The relative quantification (RQ) values were LY3023414 cost calculated by the following formula: RQ = 2− [ΔCT(mutant) − ΔCT(wild type)][39, 40]. Q-PCR analysis was performed in duplicate (technical replicates) on three independent RNA isolations (biological replicates). β-galactosidase assays To study the
mbo operon expression in different genetic backgrounds, the mbo operon promoter (P mboI ) cloned into pMP220 [19] as previously described [6] was used. The derivative mutants in mgoA, gacA and gacS genes were transformed with plasmid pMP::P mboI which contains the P mboI . The plasmid BMN 673 clinical trial pLac-mgoBCAD (harboring the mgo operon) was also used to complement the mgoA, gacA and gacS mutants and finally the β-galactosidase activity of P mboI was measured. In order to evaluate the effect of the mgo operon on the activity of P mboI , P. protegens Pf-5 was used due to the LCZ696 clinical trial absence of the two operons in its genome. First, P. protegens Pf-5 was transformed with the pMP::P mboI and the promoter activity was measured, and secondly to measure the effect on the mbo operon transcription, this strain containing the plasmid pMP::P mboI , was also transformed with
the plasmid pLac-mgoBCAD (mgo operon under pLac regulation). As a negative control the β-galactosidase activity was measured for the wild type strain P. syringae pv. syringae UMAF0158 and each strain used in this assay, transformed with empty vector pMP220. β-galactosidase activities were quantified by the Miller method [41]. Briefly, an overnight culture obtained as previously described in growth curve and toxins assay section were prepared. The samples were collected at 18 h, and the cells were harvested and suspended in assay buffer to eliminate any error in the detection of β-galactosidase activity due to the effects of different carbon sources present in the growth medium. The results presented are from three separate experiments, each conducted in triplicate. Phylogeny of the mgoA gene In order to identify the presence of the mgoA gene in the different genomes of Pseudomonas
strains, the mgoA gene from P. syringae pv. syringae Sunitinib in vitro UMAF0158 was used in BLASTP [42] comparisons with whole genome sequences of Pseudomonas spp. available in the databases. Once the amino acid sequences of all the orthologous mgoA genes were obtained, the putative adenylation domains were identified using the PKS/NRPS Analysis Web-site (http://nrps.igs.umaryland.edu/nrps) [43]. Other adenylation domains of which the activated amino acid is already known were obtained from the database and from De Bruijn met al. [44]. Two phylogenetic analyses were done, the first was using the adenylation domain of all the NRPSs (328 residues) and the second was using the almost entire sequence of MgoA (1015 residues).
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