HU decreases diffusible supercoiling density in bacterial chromosome

Abstract

HU is an abundant protein in bacterial chromosome. It binds DNA and constrains negative supercoiling as well. In E.coli and Salmonella, it exists as a heterodimer composed of alpha and beta subunits. To study in vivo supercoiling that exists in hupA, hupB and hupAhupB double mutants, we employed the gamma delta resolution assay. Supercoil sensors were placed eight sites around the chromosome and resolution assay done to determine diffusible supercoil densities at these sites. Our studies showed that in addition to constraining supercoiling HU also enhances diffusible supercoiling either by its direct interaction with the chromosome or through gyrase activity. Supercoil density was measured to be − 0.035±0.002 in WT then reduced to −0.027±0.006, −0.022±0.007 and 0.013±0.004 in hupA, hupB and HupAhupB double mutants respectively. We observed significant differences between hupA and hupB subunits as well with hupA dimers constrains supercoiling better than hupB. We then measured transcription elongation rates in these mutants using beta galactosidase assay. HU double mutants have reduced transcription elongation rates 26 nuc/sec from WT 38–70 nuc/sec. Though hupA and hupB have similar reduced elongation rates (37 – 46 nuc/sec) alpha‐dimers had a slightly higher rate than beta‐dimers. Taken together, we were surprised that unlike eukaryotes where histone loss activates transcription, in bacteria this seems the opposite.This work was supported by National Science Foundation