The extracellular matrix of bacterial biofilms consists of diverse components including polysaccharides, proteins and DNA. There has been a shift recently in extracellular DNA (eDNA) research towards describing factors allowing DNA to form network structures, specifically in terms of higher-order structures and associations with other biopolymers (extracellular RNA (eRNA), proteins, etc.). This becomes crucial due to differences in the physical behaviour of eDNA outside cells compared to intracellular DNA. SCELSE is investigating the structural role, physical property and nature of eDNA in the biofilm matrix of P. aeruginosa biofilms and its interactions with eRNA by employing a series of biophysical, biochemical and confocal microscopy techniques.

Extracellular DNA (eDNA) is a critical biofilm component, however, elucidating how it forms networked matrix structures within a biofilm matrix has been hampered by a lack of non-destructive isolation methodology. To overcome this obstacle, SCELSE has developed a protocol that uses ionic liquids to isolate eDNA from static-culture Pseudomonas aeruginosa biofilms and preserve its biophysical signatures of fluid viscoelasticity and the temperature dependency of DNA transitions.

The research discovered a loss of eDNA network structure resulted from a change in nucleic acid conformation, suggesting that the ability to form viscoelastic structures is key to eDNA’s role in building biofilm matrices. Using solid-state analysis of isolated eDNA, the SCELSE team revealed 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. The occurrence of G-quadruplex structures in the extracellular matrix of intact static and flow-cell biofilms of P. aeruginosa was verified using G-quadruplex-specific antibody binding, and the loss of G-quadruplex structures was validated 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.

The overall process of extracellular DNA extraction, biochemical and biophysical characterisation. (a) P. aeruginosa biofilms with different extracellular biopolymers; (b) isolation of eDNA using ionic liquid extraction; (c) separation of other extracellular biopolymers; (d) confocal laser scanning microscopy of eDNA fibres (in green); (e) secondary order structural conformation and thermal stability; (f) biochemical characterisation of biofilm and purified nucleic acid gel using nucleic acid resonance; (g) sequencing the genes and RNA involved in biofilm matrix building.

Extracellular RNA (eRNA) is also present in the extracellular matrix of bacterial biofilms and contributes to their structural integrity. However, technical difficulties related to the low stability of RNA make it difficult to understand the precise roles of eRNA in biofilms. SCELSE has demonstrated that eRNA associates with eDNA to form matrix fibres in P. aeruginosa biofilms, and the eRNA is enriched in certain bacterial RNA transcripts. Degradation of eRNA associated with eDNA leads to a loss of eDNA fibres and biofilm viscoelasticity. Compared with planktonic and biofilm cells, the biofilm matrix is enriched in specific mRNA transcripts, including lasB (encoding elastase). The mRNA transcripts colocalise with eDNA fibres in the biofilm matrix (shown by single molecule inexpensive FISH microscopy (smiFISH). The lasB mRNA is also observed in eDNA fibres in a clinical sputum sample positive for P. aeruginosa. These results indicate that the interaction of specific mRNAs with eDNA facilitates the formation of viscoelastic networks in the matrix of Pseudomonas aeruginosa biofilms.

The findings guide our understanding of biofilm matrix formation and the viscoelastic phenotypes of biofilms and, crucially, will underpin new biofilm disruption and control strategies.

Enzymatic digestion of eRNA leads to loss of eDNA fibres and loss of biofilm viscoelasticity. 3D confocal micrographs of 5 d eDNA-specific TOTO-1-stained P. aeruginosa wildtype biofilm (green) (a) with DNase I pre-treatment and (b) RNase H treatment, (c) DNase I pre-treatment followed by RNase H. Scale bars = 10μm. Adapted from Nat Commun (2023) 14: 7772.