The Bacteroides thetaiotaomicron Protein Bacteroides Host Factor A Participates in Integration of the Integrative Conjugative Element CTnDOT into the Chromosome

Abstract

CTnDOT is a conjugative transposon found in Bacteroides species. It encodes multiple antibiotic resistances and is stimulated to transfer by exposure to tetracycline. CTnDOT integration into the host chromosome requires IntDOT and a previously unknown host factor. We have identified a protein, designated BHFa (Bacteroides host factor A), that participates in integrative recombination. BHFa is the first host factor identified for a site-specific recombination reaction in the CTnDOT family of integrative and conjugative elements. Based on the amino acid sequence of BHFa, the ability to bind specifically to 4 sites in the attDOT DNA, and its activity in the integration reaction, BHFa is a member of the IHF/HU family of nucleoid-associated proteins. Other DNA bending proteins that bind DNA nonspecifically can substitute for BHFa in the integration reaction. IMPORTANCEBacteroides species are normal members of the human colonic microbiota. These species can harbor and spread self-transmissible genetic elements (integrative conjugative elements [ICEs]) that contain antibiotic resistance genes. This work describes the role of a protein, BHFa, and its importance in the integration reaction required for the element CTnDOT to persist in Bacteroides host cells. Bacteroides species are Gram-negative, obligate anaerobes that are part of the normal microbiota in the human colon (1). When the gut is punctured, Bacteroides can act as an opportunistic pathogen that may form abscesses in other regions of the body. Treatment of the abscesses is complicated by widespread resistance to tetracycline and erythromycin carried by integrative conjugative elements (ICEs) (also called conjugative transposons [CTns]) found in Bacteroides. These elements are capable of transferring between diverse organisms by conjugation and integrate into the host bacterium's chromosome. Due to this integration, ICEs are maintained stably, even in the absence of selective pressure. As a result, the frequency of antibiotic resistance in Bacteroides has increased dramatically over the last 30 years (2). ICEs also carry genes to regulate and carry out their own transfer. Furthermore, some ICEs can mobilize coresident genetic elements that could not otherwise transfer (3). Because of their benefit to the bacterial host and their ability to transfer among organisms, ICEs are widespread in both Gram-positive and -negative bacterial populations (2).CTnDOT is a well-characterized ICE found in Bacteroides species. It carries the ermF and tetQ genes that encode resistance to erythromycin and tetracycline, respectively. Exposure to tetracycline induces the excision and transfer of CTnDOT. CTnDOT integration and excision require an integrase, IntDOT, and a hostencoded protein factor. IntDOT is a tyrosine recombinase and is in the same family of enzymes as Int, Flp, XerC, XerD, and Cre (4). IntDOT contains five of the six conserved amino acid residues that form the catalytic sites of tyrosine recombinases (5, 6). These enzymes perform strand exchanges by a site-...

The hidden life of integrative and conjugative elements

“… 2000 , 2001 ; Keeton and Gardner 2012 ; Keeton et al. 2013a , Ringwald and Gardner 2015 ). Xis2c and Xis2d are essential for the excision of CTn DOT and bind to attL and attR (Hopp, Gardner and Salyers 2015 ).…”

“…To promote site-specific insertion, it is thought that IntDOT assembles at attDOT along with the host factor BHFa, to form the integrative intasome nucleoprotein complex (Ringwald and Gardner 2015 ). This integrative intasome differs from it excisive counterpart by the absence of Xis2c, Xis2d and Exc.…”

The N-terminus of IntDOT forms hydrophobic interactions during Holliday Junction resolution

“…Previous work has shown that the Bacteroides host factor BHFa binds and bends DNA (21). It also forms a complex with the wild type IntDOT protein.…”

Resolution of Mismatched Overlap Holliday Junction Intermediates by the Tyrosine Recombinase IntDOT

“…5A). The H3 site overlaps with the entire L1 arm-type site, but only 6 of the previously BHFa protected 37 bases were changed in the L1 mutant (L1 Mut ) arm-type HJ intermediate (22). It may be that nonspecific binding of BHFa to the HJ intermediates is sufficient for resolution.…”

“…1A). Of the sites, BHFa has the highest affinity for the H2 site, less affinity for the paired H3-H4 sites, and the lowest affinity for the H1 site (22).…”