Screening of about 9000 transposon insertion derivatives of the colR mutant disclosed 27 clones with higher phenol tolerance. Sequencing of mini-transposon insertion sites revealed that phenol sensitivity of the colR-deficient strain was elevated by disruption of genes dispersed between different
functional classes (Table 1). As ColRS system is obviously involved in membrane functionality [8, 11, 12] it was expected that disruption of several membrane-related genes could complement the colR-deficiency. However, some metabolic genes were also identified as determinants of phenol tolerance (Table 1). Most of mini-transposon insertions were located in open reading frames of targeted genes, thus obviously abolishing their function. However, in case of PP1824 the mini-transposon was inserted upstream of the ATG start codon most probably changing the expression level GW786034 price of this gene. Table 1 Description of chromosomal loci of phenol tolerant mini-transposon derivatives see more of colR-deficient P. putida http://www.jcvi.org/. Locus ID Gene name NCT-501 supplier protein name Probable localization* Number of Insertions PP0145 Na+/Pi cotransporter family protein CM 1 PP1386 ttgA multidrug/solvent RND membrane fusion protein CM 4 PP1385 ttgB multidrug/solvent RND transporter CM 9 PP1384 ttgC multidrug/solvent RND outer membrane protein OM 1 PP1619 conserved hypothetical protein C 1 PP1621 pcm protein-L-isoaspartate
O-methyltransferase C 4 PP1650 gacS sensor histidine kinase-response regulator CM 3 PP1842
glutamine amidotransferase, class I C 1** PP3997 glycosyl transferase, putative C 1 PP4422 succinate-semialdehyde dehydrogenase, putative C 1 PP4798 membrane-bound lytic murein transglycosylase, putative CM 1 * Abbreviations: CM – cytoplasmic membrane; OM – outer membrane; C – cytoplasm ** insertion 3 bp upstream of ATG Disruption of ttgC enhances phenol tolerance of both colR-deficient and colR-proficient P. putida 14 out of 27 phenol tolerant minitransposon derivatives of PD184352 (CI-1040) the colR-deficient strain possessed miniTn5 insertion in the ttgABC operon (Table 1) and therefore we focused on this system. In toluene tolerant Pseudomonas putida DOT-T1E, three homologous efflux pumps TtgABC, TtgDEF and TtgGHI belonging to the RND (resistance-nodulation-cell division) family transporters contribute to solvent tolerance [28]. TtgABC efflux pump plays a major role in antibiotic resistance of this strain, and it also expels solvents and plant antimicrobials from cells [28–31]. The basal expression level of TtgABC in Pseudomonas putida DOT-T1E is relatively high being further enhanced by hydrophobic antibiotics and some plant metabolites [30, 31]. However, the expression of this efflux system does not respond to solvents [29]. TtgABC efflux pump proteins are highly similar between DOT-T1E and KT2440 strains (over 99% identity) suggesting that their substrate range and biological role could be similar.