The extracellular DNA lattice of bacterial biofilms is structurally related to Holliday junction recombination intermediates Aishwarya Devaraja, John R. Buzzoa, Lauren Mashburn-Warrena, Erin S. Gloagb, Laura A. Novotnya, Paul Stoodleyb,c,d, Lauren O. Bakaletza,e, and Steven D. Goodmana,e,1 aCenter for Microbial Pathogenesis, Abigail Wexner Research Institute at Nationwide Children’s Hospital, Columbus, OH 43205; bDepartment of Orthopedics, The Ohio State University, Columbus, OH 43210; cDepartment of Microbial Infection and Immunity, The Ohio State University, Columbus, OH 43210; dNational Centre for Advanced Tribology at Southampton, University of Southampton, Southampton SO17 1BJ, United Kingdom; and eDepartment of Pediatrics, College of Medicine, The Ohio State University, Columbus, Ohio 43210 Edited by E. Peter Greenberg, University of Washington, Seattle, WA, and approved October 30, 2019 (received for review May 28, 2019) Extracellular DNA (eDNA) is a critical component of the extracel- In addition, we have revealed that the DNABII family of pro- lular matrix of bacterial biofilms that protects the resident bacteria teins (integration host factor, IHF, and histone-like protein, HU) from environmental hazards, which includes imparting signifi- bind to and stabilize the eDNA lattice structure and are funda- cantly greater resistance to antibiotics and host immune effectors. mental to the structural stability of bacterial biofilms (5, 7–14). eDNA is organized into a lattice-like structure, stabilized by the DNABII proteins that are localized at the vertices of the eDNA DNABII family of proteins, known to have high affinity and specificity lattice within bacterial biofilms (5–8) have high affinity for branched for Holliday junctions (HJs). Accordingly, we demonstrated that the DNA structures which include Holliday junction (HJ) DNA (15, branched eDNA structures present within the biofilms formed by 16). HJs are single-strand cross-over intermediates of homologous NTHI in the middle ear of the chinchilla in an experimental otitis recombination and are common across both eukaryotes and media model, and in sputum samples recovered from cystic fibrosis prokaryotes (17). HJs appear as cross-like or cruciform struc- patients that contain multiple mixed bacterial species, possess tures with 4 double-stranded DNA (dsDNA) arms. In most an HJ-like configuration. Next, we showed that the prototypic eubacteria, the resolution of homologous recombination occurs BIOCHEMISTRY Escherichia coli HJ-specific DNA-binding protein RuvA could be through the association of HJ DNA with RuvA, RuvB, and functionally exchanged for DNABII proteins in the stabilization of biofilms formed by 3 diverse human pathogens, uropathogenic RuvC, where RuvA binds to HJ DNA with high affinity in a E. coli, nontypeable Haemophilus influenzae, and Staphylococcus structure-specific, but sequence-independent, manner (18). epidermidis. Importantly, while replacement of DNABII proteins within RuvA then recruits RuvB to the HJ, and the RuvAB complex the NTHI biofilm matrix with RuvA was shown to retain similar me- drives translocation of the junction that expands the heterodu- chanical properties when compared to the control NTHI biofilm plex region in an adenosine 5′-triphosphate (ATP)-dependent structure, we also demonstrated that biofilm eDNA matrices stabi- fashion (19). Finally, the endonuclease RuvC binds the RuvAB lized by RuvA could be subsequently undermined upon addition of complex, which results in cleavage of HJ DNA and resolution the HJ resolvase complex, RuvABC, which resulted in significant biofilm to yield 2 nicked duplexes (20). RusA, a resolvase of lambdoid disruption. Collectively, our data suggested that nature has recapitu- lated a functional equivalent of the HJ recombination intermediate to Significance maintain the structural integrity of bacterial biofilms. Most chronic and recurrent bacterial infections are the result of extracellular matrix | Holliday junction resolvase | DNABII proteins biofilms. Extracellular DNA (eDNA) is a ubiquitous and pivotal structural component of biofilms that protects the resident bac- ost bacteria in natural ecosystems prefer a biofilm lifestyle. teria from the host immune system and antimicrobial agents. It is MBiofilm bacteria are encased within a self-produced ex- of the highest priority to characterize the structure of the eDNA tracellular matrix (extracellular polymeric substances, or EPS) to understand the development of bacterial biofilm communities. comprised of extracellular DNA (eDNA), proteins, lipids, and Here, we employed the prototypic Holliday junction-specific exopolysaccharides (1). The biofilm EPS provides structural in- (HJ) DNA-binding protein RuvA and demonstrated that eDNA