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The extracellular DNA lattice of bacterial biofilms is structurally related to Holliday junction recombination intermediates.

ABSTRACT: Extracellular DNA (eDNA) is a critical component of the extracellular matrix of bacterial biofilms that protects the resident bacteria from environmental hazards, which includes imparting significantly greater resistance to antibiotics and host immune effectors. eDNA is organized into a lattice-like structure, stabilized by the DNABII family of proteins, known to have high affinity and specificity for Holliday junctions (HJs). Accordingly, we demonstrated that the branched eDNA structures present within the biofilms formed by NTHI in the middle ear of the chinchilla in an experimental otitis media model, and in sputum samples recovered from cystic fibrosis patients that contain multiple mixed bacterial species, possess an HJ-like configuration. Next, we showed that the prototypic Escherichia coli HJ-specific DNA-binding protein RuvA could be functionally exchanged for DNABII proteins in the stabilization of biofilms formed by 3 diverse human pathogens, uropathogenic E. coli, nontypeable Haemophilus influenzae, and Staphylococcus epidermidis Importantly, while replacement of DNABII proteins within the NTHI biofilm matrix with RuvA was shown to retain similar mechanical properties when compared to the control NTHI biofilm structure, we also demonstrated that biofilm eDNA matrices stabilized by RuvA could be subsequently undermined upon addition of the HJ resolvase complex, RuvABC, which resulted in significant biofilm disruption. Collectively, our data suggested that nature has recapitulated a functional equivalent of the HJ recombination intermediate to maintain the structural integrity of bacterial biofilms.

SUBMITTER: Devaraj A

PROVIDER: S-EPMC6911203 | biostudies-literature | 2019 Dec

REPOSITORIES: biostudies-literature

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The extracellular DNA lattice of bacterial biofilms is structurally related to Holliday junction recombination intermediates.

Devaraj Aishwarya A Buzzo John R JR Mashburn-Warren Lauren L Gloag Erin S ES Novotny Laura A LA Stoodley Paul P Bakaletz Lauren O LO Goodman Steven D SD

Proceedings of the National Academy of Sciences of the United States of America 20191125 50

Extracellular DNA (eDNA) is a critical component of the extracellular matrix of bacterial biofilms that protects the resident bacteria from environmental hazards, which includes imparting significantly greater resistance to antibiotics and host immune effectors. eDNA is organized into a lattice-like structure, stabilized by the DNABII family of proteins, known to have high affinity and specificity for Holliday junctions (HJs). Accordingly, we demonstrated that the branched eDNA structures presen ...[more]

PMID: 31767757

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Project description:CTnDOT is an integrated conjugative element found in Bacteroides species. CTnDOT contains and transfers antibiotic resistance genes. The element integrates into and excises from the host chromosome via a Holliday junction (HJ) intermediate as part of a site-specific recombination mechanism. The CTnDOT integrase, IntDOT, is a tyrosine recombinase with core-binding, catalytic, and amino-terminal (N) domains. Unlike well-studied tyrosine recombinases, such as lambda integrase (Int), IntDOT is able to resolve Holliday junctions containing heterology (mismatched bases) between the sites of strand exchange. All known natural isolates of CTnDOT contain mismatches in the overlap region between the sites of strand exchange. Previous work showed that IntDOT was unable to resolve synthetic Holliday junctions containing mismatched bases to products in the absence of the arm-type sites and a DNA-bending protein. We constructed synthetic HJs with the arm-type sites and tested them with the Bacteroides host factor (BHFa). We found that the addition of BHFa stimulated resolution of HJ intermediates with mismatched overlap regions to products. In addition, the L1 site is required for directionality of the reaction, particularly when the HJ contains mismatches. BHFa is required for product formation when the overlap region contains mismatches, and it stimulates resolution to products when the overlap region is identical. Without this DNA bending, the N domain of IntDOT is likely unable to bind the L1 arm-type site. These findings suggest that BHFa bends DNA into the necessary conformation for the higher-order complexes, including the L1 site, that are required for product formation.IMPORTANCE CTnDOT is a mobile element that carries antibiotic resistance genes and moves by site-selective recombination and subsequent conjugation. The recombination reaction is catalyzed by an integrase IntDOT that is a member of the tyrosine recombinase family. The reaction proceeds through ordered strand exchanges that generate a Holliday junction (HJ) intermediate. Unlike other tyrosine recombinases, IntDOT can resolve HJs containing mismatched bases in the overlap region in vivo, as is the case under natural conditions. However, HJ intermediates including only IntDOT core-type sites cannot be resolved to products if the HJ intermediate contains mismatched bases. We added arm-type sites in cis and in trans to the HJ intermediates and the protein BHFa to study the requirements for higher-order nucleoprotein complexes.

Dataset Information

The extracellular DNA lattice of bacterial biofilms is structurally related to Holliday junction recombination intermediates.

Publications

The extracellular DNA lattice of bacterial biofilms is structurally related to Holliday junction recombination intermediates.

Similar Datasets

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