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The Integration and Excision of CTnDOT

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  • Authors: Margaret M. Wood1, Jeffrey F. Gardner2
  • Editors: Phoebe Rice3, Nancy Craig4
  • VIEW AFFILIATIONS HIDE AFFILIATIONS
    Affiliations: 1: Simmons College, 300 The Fenway, Boston, MA 02115; 2: Department of Microbiology, University of Illinois at Urbana-Champaign, 601 S Goodwin Avenue, Urbana, IL 61801; 3: University of Chicago, Chicago, IL; 4: Johns Hopkins University, Baltimore, MD
  • Source: microbiolspec March 2015 vol. 3 no. 2 doi:10.1128/microbiolspec.MDNA3-0020-2014
  • Received 30 March 2014 Accepted 23 June 2014 Published 12 March 2015
  • Margaret Wood, margaret.wood@simmons.edu
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  • Abstract:

    species are one of the most prevalent groups of bacteria present in the human colon. Many strains carry large, integrated elements including integrative and conjugative elements (ICEs). One such ICE is CTnDOT, which is 65 kb in size and encodes resistances to tetracycline and erythromycin. CTnDOT has been increasing in prevalence in spp., and is now found in greater than 80% of natural isolates. In recent years, CTnDOT has been implicated in the spread of antibiotic resistance among gut microbiota. Interestingly, the excision and transfer of CTnDOT is stimulated in the presence of tetracycline. The tyrosine recombinase IntDOT catalyzes the integration and excision reactions of CTnDOT. Unlike the well-characterized lambda Int, IntDOT tolerates heterology in the overlap region between the sites of cleavage and strand exchange. IntDOT also appears to have a different arrangement of active site catalytic residues. It is missing one of the arginine residues that is conserved in other tyrosine recombinases. The excision reaction of CTnDOT is complex, involving excision proteins Xis2c, Xis2d, and Exc, as well as IntDOT and a host factor. Xis2c and Xis2d are small, basic proteins like other recombination directionality factors (RDFs). Exc is a topoisomerase; however, the topoisomerase function is not required for the excision reaction. Exc has been shown to stimulate excision frequencies when there are mismatches in the overlap regions, suggesting that it may play a role in resolving Holliday junctions (HJs) containing heterology. Work is currently under way to elucidate the complex interactions involved with the formation of the CTnDOT excisive intasomes.

  • Citation: Wood M, Gardner J. 2015. The Integration and Excision of CTnDOT. Microbiol Spectrum 3(2):MDNA3-0020-2014. doi:10.1128/microbiolspec.MDNA3-0020-2014.

Key Concept Ranking

Integrative and Conjugative Elements
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References

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33. Keeton CM, Gardner JF. 2012. Roles of Exc Protein and DNA Homology in the CTnDOT Excision Reaction. J Bacteriol 194:3368–3376. [PubMed][CrossRef]
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35. Van Duyne GD. 2002. A structural view of tyrosine recombinase site-specific recombination, p 93–117. In Craig NL, Craigie R, Gellert M, Lambowitz AM (ed), Mobile DNA II. ASM Press, Washington, DC.
36. Azaro MA, Landy A. 2002. Lambda integrase and the lambda Int family, p 119–148. In Craig NL, Craigie R, Gellert M, Lambowitz AM (ed), Mobile DNA II. ASM Press, Washington, DC.
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39. Wood MM, Dichiara JM, Yoneji S, Gardner JF. 2010. CTnDOT integrase interactions with attachment site DNA and control of directionality of the recombination reaction. J Bacteriol 192:3934–3943. [PubMed][CrossRef]
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41. Warren D, Lee SY, Landy A. 2005. Mutations in the amino-terminal domain of lambda-integrase have differential effects on integrative and excisive recombination. Mol Microbiol 55:1104–1112. [PubMed][CrossRef]
42. Lee SY, Radman-Livaja M, Warren D, Aihara H, Ellenberger T, Landy A. 2005. Non-equivalent interactions between amino-terminal domains of neighboring lambda integrase protomers direct Holliday junction resolution. J Mol Biol 345:475–485. [PubMed][CrossRef]
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55. Voziyanov Y, Pathania S, Jayaram M. 1999. A general model for site-specific recombination by the integrase family recombinases. Nucleic Acids Res 27:930–941. [PubMed][CrossRef]
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67. Numrych TE, Gumport RI, Gardner JF. 1990. A comparison of the effects of single-base and triple-base changes in the integrase arm-type binding sites on the site-specific recombination of bacteriophage lambda. Nucleic Acids Res 18:3953–3959. [PubMed][CrossRef]
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2015-03-12
2017-07-24

