The DNA-binding protein HU is a molecular glue that attaches bacteria to extracellular DNA in biofilms

Abstract

In biofilms, bacteria that possess a negatively charged surface are embedded within a matrix of polymers consisting mainly of negatively charged extracellular DNA (e-DNA). In all likelihood, a multivalent positively charged substance, for example, a basic protein, exists within biofilms to neutralize charge–charge repulsions and act as a ‘glue’ attaching negatively charged bacteria to negatively charged e-DNA; however, no protein capable of doing so has yet been identified. We decided to investigate whether a highly abundant nucleoid-associated histone-like protein (HU) happens to be the glue in question. In recent years, HU has been shown to possess qualities that could be considered desirable in the proposed glue, for example, (a) availability in association with e-DNA; (b) multivalent DNA binding; (c) non–sequence-specific DNA-binding; (d) enhancement of biofilm formation upon exogenous addition, and (e) disruption of biofilms, upon removal by HU–cognate antibodies. Geometric considerations suggest that basic residues in HU's canonical and noncanonical DNA-binding sites can interact with sugar-linked terminal phosphates in lipopolysaccharide (LPS) molecules in bacterial outer membranes. Here, using genetic, spectroscopic, biophysical–chemical, microscopy-based, and cytometry-based experiments, we demonstrate that HU's DNA-binding sites also bind to LPS, that this facilitates DNA–DNA, DNA–LPS, and LPS–LPS interactions, and that this facilitates bacterial clumping and attachment of bacteria to DNA. Exogenous addition of HU to bacteria in (nonshaken) cultures is shown to cause cells to become engulfed in a matrix of DNA, potentially arising from the lysis of bacteria with vulnerable cell walls (as they strain to grow, divide, and move away from each other, in opposition to the accreting influence of HUs).

Acetylation at Lysine 86 of Escherichia coli HUβ Modulates the DNA-Binding Capability of the Protein

“…Neutralization of the positive charge at Lys86 can also be achieved by amino acid substitution. Indeed, replacement of Lys86 with alanine reduced the DNA binding capability of E. coli HUαβ (Thakur et al, 2021). A similar effect was found with Bacillus subtilis HU, where K86A mutation reduced the DNA binding capacity to 20% of the wild-type protein (Köhler and Marahiel, 1998).…”

“…Nevertheless, a DNA-bound structure of Anabaena HU (pdb: 1P51; Swinger et al, 2003), which shares 42% sequence identity and 71% sequence similarity to E. coli HUβ, is available and was employed to generate a homology model using SWISS-MODEL (Biasini et al, 2014;Bienert et al, 2016). Based on the homology model, the β-ribbon arms (amino acid residues 56-73) and the C-terminal α-helix (amino acid residues 82-90) are involved in DNA binding (Figure 1), and this assertion is supported by previous biochemical studies of E. coli HU (Bhowmick et al, 2014;Hammel et al, 2016;Agapova et al, 2020;Thakur et al, 2021). A report of E. coli acetylome revealed acetylation at Lys3/9/18/67/86 of HUβ (Castaño-Cerezo et al, 2014).…”

“…Therefore, Lys67 and Lys86 may be acetylated at the same time, giving a compounded effect on protein properties. Indeed, Thakur et al (2021) investigated modifications of E. coli HUβ and found when either Lys83 or Lys86 was replaced with alanine, or when the DNA-binding β-arms were deleted, HU could still bind DNA albeit with reduced affinity. However, when one residue was replaced with alanine and the DNA binding loop was deleted, no DNA binding capacity remained (Thakur et al, 2021).…”

“…Indeed, Thakur et al (2021) investigated modifications of E. coli HUβ and found when either Lys83 or Lys86 was replaced with alanine, or when the DNA-binding β-arms were deleted, HU could still bind DNA albeit with reduced affinity. However, when one residue was replaced with alanine and the DNA binding loop was deleted, no DNA binding capacity remained (Thakur et al, 2021). In this case, compromise of either site was tolerated but simultaneous modification of both sites rendered HU unable to bind DNA.…”

Small Prokaryotic DNA-Binding Proteins Protect Genome Integrity throughout the Life Cycle

“…Biofilms are structured bacterial communities attached to inert or living surfaces that create a protective environment for bacterial cells, enabling the survival of physical and chemical treatments including high-dose antibiotics [ 40 ]. The E. coli HU protein has been shown to act as a molecular glue attaching bacteria to extracellular DNA in biofilms [ 41 ].…”