Single‐molecule tracking reveals that the nucleoid‐associated protein HU plays a dual role in maintaining proper nucleoid volume through differential interactions with chromosomal DNA

Abstract

HU (Histone‐like protein from Escherichia coli strain U93) is the most conserved nucleoid‐associated protein in eubacteria, but how it impacts global chromosome organization is poorly understood. Using single‐molecule tracking, we demonstrate that HU exhibits nonspecific, weak, and transitory interactions with the chromosomal DNA. These interactions are largely mediated by three conserved, surface‐exposed lysine residues (triK), which were previously shown to be responsible for nonspecific binding to DNA. The loss of these weak, transitory interactions in a HUα(triKA) mutant results in an over‐condensed and mis‐segregated nucleoid. Mutating a conserved proline residue (P63A) in the HUα subunit, deleting the HUβ subunit, or deleting nucleoid‐associated naRNAs, each previously implicated in HU’s high‐affinity binding to kinked or cruciform DNA, leads to less dramatically altered interacting dynamics of HU compared to the HUα(triKA) mutant, but highly expanded nucleoids. Our results suggest HU plays a dual role in maintaining proper nucleoid volume through its differential interactions with chromosomal DNA. On the one hand, HU compacts the nucleoid through specific DNA structure‐binding interactions. On the other hand, it decondenses the nucleoid through many nonspecific, weak, and transitory interactions with the bulk chromosome. Such dynamic interactions may contribute to the viscoelastic properties and fluidity of the bacterial nucleoid to facilitate proper chromosome functions.

Separate physiological roles of specific and non-specific DNA binding of HU protein in Escherichia coli

“…We observed cooperative binding of wild-type HUα 2 to both the linear duplex and cruciform DNA in solution with similar calculated Hill coefficients ( h ) and dissociation constants ( K d ) (non-specific binding: h = 3.2 and K d = 0.101 µM; structure-specific binding: h = 2.8 and K d = 0.096 µM) (Figure 1). However, the linear duplex did not migrate as discrete retarded species in EMSA, consistent with weak and transitory non-specific binding (Bettridge et al, 2020; Pinson et al, 1999), but the cruciform DNA migrated as four discrete bands, indicative of strong, cooperative binding of each cruciform DNA molecule to four molecules of HUα 2 as suggested previously (Bonnefoy et al, 1994). The P63A mutation reduced the cooperative binding to cruciform DNA with K d = 0.149 µM, h =1.6, validating the importance of P63 in binding to a distorted DNA as suggested by crystal structures of HU-structure-specific DNA complexes (Guo and Adhya, 2007; Swinger et al, 2003), but it did not affect the binding to the linear DNA duplex ( K d 0.106 µM, h = 3.0).…”

“…Here the HU structure-specific binding indirectly impacts the chromosome partitioning through alteration of gene expression. We found that only the non-specific binding participated in a major role of HU - its association with the chromosomal DNA that dictates the chromosome structure and organization. By tracking single molecule analysis in live cells, we have previously demonstrated (Bettridge et al, 2020) and further confirmed here that the non-specific binding of HU is responsible for this interaction with rapid association and dissociation kinetics that keep most HU within the nucleoid. Mutating the three lysine residues to alanine dramatically altered the interacting dynamics of HUα 2 but mutating P63 to alanine did not have any effect (Bettridge et al, 2020).…”

“…By tracking single molecule analysis in live cells, we have previously demonstrated (Bettridge et al, 2020) and further confirmed here that the non-specific binding of HU is responsible for this interaction with rapid association and dissociation kinetics that keep most HU within the nucleoid. Mutating the three lysine residues to alanine dramatically altered the interacting dynamics of HUα 2 but mutating P63 to alanine did not have any effect (Bettridge et al, 2020). We do not rule out that structure-specific binding of HU at some defined sites may also play some architectural role in the chromosome as well. In summary, our findings demonstrate that non-specific and structure-specific DNA binding modes of HU plays distinct roles: (a) HU has many targeted roles in various DNA metabolic processes like gene transcription, DNA supercoiling, chromosome positioning and partitioning.…”

“…The structure-specific binding, while not necessary for overall chromosome structure, is critical for the regulation of specific gene expressions by HU. On the contrary, HU binds to DNA as part of the chromosome structure primarily through non-specific binding as we previously suggested from single cell analysis (Bettridge et al, 2020). Our findings reveal distinct physiological roles of both structure-specific and non-specific DNA binding modes of HU.…”

“…By consolidating these multiple lines of evidence, we argue that the multimerization of HUα 2 molecules non-specifically bound to numerous positions on the chromosomal DNA brings distant genomic loci together. The rapid interacting dynamics of HU with DNA (Bettridge et al, 2020) may be necessary for the newly replicated genome to dynamically reorganize as the genome progressively segregate during DNA replication (Nielsen et al, 2006). Most target specific DNA binding proteins use base sequences specific motifs; their non-specific DNA binding abilities are relegated to as background noise.…”