strain: TBCF10839 wild type genotype: wild type growth protocol: cultivated in the presence of polymorphonuclear neutrophils growth phase: stationary phase stress: PMN exoproducts
Treatment protocol
To examine the effect of exoproducts of PMNs on P. aeruginosa strains, stationary phase-grown bacterial cultures (1 x 10e10 cells) were pelleted and resuspended in 2 ml of RPMI 1640 medium (Invitrogen, Darmstadt, Germany), placed into a dialysis bag (300 kDa) and incubated in presence of 1x 10e7 PMNs in 15 ml RPMI 1640 medium containing 10 % (v/v) human AB blood serum, at 200 rpm on a rotary shaker for 2 h (37°C).
Growth protocol
P. aeruginosa strains were routinely grown overnight as shaken cultures (230 rpm) in Luria broth (LB) medium at 37°C. Five mL LB broth were inoculated with a toothpick of frozen bacterial stock solution and incubated for 16-48 h. Aliquots of these precultures served as inoculates for the main cultures. Stationary phase-grown cultures (3 x 10e10 cells) were resuspended in fresh Luria broth and kept in a dialysis tube (14-kDa cutoff, 25 mm) with an effective length of 6 cm for the exchange of fluids. Then the dialysis tube was resuspended in a 1-liter Erlenmeyer flask containing 600 ml of LB. The flasks were incubated at 37°C and 200 rpm on a rotary shaker for 2 h.
Extracted molecule
total RNA
Extraction protocol
RNA isolation: Bacterial cells were harvested by centrifugation at 3800 X g for 2 min at 4 oC. Total RNA from approximately 3 x 1010 cells was extracted with a modified hot phenol method (Tao et al., 1999). Bacteria were quickly resuspended in 0.5 ml of distilled water and lysed in 7.5 ml of preheated (65 oC) phenol/lysis-buffer mix 5 ml phenol (pH 5.5); 2.5 ml 2 % SDS, 30 mM Na-acetate, 3 mM EDTA, pH 5.5 with vigorous shaking for 10 min. The cell lysate was centrifuged at 3,800 X g for 20 min and the supernatant was extracted with 3 ml of phenol:chloroform:isoamyl alcohol (25:24:1, v/v) and then subsequently with 3 ml of chloroform:isoamyl alcohol (24:1, v/v). To pellet the nucleic acids, 0.1 volume of 3 M Na-acetate (pH 5.2) and 2.5 volumes of ethanol were added, incubated at –20oC overnight and centrifuged for 30 min at 3,800 X g. The pellet was washed with 5 ml 70 % ethanol and resuspended in 175 µl diethyl pyrocarbonate-treated water. DNA was digested by the addition of 40 units of DNaseI (Roche) and 20 units of SUPERaseIn (Ambion, Cambridgeshire, UK) in DNaseI buffer (50 mM Na-acetate, 10 mM MgCl2, 2 mM CaCl2, pH 6.5) for 30 min at 37 oC in a total volume of 200 µl. Then the RNA was purified with the use of RNeasy columns (Qiagen, Hilden, Germany) according to the manufacturer’s instructions and the yield of total cellular RNA was quantified by measuring the light absorption at 260 nm. RNA with a size below 200 bp (e.g., tRNAs, 5S rRNA) is below the cutoff of the column and therefore could not be recovered. All the steps were carried out at 4 oC or RNA was also incubated on ice intermittently during the whole RNA isolation procedure. RNA samples were separated electrophoretically in 1.2 % agarose with 2 % formaldehyde as the denaturing reagent and 1x MOPS (20 mM 4-morpholinepropanesulfonic acid, 10 mM Na-Acetate, 1 mM EDTA, pH 7.0) buffer was used as the running buffer. The agarose gels were stained with ethidium bromide to check the purity and integrity of RNA preparation, with the 16S and 23S ribosomal bands as reference. cDNA generation, fragmentation and labelling: To reduce variations in the abundance of specific mRNAs due to slight differences in growth conditions and RNA preparation, we pooled equal amounts of RNA from three cultures to get a final amount of 10 µg. The subsequent steps of cDNA generation and biotin-ddUTP terminal labeling were performed as described in the manufacturer's instructions for the P. aeruginosa GeneChip. Ten micrograms of total RNA was mixed with random primers (Invitrogen, Darmstadt, Germany) and control in vitro transcripts of 10 non-Pseudomonas gene sequences (kindly provided by S. Lory and coworkers) and incubated for 10 min at 70°C followed by 10 min at 25°C. Then cDNA reaction mix was added, which consisted of 5x first strand buffer (10 mM dithiothreitol, 0.5 mM deoxynucleoside triphosphates [dNTPs], 25 U/µl SuperScript II [all from Invitrogen], and 0.5 U/µl SUPERaseIn [Ambion]). This was followed by incubation for 10 min at 25°C, 60 min at 37°C, 60 min at 42°C, and 10 min at 70°C. RNA was hydrolyzed by adding 1 N NaOH and incubation for 30 min at 65°C; 1 N HCl neutralized the reaction. The cDNA was purified with the QIAquick column (QIAGEN) and quantified by A260. cDNA was fragmented in One Phor-All buffer with 0.5 U of DNase I per µg of cDNA for 10 min at 37°C and subsequent inactivation for 10 min at 98°C. To check if the majority of cDNA fragments were in a 50- to 200-bp range, 5 µl was loaded on a 2% (wt/vol) agarose gel stained with SYBR Green.
