Computational protocol: Phage ϕC2 Mediates Transduction of Tn6215, Encoding Erythromycin Resistance, between Clostridium difficile Strains

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Protocol publication

[…] The mobile element containing erm(B) was amplified from CD80 and CD062E1 in four parts and sequenced by the Sanger method (Eurofins MWG Operon, Germany). Primers SG2F/SG3R and TRR4/TPLHSout1 were used with gDNA, while primers invF2/invR2, invF2.1/invR2.1, and invF3/invR3 were used in ligation-assisted PCR (see in the supplemental material). CD80 gDNA (0.5 to 1 µg) was completely digested with either Hpy99I or BsrGI (New England Biolabs) and then self-ligated with T4 DNA ligase (10 U per reaction mixture volume; Fermentas) at 16°C for 18 h, and 16 to 32 ng was used in PCR. Sequences were analyzed and assembled into contigs with CLC Workbench version 6, ORFs were predicted with GeneMark, sequence alignments and homology searches were carried out using BLASTN, BLASTP, and ClustalW, and repeats/palindromes were searched for using einverted (, Inverted Repeats Finder (, and REPFIND ( integration site of the erm(B) mobile element in transductants was confirmed by Southern hybridization as described below. Primers P5/P6 (; see in the supplemental material) were used to detect the integrated erm(B) mobile element and regenerated or empty integration sites in the donor, recipient, and transductants. Circular molecules of the erm(B) mobile element in transductants were searched for using four primer pairs (P2/P4, P1/P4, P2/P3, and P1/P3) reading out of the ends of the element (; ). [...] As the donor and recipient strains were highly similar, we needed to find genetic markers for a PCR assay to determine real transconjugants after filter matings. Genes unique to the donor and recipients were found by whole-genome sequencing using the Illumina genome analyzer IIx at low (~100×) coverage. Initially, the sequence reads from the recipients were filtered by excluding those found to align to the donor. This was done using the BWA alignment package (). The remaining reads were assembled using the A5 pipeline (), and contigs were annotated against the genome of CD630 using the xBASE annotation pipeline (–). A similar process was followed to obtain donor-specific contigs. Candidate marker genes were selected to exclude hypothetical genes without a known function and genes on known transposons or phages. Finally, primers CD80C3U57F/CD80C3U57R () were designed for a candidate marker gene with 99% (1,035/1,038) sequence similarity to a gene in CD630 encoding an ABC-type transport system (CD630_08760), present only in the donor, and experimentally verified by PCR as described above. The PCR assay was used to distinguish transconjugants from spontaneous rifampin-resistant mutants of the donor after filter matings. […]

Pipeline specifications

Software tools GeneMark, BLASTN, BLASTP, Clustal W, EMBOSS, IRF, BWA
Databases xBASE
Applications Genome annotation, WGS analysis
Organisms Clostridioides difficile, Homo sapiens
Chemicals Erythromycin