pickettii ULC193, ULC194, ULC277, ULC297, ULC298, ULC224, ULC421 Microbiology laboratory of Limerick Regional Hospital (Cystic Fibrosis
Patients) R. pickettii ULI785, ULI788, ULI790, ULI791, ULI796, ULI798, ULI800, ULI801, ULI804, ULI806, ULI807, ULI818, ULI159, ULI162, ULI165, ULI167, ULI169, Fulvestrant in vitro ULI171, ULI174, ULI181, ULI187, ULI188, ULI193 Isolated from various Millipore Purified water systems (Ireland) R.insidiosa ULI821, ULI797, ULI785, ULI181, ULI794, ULI185, ULI166, ULI819, ULI784, ULI163, ULI795 Isolated from various Millipore Purified water systems (Ireland) R. pickettii ULM001, ULM002, ULM003, ULM004, ULM005, ULM006 Isolated from various Millipore Purified water systems (France) R. pickettii ULM007, ULM008, ULM009, ULM010, ULM011 Isolated from various Millipore Purified water systems (Ireland) R.insidiosa ULM008, ULM009 Isolated from various Millipore Purified water systems (Ireland) Molecular analysis of genes of Tn4371-like ICEs PCR primers were designed based on the conserved aligned scaffold common to all ICEs characterised in this study and
from the consensus sequence of the Ralstonia pickettii 12J Tn4371 ICE using the Primer 3 program [[67], http://frodo.wi.mit.edu/]. All primers are listed in Table 5. The cycling conditions were as follows: initial denaturation (98°C, 2 min); DMXAA supplier 35 cycles consisting of denaturation [98°C for 15 s], primer annealing [TA [estimated primer annealing temperature], 1 min], and extension [72°C, 1 min/kb]; followed by a final extension step [72°C, 10 min]. Amplification was carried out with a GC buffer [in a total reaction of 100 μL containing 0.2 mM deoxynucleoside triphosphates, 100 pmol of each primer, 8 μL of genomic template DNA, Resminostat and 3 units of Phusion polymerase [New England Biolabs, UK]. Amplification was carried out using a GeneAmp 2400 Thermocycler. Bacterial DNA for PCR amplification was extracted
according to Ausubel et al. [68]. Amplicons to be sequenced were directly purified from the PCR reaction by the NucleoSpin Extract II kit [Macherey-Nagel, Düren] according to the manufacturer’s instructions. Sequence analysis was performed by Euorfins-MWG [Germany] using both the forward and reverse primers listed in Table 3. Bioinformatic Analysis of the Tn4371-like ICEs in genomes All analysed DNA sequences were retrieved from the GenBank database http://www.ncbi.nlm.nih.gov. DNA and protein sequences similar to Tn4371 [[13], AJ536756] were detected within the NCBI nonredundant nucleotide and protein databases http://www.ncbi.nlm.nih.gov via blastp and blastn analysis using the original Tn4371 sequence as a probe [69]. Assembly and comparison with other Tn4371-like sequences was performed with the Artemis Comparison Tool [ACT] [[70], http://www.sanger.ac.uk/Software/ACT]. The complete DNA sequences were also manually annotated to verify the deposited sequence.