For acute stress assays, nine culture flasks containing standard DSM457 medium were inoculated with a single stationary-phase culture of strain RW1. After cultures reached the mid-exponential phase (OD600 of 0.2 to 0.3), three cultures each were diluted into 180 mL of sodium chloride-amended DSM457 medium (solute potential decreased by 0.25 MPa), into polyethylene glycol (MW of 8000)-amended DSM457 medium (matric potential decreased by 0.25 MPa), or into standard DSM457 medium (control cultures). The cultures were then incubated for 30 min, cells were collected by vacuum filtration, and the filters were frozen with liquid nitrogen and stored at -80°C until further processing.For chronic stress assays, three culture flasks containing sodium chloride-amended DSM457 medium (solute potential decreased by 0.25), three culture flasks containing polyethylene glycol (MW of 8000)-amended DSM457 medium (matric potential decreased by 0.25 MPa), and three culture flasks containing standard DSM457 medium (control cultures) were inoculated with a single stationary-phase culture of strain RW1. After inoculation, the cultures were grown for approximately 24 hours until reaching the mid-exponential phase (OD600 of 0.2 to 0.4). Cells were then collected by vacuum filtration and the filters were frozen with liquid nitrogen and stored at -80°C until further processing.For sand desiccation experiments, seven culture flasks containing standard DSM457 medium were inoculated with a single stationary-phase culture of RW1. After the cultures reached the mid-exponential phase (OD600 of 0.2 to 0.3), cells were collected by centrifugation and the cell pellets were suspended in 100 µl of the remaining medium. Four of the 100-µL cell suspensions were then each inoculated into a 15-mL plastic tube containing two grams of dry sand and homogenized by mixing. The amount of liquid added was approximately 10% of the water holding capacity of the sand, at which level more than 70% of the inoculated RW1 cells remained viable after 60 min of incubation in the sand. The remaining three 100-µL cell suspensions were each inoculated into a 15-mL plastic tube that did not contain sand to serve as controls. All seven 15-mL tubes were then incubated for 60 min on a tube roller at 50 rpm and room temperature. After incubation, cells were extracted from the four sand treatments by adding five mL of a saline solution to each tube, vortexing the mixture, and filtering the suspension through a 70 µm pore-size cell strainer. Cells were then collected from the strainer filtrate by vacuum filtration and the filters were frozen with liquid nitrogen and stored at -80°C until further processing. For the three control cultures, two grams of sand and five mL of saline solution were added simultaneously to the samples after incubation for 60 min and cells were then immediately collected as described for the sand treatment samples.
Growth protocol
Sphingomonas wittichii strain RW1 was obtained from Rolf-Michael Wittich. All cultures were grown in 100-mL culture flasks containing 20 mL of a phosphate-buffered mineral medium (medium DSM457 from the German Resource Centre for Biological Material, Braunschweig, Germany) and 5 mM of sodium salicylate as the sole carbon source. Bacterial growth was assessed from culture turbidity at 600 nm (OD600). Cells were collected from exponential phase liquid cultures (OD600 of 0.2 to 0.3) or from cells after inoculation into dry sand. Cells were collectd by vacuum filtration and the filters were stored at -80°C until processing.
Extracted molecule
total RNA
Extraction protocol
RNA was extracted from frozen filters using a previously described acid-phenol method (Johnson et al., 2005, Appl. Environ. Microbiol. 71:3866-3871; Johnson et al., 2008, Appl. Environ. Microbiol. 74:3533-3540). RNA samples were purified from contaminating DNA using the DNA-free kit from Ambion. The quantity and quality of the purified RNA was assessed using the A260 and A 280 values measured with the MICROARRAY function on a NanoDrop spectrophotometer (Witec AG). The integrity of RNA was further verified by agarose gel electrophoresis and the absence of DNA was verified by PCR.
Label
Cy3
Label protocol
cDNA was synthesized from total RNA and directly labeled with cyanine-3-dCTP using a modification of a protocol described elsewhere (Gaillard et al., 2010, BMC Microbiol. 10:153). Briefly, each 50-µL reaction contained 10 µg of total RNA, 1.25 µg of random hexanucleotide primers (Promega), 100 µM each of unlabeled dATP, dGTP, and dTTP (Invitrogen), 25 µM of unlabeled dCTP (Invitrogen), 25 µM of cyanine-3-labeled dCTP (Perkin-Elmer), 25 U SUPERase•In (Ambion), and 400 U Superscript II reverse transcriptase (Invitrogen). Reactions were performed by heating at 42ºC for 2 hours followed by 70ºC for 10 min. RNA was then removed by adding 100 mM NaOH, heating to 65ºC for 20 min, and neutralizing with 100 mM HCl and 300 mM sodium acetate (pH 5.2). Labeled cDNA products were purified using the MinElute PCR purification kit (Qiagen) and the quantity and incorporation frequency of cyanine-3-labeled dCTP were calculated using the MICROARRAY function on a NanoDrop Spectrophotometer.
Hybridization protocol
Sixty ng of labeled cDNA was loaded onto each microarray, hybridized for 17 hours at 65ºC, and washed and scanned as described for labeled cRNA in the One-Color Microarray-Based Gene Expression Analysis Manual (Agilent). The fragmentation step (heating to 60ºC for 30 minutes) was omitted.
Scan protocol
Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner (G2505B) using one color scan setting for 8x15k array slides (Scan Area 61x21.6 mm, Scan resolution 5um, Dye channel is set to Green and Green PMT is set to XDR: Hi:100% and XDR Lo:10%.
Description
Gene expression 30 min control
Data processing
The scanned images were analyzed with Feature Extraction Software 10.5.1.1 (Agilent) using default parameters (protocol GE1-v5_95_Feb07 and Grid: 021656_D_F_20081007) to obtain background subtracted and spatially detrended Processed Signal intensities.
