4.7.2 software. The Read Mapper Tool maps reads and calculates average coverage at single nucleotide resolution. The Probabilistic Variant Caller identifies variants by using a probabilistic model built from read mapping data. Based on a combination of a Bayesian model and a Maximum Likelihood approach the algorithm calculates prior and error probabilities for the Bayesian

model. By using the Probabilistic Variant Caller Foretinib nmr software and defining various parameters, such as sequence frequency, size of mutated areas and mutation abundance, lists of SNPs and DIPs were created. A frequency of more than 30 reads was required for all fragments. The maximum number of allel-variations was restricted to two, and the threshold of the frequency of the allel-variations was set at a minimum of 30%. These lists were compared for the wild type strain Selumetinib datasheet PD0325901 in vivo and the pooled resistant mutants, and SNPs that

are unique for the mutants were identified. Colony PCR and sequencing The 15 resistant mutants were analyzed individually to determine whether they carry the point mutation on position 848 of the kdpD gene. Individual colonies were heated in 36.5 μl of water for 5 min at 95°C. 1 μl of dNTPs (stock solution 10 mM), 2.5 μl of primers VC_A0531_forw2 and VC_A0531_rev2 (stock solution 100 pmol/μl), 5 μl 10× PCR buffer and 2.5 μl RED Taq polymerase (1 U/μl) were added. After the PCR procedure, the products had the expected size of 915 bp. They were purified and sequenced in the sequencing facility of the HZI using the above primers. Construction of the point-mutant KdpD T283M in strain NM06-058 The gene VC_A0531 has

a size of 1,494 base pairs (coding for 497 amino acids plus stop codon). The base cytosine, which was changed to tyrosine in the predominant resistant mutants, is located on position 848. Site-directed mutagenesis Aprepitant was used for the incorporation of this modification into the wild type strain NM06-058. Two overlapping amplicons with a size of 525 and 616 bp were generated from the gene of the wild type strain NM06-058. Fragment one was amplified using the primer pair (i) Mut_forw_1/Mut_rev_1, and the second fragment was amplified with primer pair (ii) Mut_forw_2/Mut_rev_2. The primers Mut_rev_1 and Mut_forw_2 carried the point-mutation (Table  3, bold nucleobases). Primers Mut_forw_1 and Mut_rev_2 contained specific recognition nucleotide sequences for the restriction enzymes XbaI and HindIII. Both amplicons were mixed at equimolar ratio and a re-PCR was performed with the primers Mut_forw_1 and Mut_rev_2 to generate an amplicon with a size of 1,114 bp. This amplicon and the plasmid pEX18Ap were restricted with XbaI and HindIII. Insert and plasmid were ligated and transformed into chemically competent E. coli strain S17-1. Amp (100 μg/ml) was incorporated into the agar of the plate for selection of pEX18Ap containing transformants.