0 (Applied Biosystems). The fluorescence of SYBR Green is measured against ROX at the end of each PCR cycle in

the ABI 7500 Fast Real-Time PCR System. The comparative CT method (2-ΔΔCT) was used to calculate the relative quantities of nucleic acid sequence of target genes in each sample [22]. CT (threshold cycle) is the fractional cycle number at which the SYBR Green fluorescence passes the baseline signal [22]. The expression levels of target genes were normalized against that of the 16S rRNA gene (endogenous control). RNA obtained from P. fluorescens cLP6a cultures grown at 28°C to stationary phase was used as the calibrator sample in this study. Statistical Panobinostat manufacturer analysis of data was performed using ANOVA (Excel 2007). Membrane GW4869 concentration integrity assay Membrane integrity of P. fluorescens cLP6a cells grown to stationary phase at 10°C, 28°C or 35°C was determined using a modification of the method described by Niven and Mulholland [23].

Cell samples (1 ml) were harvested by centrifugation, re-suspended in 1 ml of phosphate-buffered saline and adjusted to an OD600 of 1.0. Propidium iodide (PI; Invitrogen), either alone or with the membrane-disrupting agent cetyltrimethylammonium bromide (CTAB; Sigma), were added to final concentrations of 30 μmol l-1 and 1 μmol l-1 respectively; untreated cells were included as parallel controls. After 30 min incubation at room temperature, fluorescence of 100-μl cell samples was measured in a 96-well AMN-107 purchase microplate using a Synergy HT Multi-mode Microplate Reader (BioTek) at excitation and emission wavelengths of 500 nm and 600 nm respectively.

Phospholipid fatty acid (FA) extraction and identification Total cell lipids were extracted using the Bligh-Dyer method [24] modified by White and Ringelberg [25] from 10 mg lyophilized cLP6a or cLP6a-1 cells grown to stationary phase at different temperatures or in the presence of antibiotics (at 1/4 MIC) or PAHs (5 mmol l-1). Fatty acid methyl esters (FAME) were prepared from extracted Glycogen branching enzyme total lipids using mild alkaline methanolysis [26], dried under a stream of N2 and re-dissolved in 500 μl chloroform (HPLC grade, Fisher Scientific). FAME were analysed by gas chromatography with mass spectrometry (GC-MS) on an Agilent 6890N GC with a model 5973 inert mass selective detector (Agilent) fitted with an Agilent HP-5MS capillary column (30 m × 0.25 mm ID, 0.25 μm film thickness; J + W Scientific). Helium was used as the carrier gas with a temperature program of 150°C (1 min) increasing to 190°C at 1.5°C min-1, then 25°C min-1 to 290°C (held for 4 min). Sample peaks were compared to Bacterial Acid Methyl Ester Mix standards (Supelco, Sigma Aldrich) and quantified by calculating individual FAME peak areas as a percentage of the total FAME in each sample [27]. Free FA assay P. fluorescens strains cLP6a and cLP6a-1 cultures grown to stationary phase at 10°C, 28°C or 35°C were harvested by centrifugation. The culture supernatants were filtered using a 0.