The biofilm matrix scaffold of Pseudomonas species contains non-canonically base paired extracellular DNA and RNA

Abstract

17While the array of emergent properties assigned to biofilms is extensive (e.g. antimicrobial 18 tolerance), the mechanisms that underpin these are largely unknown. In particular, the 19 extracellular matrix, a defining feature of biofilms, remains poorly understood in terms of its 20 composition and contribution to biofilm structure and function. Here we demonstrate that 21 extracellular DNA exists in a complex with RNA that forms the main cross-linking exopolymer 22 of Pseudomonas biofilms, and explains biofilm elasticity. The RNA has a high purine content 23 and our solid-state NMR data indicate the formation of Hoogsteen guanine base pairs. This may 24 suggest the presence of G-quadruplexes, which is also corroborated by the enhancement of 25 biofilm formation in the presence of potassium. The finding that non-canonical interactions 26 mediate networking of matrix-forming extracellular nucleic acids addresses how eDNA is 27 organized and contributes to matrix biophysical properties. This understanding will allow for the 28 development of more effective biofilm control strategies. 29 30 42 traits in P. aeruginosa biofilms, including three exopolysaccharides (Colvin et al., 2012), four 43 proteins (Allesen-Holm et al., 2006; Borlee et al., 2010; Seviour et al., 2015a) and extracellular 44 DNA (eDNA) (Okshevsky and Meyer, 2015). Each putative exopolysaccharide (Colvin et al., 45 2012) has been identified as a primary structural agent, suggesting the existence of functional 46 redundancy. Other exopolymers have multiple roles (Irie et al., 2012) and a wide range of 47 2014). We undertook to identify the foundation polymer/s in P. aeruginosa biofilms, which are 54 defined here as those that either dominate biofilm elasticity or constitute the primary structural 55 agent/s. 56 Results 57 eDNA dominates the elastic response of Pseudomonas aeruginosa. 58 To characterize the foundation polymer of P. aeruginosa biofilms we exploited the reported 59 ability of the ionic liquid 1-ethyl-3-methyl-imidazolium acetate (EMIM-Ac) to dissolve a range 60 of recalcitrant biopolymers, including DNA(Zhao, 2015) and cellulose (Vitz et al., 2009), which 61 led us to demonstrate this also for P. aeruginosa biofilm exopolymers (Seviour et al., 2015b). 62Here, when P. aeruginosa biofilms were dissolved in EMIM-Ac, the subsequent fluid was highly 63 viscoelastic. We measured non-linear elasticity as a shear-rate dependent high normal stress 64 difference (N 1 -N 2 ), where N 1 and N 2 are primary and secondary normal stress differences 65 respectively. Elasticity dominated the viscous flow properties for the wild type biofilm in 66 EMIM-Ac, with (N 1 -N 2 ) an order of magnitude greater than shear stress ( Figure 1A; Wild type). 67 The solvent (EMIM-Ac) alone exhibited no elasticity, indicating that the elastic properties are 68 transferred to the EMIM-Ac from the biofilm matrix. Viscosity was slightly shear-thinning 69 (Supplementary Figure 1A and Supplementary Table 1), which would be expected from dilute 70 polymer soluti...

Extracellular Nucleic Acids Present in the Candida albicans Biofilm Trigger the Release of Neutrophil Extracellular Traps

“…In the case of strain ATCC 10231, we observed cell growth mainly in the form of pseudohyphae, while the SC5314 strain forms typical long, thin hyphae which promotes increased tissue penetration and increases virulence ( Thewes et al., 2008 ). It can be assumed that the pseudohyphal form may favor the release of eDNA and eRNA, which can form a gel-like structure of the ECM, as was proposed for the biofilm of P. aeruginosa ( Seviour et al., 2019 ). Such conception could support the finding that the ATCC10231 biofilm was more resistant to substances affecting cell membrane integrity ( Lupetti et al., 2002 ).…”

“…(2015) who showed that short fragments (<250 nucleotides) of C. albicans RNA may be transported by MVs, supporting our finding of a significant amount of eRNA in ATCC 10231 biofilm. In turn, the results obtained in the case of bacterial biofilm indicated that DNA and RNA molecules may form complexes by Watson-Crick interactions and nonspecific bonds ( Seviour et al., 2019 ). Such structures may favor the formation of a dense ECM, stabilizing the bacterial biofilm.…”

The Role of DNA in the Extracellular Environment: A Focus on NETs, RETs and Biofilms

“…Apart from the structural role discussed above, other specific functions of exDNA in biofilms have been suggested. ExDNA may act as facilitator of the initial adhesion of cells to the surface (Das et al, 2010), in support of EPS gelification (Seviour et al, 2019), in the maintenance of specific cell orientations (Gloag et al, 2013), in the control of viscoelastic relaxation of the biofilm in mechanical stress conditions (Peterson et al, 2013) and in the induction of the morphological changes from yeast to hyphal growth, during Candida albicans biofilm development (Payne and Boles, 2016).…”

“…,19 Das et al (2010),20 Seviour et al (2019),21 Gloag et al (2013),22 Peterson et al (2013),23 Payne and Boles (2016),24 Gallo et al (2015),25 Halverson et al(2015),26 Baums and von Köckritz-Blickwede (2015),27 Tsourouktsoglou et al(2020),28 Tran et al(2016),29 Driouich et al(2019),30 Plancot et al(2013),31 Hawes et al(2011),32 Chiang et al(2013),33 Mulcahy et al (2008),34 Savchenko et al(2014),35 Shah et al(2020),36 Krysko et al (2011),37 Demers et al(2012),38 Brinkmann(2018),39 Daniel et al (2019),40 Erpenbeck et al (2019),41 Leppkes et al (2020),42 Arazna et al (2013),43 Wang et al(2015),44 Mazzoleni et al (2015a),45 Mazzoleni et al (2015b),46 Barbero et al (2016), 47 Duran-Flores and Heil(2018),48 Vega-Muñoz et al (2018),49 Lorenz and Wackernagel (1994),50 Merod and Wuertz (2014),51 Orwin et al (2018), and 52 Paungfoo-Lonhienne et al (2010).…”

Selective, pH‐Dependent Colorimetric and Fluorimetric Detection of Quadruplex DNA with 4‐Dimethylamino(phenyl)‐Substituted Berberine Derivatives

“…Although numerous fluorescent probes for G4‐DNA are known, [9–19] to the best of our knowledge, no examples have been reported so far with these particular features. Namely, only a few fluorescent probes for G4‐DNA have been reported that operate at lower pH, [61] but these probes do not exhibit the dual‐mode response of compound 3 a . In conclusion, the derivatives 3 a and 3 b provide a promising starting point for the development of selective DNA‐targeting fluorescent probes that combine the favorable biological properties of berberine with tailor‐made, medium sensitive photophysical properties.…”