Newly-discovered behaviour in the bacterial histone-like protein, HU

Abstract

This paper summarizes the contents of a talk delivered at the MS University of Baroda (Vadodara, Gujarat) on 3^rd March, 2023, at a conference held to celebrate proteins in commemoration of the birth centenary of Prof. G. N. Ramachandran. Here, we review several recent discoveries and applications from our group that relate to HU, a DNA-binding nucleoid-associated protein found in bacteria: (1) HU uses its DNA-binding sites to bind to lipopolysaccharide (LPS) upon bacterial cell surfaces, in the extracellular milieu in biofilms, thus working as a glue to attach bacteria to extracellular DNA; (2) HU and DNA perform mutual macromolecular crowding, as well as mutual charge neutralization, to together undergo condensation into nucleoids that appear to maintain DNA in a compacted state in bacterial genomes through liquid-liquid phase separation (LLPS); (3) HU appears to have evolved to avoid use of the amino acid residue, tryptophan, to avoid damage to bacterial genomic DNA by a combination of sunlight and photosensitized oxidation by tryptophan photodecomposition products; (4) HU’s N-terminal (positive) charge destabilizes hydrophobic inter-subunit interactions between beta strands in HU dimers and promotes subunit exchange between HU-A and HU-B (the two isoforms of HU in gut bacteria), thus hindering the facile dissociation of subunits if an N-terminal 6xHis affinity tag is present; (5) HU-A and HU-B can be genetically fused to generate a simulacrum of an HU heterodimer; (6) HU’s DNA-binding regions from two bacterial homologs (one mesophilic and the other thermophilic) can be isolated and genetically fused to generate a novel thermostable DNA-binding protein; (7) HU’s ability to titrate onto the bacterium’s nucleoid can be exploited to deploy fluorescent protein-labelled HU in cells to test for leaky expression from bacterial promoters, using fluorescence microscopy.