To make the transcriptional fusion of gtsA (PP1015) with lacZ reporter gene, we used the promoter probe plasmid p9TTBlacZ.

The 980-bp-long gtsA promoter region was amplified from P. putida PaW85 chromosome see more using oligonucleotides PP1014kesk (5′-GCTGTCGACGCCAATACGCT) and PP1015alg (5′-GCATCTAGACGAAGCGTGGAATTCATC). The PCR-amplified DNA fragment was cleaved with HincII and XbaI and ligated into SmaI-XbaI-opened p9TTBlacZ, yielding p9TT1015. β-galactosidase assay β-galactosidase activities were measured either from solid or liquid medium-grown bacteria. For the analysis of gtsA promoter, total enzyme activity was measured using permeabilized cells as described elsewhere [33]. Cell lysis assay To evaluate the cell lysis of the colR mutant, we have previously used so-called unmasked β-galactosidase assay which relies on the detection of a cytoplasmic enzyme β-galactosidase leaked out from the cells [25, 34]. In this assay we measured the β-galactosidase activity in suspension of cells permeabilized Protein Tyrosine Kinase inhibitor with SDS and chloroform (total activity), and also in intact, non-permeabilized cells. The percentage of unmasked β-galactosidase activity was calculated from equation: xn/xp × 100%, where xp is β-galactosidase

activity measured in SDS and chloroform-treated cells, and xn is β-galactosidase activity measured in non-permeabilized cells. We have shown earlier that in case of ColR-deficiency-dependent cell lysis, unmasked β-galactosidase values are above 5% [25]. As a source of β-galactosidase, the plasmid pKTlacZS/C containing the lacZ gene, was used [35]. Bacteria were grown for 24, 48, or 72 hours on glucose (0.2, 0.4, or 0.8%) or gluconate (0.2%) M9 minimal media. To enhance lysis, 1 mM phenol was added to the growth medium in some experiments. Bacteria were scraped off the agar plate using plastic Cobimetinib chemical structure swabs and suspended in M9 solution. Optical density of the cell suspension was determined

at 580 nm and β-galactosidase activity was measured [34]. Isolation of outer membrane proteins For the isolation of outer membrane proteins (OMPs) bacteria were grown for 24 hours on two Petri plates. Bacteria were scraped off the agar and suspended in 3 ml of 10 mM HEPES buffer (pH 7.4). For the analyses of peripheral and central subpopulations, bacteria were grown on agar plate in sectors as pictured in Results. To collect enough cells from the sectors, five to ten plates were used, i.e., cells from 15 to 30 sectors per strain were collected and suspended in 3 ml of 10 mM HEPES buffer (pH 7.4). Cells were disrupted by ultrasonication and the cell debris was pelleted by centrifugation at 10 000 g at 4°C for 10 minutes. The supernatant was then centrifuged at 100 000 g at 4°C for 1 hour to pellet membrane proteins.