segregation and lead to, for example, the spontaneous de-mixing of daughter strands during cell division [4]. We are just beginning to understand how the biomechanical and dynamical properties of the chromosome affect cell function
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