The biofilm matrix scaffold of Pseudomonas aeruginosa contains G-quadruplex extracellular DNA structures

Thomas Seviour,Anh Tuân Phan,Yong Hwee Foo,Sunil S Adav,Jason R Stokes,Sudarsan Mugunthan,Gurjeet Singh Kohli,Staffan Kjelleberg,Scott A Rice,Heather M Shewan,Fernaldo Richtia Winnerdy,Sujatha Subramoni,Lan Li Wong,Remi Castaing,Xiangyan Shi

doi:10.1038/s41522-021-00197-5

Abstract

Extracellular DNA, or eDNA, is recognised as a critical biofilm component; however, it is not understood how it forms networked matrix structures. Here, we isolate eDNA from static-culture Pseudomonas aeruginosa biofilms using ionic liquids to preserve its biophysical signatures of fluid viscoelasticity and the temperature dependency of DNA transitions. We describe a loss of eDNA network structure as resulting from a change in nucleic acid conformation, and propose that its ability to form viscoelastic structures is key to its role in building biofilm matrices. Solid-state analysis of isolated eDNA, as a proxy for eDNA structure in biofilms, reveals non-canonical Hoogsteen base pairs, triads or tetrads involving thymine or uracil, and guanine, suggesting that the eDNA forms G-quadruplex structures. These are less abundant in chromosomal DNA and disappear when eDNA undergoes conformation transition. We verify the occurrence of G-quadruplex structures in the extracellular matrix of intact static and flow-cell biofilms of P. aeruginosa, as displayed by the matrix to G-quadruplex-specific antibody binding, and validate the loss of G-quadruplex structures in vivo to occur coincident with the disappearance of eDNA fibres. Given their stability, understanding how extracellular G-quadruplex structures form will elucidate how P. aeruginosa eDNA builds viscoelastic networks, which are a foundational biofilm property.

Highlights

Biofilms are key microbial ecosystems that, for example, contribute to bacterial pathogenicity[1], disrupt the flow in water filtration systems[2] and facilitate wastewater treatment bioprocesses[3]
We address the question of how extracellular DNA (eDNA) is assembled in the extracellular matrix of biofilms by describing atomic-level interactions between the nucleic acids using solid-state nuclear magnetic resonance (SSNMR)
The extracted eDNA gel isolate is an accurate proxy for understanding the intermolecular interactions that stabilise the extracellular matrices of P. aeruginosa biofilms

Summary

INTRODUCTION

Biofilms are key microbial ecosystems that, for example, contribute to bacterial pathogenicity[1], disrupt the flow in water filtration systems[2] and facilitate wastewater treatment bioprocesses[3] They represent bacterial adaptation strategies allowing for increased antibiotic tolerance[4], enhanced resource capture[5] and the establishment of ecological microniches[6]. While progress has been made towards describing the structures and identities of extracellular polysaccharides and proteins by applying classical chemical and molecular approaches, important questions regarding eDNA remain, for example, how does it differ structurally from chromosomal DNA (cDNA) and what enables it to induce structure-dependent functions in the biofilm matrix? To investigate whether the viscoelasticity of DNA during extraction by EMIM-Ac from P. aeruginosa biofilms relies on Pel and Psl, as described by Chew et al.[24], we used static biofilms of alginate over-expressing strain PDO300

RESULTS

DISCUSSION

Findings

METHODS