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Category: Clinical Microbiology
The Integration and Excision of CTnDOT, Page 1 of 2
< Previous page | Next page > /docserver/preview/fulltext/10.1128/9781555819217/9781555819200_Chap08-1.gif /docserver/preview/fulltext/10.1128/9781555819217/9781555819200_Chap08-2.gifAbstract:
Bacteroides spp. are one of the more prevalent members of the human colonic microbiota, representing approximately 40% of the bacterial community ( 1 ). Bacteroides spp. are normally in symbiosis with their human hosts. Although they are usually harmless members of the gut microbiota, they can become opportunistic pathogens if released from the colon ( 2 , 3 ). This most commonly occurs due to surgery, trauma or disease such as gangrenous appendicitis or malignancies ( 4 ). Among anaerobic bacteria, Bacteroides spp. are the pathogens most commonly isolated from clinical samples, including blood ( 2 ). The treatment of Bacteroides infections has become more challenging as they have acquired a variety of genes that encode resistances to antibiotics. In the 1970s, only 20 to 30% of Bacteroides spp. clinical isolates were resistant to tetracycline. By the 1990s, the prevalence of tetracycline resistance had increased to 80% ( 5 ). This increase in tetracycline resistance can be attributed to the presence of integrative and conjugative elements (ICEs) that encode antibiotic resistance genes.
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A schematic of CTnDOT. The genes xis2c, xis2d, orf3, and exc (shown in blue) are part of the excision operon. The genes shown in orange (tetQ, rteA, and rteB) are involved in regulating CTnDOT excision.
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 attL and attR 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 attDOT site of the circular intermediate as well as the attB 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.
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.
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.
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.
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.
(A) The core-type sites and overlap regions of wild-type attDOT and attB. The horizontal arrows denote the core-type sites D and D′ on attDOT and B and B′ on attB. 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 attB 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.
(A) The core-type sites and overlap regions of wild-type attDOT and attB. The horizontal arrows denote the core-type sites D and D′ on attDOT and B and B′ on attB. 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 attB 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.
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 attL site is shown with a purple line, the attR is shown with a gray line, the attB is denoted with a blue line, and attDOT is denoted with a red line. Resolution at the sites of the black arrows form the attDOT and attB substrates while resolution at the sites of the gray arrows forms the attL and attR 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 attDOT and attB substrates.
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.