Abstract:

species are one of the most prevalent groups of bacteria present in the human colon. Many strains carry large, integrated elements including integrative and conjugative elements (ICEs). One such ICE is CTnDOT, which is 65 kb in size and encodes resistances to tetracycline and erythromycin. CTnDOT has been increasing in prevalence in spp., and is now found in greater than 80% of natural isolates. In recent years, CTnDOT has been implicated in the spread of antibiotic resistance among gut microbiota. Interestingly, the excision and transfer of CTnDOT is stimulated in the presence of tetracycline. The tyrosine recombinase IntDOT catalyzes the integration and excision reactions of CTnDOT. Unlike the well-characterized lambda Int, IntDOT tolerates heterology in the overlap region between the sites of cleavage and strand exchange. IntDOT also appears to have a different arrangement of active site catalytic residues. It is missing one of the arginine residues that is conserved in other tyrosine recombinases. The excision reaction of CTnDOT is complex, involving excision proteins Xis2c, Xis2d, and Exc, as well as IntDOT and a host factor. Xis2c and Xis2d are small, basic proteins like other recombination directionality factors (RDFs). Exc is a topoisomerase; however, the topoisomerase function is not required for the excision reaction. Exc has been shown to stimulate excision frequencies when there are mismatches in the overlap regions, suggesting that it may play a role in resolving Holliday junctions (HJs) containing heterology. Work is currently under way to elucidate the complex interactions involved with the formation of the CTnDOT excisive intasomes.

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FIGURE 1

A schematic of CTnDOT. The genes , , , and (shown in blue) are part of the excision operon. The genes shown in orange (, , and ) are involved in regulating CTnDOT excision. doi:10.1128/microbiolspec.MDNA3-0020-2014.f1

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FIGURE 2

A diagram showing the integration and excision reactions of CTnDOT. The D and D′ core-type sites are located on CTnDOT (red), while B and B′ core-type sites are located in the bacterial chromosome (purple). IntDOT makes staggered cuts 7 bp apart (vertical arrows) on and to excise CTnDOT. The element forms a closed circular intermediate containing a 5 bp heteroduplex known as the coupling sequence. The heterology is likely resolved following conjugative transfer into a recipient cell. During integration into the bacterial chromosome, IntDOT makes staggered cuts 7 bp apart on the site of the circular intermediate as well as the target sequence, and CTnDOT integrates into the bacterial chromosome. The 5 bp heteroduplexes that form following integration are resolved by DNA replication or repair in the recipient cell. doi:10.1128/microbiolspec.MDNA3-0020-2014.f2

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FIGURE 3

The location of IntDOT substitution mutants isolated from hydroxylamine random mutagenesis experiments (red) or site-directed mutagenesis (black). The circled residue is the catalytic tyrosine, which was mutated to phenylalanine via site-directed mutagenesis. The N domain is shown in purple, the CB domain is shown in dark blue, and the CAT domain is shown in light blue. doi:10.1128/microbiolspec.MDNA3-0020-2014.f3

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FIGURE 4

Alignment of the amino acid sequences and secondary structures of the N domains of lambda Int (top) and IntDOT (bottom). The helix α H2 is the lambda Int coupler. The bold arrows pointing to the lambda Int sequence denote residues R30 and D71 that form a protein–protein interaction. The V95 residue of IntDOT (indicated by a bold arrow on the bottom sequence) is located in the putative IntDOT coupler. Thinner arrows denote aspartic acid residues that were mutated to lysines to examine possible charge interactions between IntDOT monomers. doi:10.1128/microbiolspec.MDNA3-0020-2014.f4

