B. petrii was routinely grown in LB broth, while B. bronchiseptica strains were cultured on BG-agar plates or in SS-liquid selleck kinase inhibitor medium, as previously described [26]. If necessary, antibiotics were added to the culture media in the following concentrations: tetracycline, 12.5 μg/ml; kanamycine, 50 μg/ml. Conjugation experiments were carried out by filter mating as described previously [26]. The long time survival experiments were carried out as described by Preston and Wardlaw [18]. For this purpose sterile filtered river water from the river Main was inoculated with bacteria B. bronchiseptica BB7866 and B. petrii (2,000 CFU/ml), respectively,

and incubated at 37°C. Samples were taken at different time intervals up to 263 days after inoculation and bacterial number was LGX818 ic50 counted by plating out serial dilutions of the bacteria. Molecular genetic tools DNA manipulations including cloning, restriction analysis, DNA-sequence analysis, preparation of genomic DNA, Southern blots were carried out according to standard procedures. selleck inhibitor In all cases, chromosomal

DNA used for PCR reactions or for whole genome hybridization analyses was purified from bacterial cultures inoculated from single colonies on agar plates. Pulsed field electrophoresis was carried out with the BioRad CHEF-DRII system as described previously [5]. B. petrii DNA microarray specifications and hybridization conditions Sixty-mer oligonucleotides sequences were designed as described previously using OligoArray2.0[27]. Lyophilised 5′-aminated oligonucleotides (Sigma Aldrich) were then resuspended in SciSPOT AM 1× buffer at 20 μM final concentration before being spotted on aldehyde coated Nexterion slides AL (Schott) using QArray2 (Genetix) spotter. Slides were then incubated at room temperature in a humidity chamber (> 90% relative

Methocarbamol humidity) and then in an oven at 120°C during 1 hour. Slide surface was then blocked twice for 2 min in 0.2% SDS solution, then twice for 2 min in RNase-DNase free water. The slides were then incubated at room temperature during 15 min in 125 mM NaBH4 prepared extemporally in a 3:1 (vol/vol) PBS:Ethanol mixture. The slides were then rinsed twice for 2 min in 0.2% SDS, then twice for 2 min in RNase-DNase free water and dried before hybridisation. Genomic DNA extraction and labelling Genomic DNA used for the microarray experiments was prepared by using the Genomic-tip 100/G anion exchange columns (Qiagen), following the manufactor’s recommendation. 20 μg of the genomic DNA was digested with MboI restriction enzyme (2 U/μg, Fermentas) at 37°C for 2 hours and complete restriction was confirmed by agarose gel electrophoresis. The fragmented genomic DNA was purified with phenol-chloroform-isoamyl alcohol (25:24:1). Aliqouts of 2 μg of genomic DNA were labelled using the Amersham Nick Translation Kit N5500 (GE Healthcare) in the presence of 91.3 μM dATP, 91.3 μM dGTP, 91.3 μM dTTP, 26.1 μM dCTP, and 33 μM Cy3-dCTP or Cy5-dCTP.