salmonicida subsp. salmonicida JF2267 were loaded for normalization. DNA bands were stained with ethidium bromide for control and transferred onto a nylon membrane (Roche Diagnostics,
Mannheim, Germany) with a VacuGene apparatus (GE Healthcare Bio-Sciences). The IS630 probe was prepared by PCR using primers Clust_asa1052_S6 (5′- AGGCAGAACTTGGGGTTCTT-3′) and Clust_asa1052_R4 (5′- ACAAAAGCGGGTTGTCACTC-3′) EVP4593 and DNA of A. salmonicida subsp. salmonicida JF2267 as a template. PCR was performed in 30 μL which contained 0.5 μL of Taq DNA polymerase (5 units/μL) (Roche Diagnostics, Mannheim, Germany), 300 nM of each primer, 1.75 mM MgCl2, 200 μM concentrations of each dNTP and 1 μl of the Digoxigenin-11-dUTP (1 nmol/μL) (Roche Diagnostics, Mannheim, Germany). Ruboxistaurin Each reaction involved a denaturing step at 94°C for 5 min followed by 30 cycles of 10 sec at 94°C, 30 sec at 54°C and 60 sec at 72°C and a final extension step of 7 min at 72°C. Bioinformatic analysis The hybridization patterns were scanned and the data were analyzed using the BioNumerics software version 6.6 (Applied Maths, Kortrijk, Belgium). Bands automatically assigned by the computer
were checked visually and corrected manually. Cluster analysis of the IS-RFLP patterns was done by the unweighted pair group method with average linkages (UPGMA) using the Dice coefficient and the following GW786034 mw parameters: 0.5% Optimization, 0% Band filtering, 0.5% Tolerance and ignore uncertain bands. Prediction of T3SS effectors was performed with the Modlab® online software (http://gecco.org.chemie.uni-frankfurt.de/T3SS_prediction/T3SS_prediction.html) [45]. Stability of IS630 in cultured A. salmonicida subsp. salmonicida The stability of IS630 under growth conditions in TSB medium was assessed by daily 100x dilution of a culture of strain JF2267 at 18°C and at 25°C during 4 days to reach 20 generations. Every day DNA was extracted
from 109 bacteria, digested with XhoI and submitted to southern blot hybridization. Acknowledgements This research Mirabegron was funded by the Swiss National Science Foundation grant no. 31003A-135808. Electronic supplementary material Additional file 1: Table S1: Table showing for each A. salmonicida A449 IS630 copy, the size of the XhoI-digested DNA fragment containing the IS, the inter- or intragenic localization, the characteristics of the adjacent genes, and the association to a region of variability or to other IS elements. (DOC 90 KB) Additional file 2: Table S2: Profound analysis and comparison of published Aeromonas genomes used for Figures 3 and 4. Grey: conserved ORFs; light green: ORFs specific of the species; yellow: IS630; pink: other IS elements; red: putative or characterized virulence factors; mauve: ORFs for resistance to antibiotic or heavy metal; dark green: ORFs associated to pili, fimbriae or flagella; blue: ORFs associated to phage; cyan: tRNA and rRNA; orange: ORFs with homology to eukaryotic genes.