tegrity; protects resident bacteria against physical, chemical, and within biofilms formed by 3 human pathogens, uropathogenic environmental stresses that includes host effectors and antimi- Escherichia coli, nontypeable Haemophilus influenzae,andStaph- crobial therapies; and affects gene regulation and nutrient ad- ylococcus epidermidis was structurally related to HJ recombina- sorption (reviewed in ref. 2). Hence, it is of utmost importance to tion intermediates. We further demonstrated that this HJ-like characterize not only the EPS components but also their sub- structure was critical to the structural and mechanical integrity of sequent structure to gain insight into the development of bac- the bacterial biofilm matrix. terial biofilm communities and, consequently, for pathogenic biofilms, identify the means to undermine them. Author contributions: A.D., L.O.B., and S.D.G. designed research; A.D., J.R.B., L.M.-W., E.S.G., and P.S. performed research; L.A.N. contributed new reagents/analytic tools; eDNA is a key structural component of the EPS and therefore A.D., J.R.B., L.M.-W., E.S.G., L.A.N., P.S., L.O.B., and S.D.G. analyzed data; and A.D., an attractive target for the control of bacterial biofilms. Al- E.S.G., P.S., and S.D.G. wrote the paper. though the importance of eDNA in the biofilm matrix has been The authors declare no competing interest. established, the structure of the eDNA itself has not been well This article is a PNAS Direct Submission. characterized. We have previously shown that eDNA in biofilms Published under the PNAS license. formed by nontypeable Haemophilus influenzae (NTHI) within a 1 To whom correspondence may be addressed. Email: steven.goodman@ chinchilla middle ear (3) and by Pseudomonas aeruginosa in a nationwidechildrens.org. murine lung model (4), as well as biofilms in pediatric sputum This article contains supporting information online at https://www.pnas.org/lookup/suppl/ and otorrhea samples that were culture-positive for multiple doi:10.1073/pnas.1909017116/-/DCSupplemental. mixed bacterial species (5–7), was present in a lattice structure. www.pnas.org/cgi/doi/10.1073/pnas.1909017116 PNAS Latest Articles | 1of10 Downloaded by guest on September 29, 2021 phage origin, binds HJ in a sequence-independent manner and molar) (15, 16), we hypothesized that these branched structures cleaves the phosphodiester bond 5′ of CC dinucleotides to re- were related to HJ recombination intermediates. Toward this goal, solve HJ into nicked duplexes (21). we assessed whether the HJ-like structure was found within bacterial Because HJ DNA is necessarily bent, it serves as an excellent biofilms that had formed in vivo with a monoclonal antibody specific substrate for the DNABII family that bind bent DNA with high for cruciform DNA that exclusively binds to the elbow region of an affinity (15, 16). We therefore hypothesized that the structure of HJ DNA structure (22). Immunohistochemistry analysis of middle eDNA at the vertices was comprised of HJs. To test this hypothesis, ear sections from chinchilla infected with NTHI and sputum solids we employed antibodies that are highly specific for HJ DNA as well from CF patients that contained multiple mixed bacterial species as proteins that bind to and resolve HJ DNA. First, we demonstrated revealed a complex lattice-like eDNA structure as indicated in that the lattice structure found within the EPS of biofilms formed in green, with punctate labeling for cruciform DNA in white, at the vivo by NTHI (in the chinchilla middle ear during experimental otitis majority of the crossed strands of the eDNA (Fig. 1). Given the media) and in polymicrobial sputum samples recovered from cystic specificity of the monoclonal antibody against cruciform DNA (SI fibrosis (CF) patients was recognized by the these highly specific HJ- Appendix,Fig.S1) (22), these data confirmed the presence of HJ directed antibodies. Further, we took advantage of the proteins in- DNA within the EPS of single and multispecies biofilms in vivo. volved in the resolution of HJ DNA, RuvABC complex, and RusA to demonstrate that the HJ DNA-binding protein RuvA functionally The Prototypic HJ DNA Binding Protein RuvA Compensated for the complemented DNABII proteins within the EPS and stabilized Removal of DNABII Proteins in Structural Stabilization of UPEC, NTHI, biofilms formed by uropathogenic Escherichia coli (UPEC), NTHI, and S. epidermidis Biofilms. To further confirm the presence of HJ-like and Staphylococcus epidermidis in vitro. We further showed that structure within the EPS of bacterial biofilms, we employed 3 op- NTHI biofilms stabilized by RuvA were biophysically indistinguish- portunistic pathogens, UPEC, NTHI, and S. epidermidis, all of able from the control NTHI biofilms as measured by