Label
biotin
Label protocol
The fragmentation product was end labeled with the Enzo BioArray terminal labeling kit with biotin-ddUTP (Affymetrix, Santa Clara, CA.).
Hybridization protocol
MES hybridization buffer (100 mM MES [morpholineethanesulfonic acid], 1 M NaCl, 20 mM EDTA, 0.01% Tween), 50 pM B2 control oligonucleotide (Affymetrix), 0.1 mg/ml herring sperm DNA (Promega), 0.5 mg/ml BSA (Invitrogen), and 7% (wt/vol) dimethyl sulfoxide (DMSO) were added to the labeled cDNA and loaded onto a P. aeruginosa PAO1 GeneChip (Affymetrix). After incubation for 16 h at 50°C at 60 rpm in an Affymetrix hybridization oven, the GeneChips were put into the Affymetrix fluidics station for washing. First the GeneChips were washed 20 times with nonstringent buffer (6x SSPE [1.08 M NaCl, 60 mM NaH2PO4, 1 mM EDTA; pH 7.7] and 0.01% [vol/vol] Tween 20) at 25°C and then they were washed 60 times with stringent buffer (100 mM MES, 0.1 M NaCl, 0.01% [vol/vol] Tween 20) at 50°C. Then a streptavidin solution mix, consisting of 10 µg/ml streptavidin, 2 mg/ml bovine serum albumin (BSA), 100 mM MES, 0.1 M NaCl, and 0.01% (vol/vol) Tween 20, was applied for 10 min at 25°C to stain bound cDNA. After washing for 40 times with nonstringent buffer at 30°C, the GeneChip was subjected to a secondary stain (5 µg/ml biotin-antistreptavidin antibody [Vector Laboratories, Burlingame, CA.], 100 µg/ml normal goat immunoglobulin G, 2 mg/ml BSA, 100 M MES, 0.1 M NaCl, 0.01% [vol/vol] Tween 20) for 10 min at 25°C and to a third stain (10 µg/ml streptavidin-phycoerythrin [Molecular Probes], 2 mg/ml BSA, 100 M MES, 0.1 M NaCl, 0.01% [vol/vol] Tween 20) for 10 min at 25°C. Finally excess label was removed by 60 washings with nonstringent buffer at 30°C.
Scan protocol
GeneChips were scanned at 570 nm with 3 µm resolution by the HP Affymetrix GeneChip scanner (Affymetrix Microarray Suite 5.0)
Description
n/a
Data processing
Data analysis was performed using the Affymetrix Microarray Suite software 5.0 with Affymetrix default parameters. The average microarray hybridization signal intensity was scaled to 150 and MAS5.0 was used as transformation algorithm. Experiments were carried out in duplicate. Two GeneChips for each mutant and wild type were compared by the four-comparison survival method (Chen et al., 2000). The data were imported into a Microsoft Access database capable of searching for genes, which were found in all four pairings defined by the Affymetrix Microarray Suite Software as having significant changes in their signal intensities. The arithmetic average and the standard deviation (SD) were calculated. Finally, a Bonferroni correction for multiple testing was applied as a rigorous criterion for significantly changed signal intensities (Salunkhe et al, 2002). Data were combined with the latest annotation (10 April 2010) from the web site of the P. aeruginosa PAO1 sequence and the community annotation project provided at http://www.pseudomonas.com.
