Single molecule tracking reveals the role of transitory dynamics of nucleoid-associated protein HU in organizing the bacterial chromosome

Abstract

13 HU is the most conserved nucleoid-associated protein in eubacteria and has been implicated as a 14 key player in global chromosome organization. The mechanism of HU-mediated nucleoid 15 organization, however, remains poorly understood. Using single molecule tracking coupled with 16 genetic manipulations, we characterized the dynamics of HU in live Escherichia coli cells. We 17 found that native HU dimers bind and unbind chromosomal DNAs weakly and transitorily across 18 the entire nucleoid volume but remain nucleoid-localized, reminiscent of random diffusion in a 19 liquid phase-separated, membrane-less "macro-compartment" distinct from the remaining 20 cytosol. Mutating three key surface lysine residues of HU nearly entirely abolished the weak and 21 transitory interactions of HU with DNA and led to severe cell growth and DNA segregation 22defects, suggesting the importance of HU's interactions with chromosomal DNA mediated by 23 the positively charged surface. A conserved proline residue important for recognizing bent and 24 cruciform DNAs such as that in recombination intermediates, similarly abolished HU's rapid and 25 transitory DNA interaction dynamics but had little impact on its apparent binding stability with 26 nonspecific chromosomal DNAs. Interestingly, the proline residue appeared to be important for 27HUαβ dimer formation as mutating this residue makes HUαβ behave similarly to HUα 2 dimers. 28Finally, we find that while prior evidence has found HU capable of depositing nucleoid-29 associated noncoding RNAs onto cruciform DNA structures, deletion of these specific naRNAs 30 or inhibition of global transcription had a relatively minor effect on HU dynamics irrespective 31 altered nucleoid compaction. Our results suggest a model of chromosome organization mediated 32 by weak, transient interactions of HU, a substantial deviation from nucleoid-like proteins such as 33 histones. Such collective sum of the numerous weak, transitory binding events of HU with 34 nonspecific chromosome DNAs could generates a "force" to maintain a dynamic, fluid nucleoid 35 with enough flexibility to rapidly facilitate global topological processes such as replication or 36 nucleoid segregation. 37 termed the nucleoid and organized into six spatially isolated macrodomains (MD) 1-5 . The precise 41 molecular mechanism of how this nucleoid organization is achieved is not well understood, but 42 many key players have been identified. A group of proteins known as nucleoid-associated 43 proteins (NAPs), such as H-NS, IHF, Fis, and HU have been shown to play important roles 44 (reviewed in 3 ). Among these NAPS, HU is the most conserved across eubacteria and the one of 45 the most abundant in E. coli ( ~30,000 copies per cell during middle exponential phase growth 6,7 ). 46 E. coli HU has two subunits of ~9 kDa each, α and β, which form either HUα 2 homodimers or 47HUαβ heterodimers depending on the growth phase (HUβ 2 homodimers were negligible under 48 log-phase growth) 7 . Monomers of HU are not detectable 6,7 . HU was origina...