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FIGURE 5

Locations of amino acids that, when mutated, yielded cleavage-defective phenotypes. N183 is shown in cyan, H143 is shown in blue, T183 is shown in green, and T194 is shown in purple. N183, H143, and T183 interact with the cleaved DNA strand, shown in yellow. T194 is proximal to the uncleaved strand, shown in gray. The scissile phosphate is indicated with a bold black arrow. doi:10.1128/microbiolspec.MDNA3-0020-2014.f5

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FIGURE 6

Predicted amino acid residues and active site of IntDOT. The DNA backbone of core-type site DNA is shown in gray. The protein backbone is shown in blue and yellow. Residues 279 to 295, which contain the active site residues and interact with DNA, are represented in yellow. Alpha helices are shown as cylinders, beta sheets are shown as arrows, and regions lacking predicted secondary structure are shown as tubes. The side chains for the predicted active site residues are shown in orange except R285, which is shown in red. The “Arg I” residue from lambda Int (R212) has been superimposed on the IntDOT structure and is shown in purple. R212 aligns with S259 in IntDOT, however S259 is not involved in catalysis. The model suggests that R285 and R212 enter the active site from different positions on the peptide backbone but interact with the similar regions of DNA. doi:10.1128/microbiolspec.MDNA3-0020-2014.f6

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FIGURE 7a

(A) The core-type sites and overlap regions of wild-type and . The horizontal arrows denote the core-type sites D and D′ on and B and B′ on . The blue boxes designate the overlap regions, also marked with “O.” The GC dinucleotides are shown in red. The vertical arrows indicate the cleavage sites. (B) Simplified schematics of the wild-type, inverted overlap and symmetric substrates utilized in homology studies. The blue boxes indicate the heterology in the overlap regions, and the red boxes indicate positioning of the GC dinucleotides in the substrates. doi:10.1128/microbiolspec.MDNA3-0020-2014.f7a

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FIGURE 7b

(A) The core-type sites and overlap regions of wild-type and . The horizontal arrows denote the core-type sites D and D′ on and B and B′ on . The blue boxes designate the overlap regions, also marked with “O.” The GC dinucleotides are shown in red. The vertical arrows indicate the cleavage sites. (B) Simplified schematics of the wild-type, inverted overlap and symmetric substrates utilized in homology studies. The blue boxes indicate the heterology in the overlap regions, and the red boxes indicate positioning of the GC dinucleotides in the substrates. doi:10.1128/microbiolspec.MDNA3-0020-2014.f7b

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FIGURE 8

Synthetic Holliday junction substrates. (A) A Holliday junction containing core-type sites (not shown) and identical (homologous) overlap sequences shown with the GC dinucleotide denoted in red. The site of first cleavage and strand exchange is denoted with black arrows, while the site of second strand cleavage and strand exchange is indicated with gray arrows. The site is shown with a purple line, the is shown with a gray line, the is denoted with a blue line, and is denoted with a red line. Resolution at the sites of the black arrows form the and substrates while resolution at the sites of the gray arrows forms the and products. (B) A synthetic Holliday junction with two bp of homology at the GC dinucleotide and mismatches in the rest of the overlap sequences. Resolution occurs only at the sites of the black arrows forming the and substrates. doi:10.1128/microbiolspec.MDNA3-0020-2014.f8

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FIGURE 9

The IntDOT arm-type sites and the Xis2d binding sites. Boxes denote arm-type sites and Xis2d binding sites, and ovals denote core-type sites. Binding sites shown in red are required for recombination. Blue boxes denote arm-type sites that have a stimulatory effect on the integration reaction. White boxes indicate binding sites that are not required for the given reaction. Figure not drawn to scale. doi:10.1128/microbiolspec.MDNA3-0020-2014.f9

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