coli and bezylpenicillin (1500 μg ml-1), kanamycin (50 μg ml-1), or streptomycin (200 μg Ceritinib ic50 ml-1) for P. putida. Selection strategy of phenol tolerant mutants in colR-deficient P. putida strain For identification of genes affecting phenol sensitivity, the colR-deficient strain was subjected to mutagenesis by Tn5 based mini-transposon containing streptomycin resistance marker. A mini-transposon-carrying plasmid mTn5SSgusA40 [21] was conjugatively transferred from E. coli CC118 λpir [16] into a P. putida colR-deficient strain with the aid of a helper plasmid pRK2013 [18]. Transconjugants with random chromosomal insertions of the mini-transposon were first selected on glucose minimal

plates supplemented with kanamycin and streptomycin. After colonies were grown for three days at 30°C, they were replicated onto glucose minimal plates containing 8 mM phenol. Although a single Z-VAD-FMK mw colR-deficient cell could not form a colony on these plates, replication of big and closely located colonies of colR-deficient bacteria enabled their growth on replica plates. After another three days, growth of replicated clones in the presence of phenol was evaluated. About 150 transconjugants out of approximately 9000 transposon mutants grew better than colR-deficient P. putida and they were subjected to secondary assay of phenol tolerance. In order to avoid spontaneous phenol tolerant mutants, the clones

of interest were picked up from glucose

plates of initial selection. The secondary screen yielded 34 clones with higher phenol tolerance than the parental colR-deficient strain. Finally, siblings were eliminated through analysis of clones by arbitrary PCR and sequencing, resulting in 27 independent transposon insertion mutants with elevated phenol tolerance. Arbitrary PCR To identify chromosomal loci interrupted by insertion of mini-transposon in selected clones arbitrary PCR and sequencing were used. PCR products were generated by two rounds of amplifications as described elsewhere [22]. In the first round, a primer specific for the Sm gene (Smsaba – 5′-GAAGTAATCGCAACATCCGC-3′) or for the gusA gene (Gus2 CYTH4 – 5′-ACTGATCGTTAAAACTGCCTGG) and an arbitrary primer were used (Arb6 – 5′-GGCCACGCGTCGACTAGTACNNNNNNNNNNACGCC-3′). Second-round PCR was performed with primers Smsaba or Gus2 and Arb2 (5′-GGCCACGCGTCGACTAGTAC-3′). Cloning procedures and construction of bacterial strains To inactivate the ttgC gene in both wild-type and colR-deficient backgrounds the ttgC gene was first amplified using oligonucleotides ttgCalgus (5′-GAAGAATTCGTCACCCCTGAAAATCC-3′) and ttgClopp (5′-CCGAATTCGGTGGGCTTTCTGCTTTT-3′) and inserted into EcoRI-opened pUCNotKm (R. Teras). For disruption of the ttgC gene in pUC/ttgC, a central 315-bp Eco255I fragment of ttgC was replaced with Smr gene from the pUTmini-Tn5Sm/Sp [23].