At exponential growth phase (OD600 = 0.4) diacetyl was added at a concentration of 5 mM. After 30 min, the equivalent of 10 OD600 units of volume (volume in mL multiplied by OD600) was taken. Cells were collected by centrifugation in 50 mL Greiner tubes in an Eppendorf 5810R centrifuge (Eppendorf AG, Hamburg, Germany), during 10 min centrifugation, speed 10,000 rpm, at 4 °C.
Growth protocol
The bacterial strain Lactococcus lactis was grown as standing cultures at 30 °C in M17 broth (DifcoTM BD, NJ, USA) supplemented with glucose (GM17; Sigma-Aldrich, MO, USA) at a concentration of 0.5 % (w/v).
Extracted molecule
total RNA
Extraction protocol
For RNA isolation, 0.5 g of glass beads (~ 100 μm in diameter), 50 μL of 10% sodium dodecylsulfate (SDS; Sigma-Aldrich, MO, USA), 500 μL of premixed phenol:chloroform:isoamylalcohol (25:24:1) and 175 μL of macaloid suspension were added to the thawed cells in the screw cap tube. Cells were disrupted using 2 cycles of 45 s of bead beating with a 1 min interval on ice. The cell lysate was cleared by centrifugation in an Eppendorf 5417R centrifuge (Eppendorf AG, Hamburg, Germany) during 10 min centrifugation, speed 10000 rpm, at 4 °C. Next, the upper phase was extracted with 500 μL of chloroform: isoamylalcohol (24:1). The two phases were resolved by centrifugation (10 min, 10000 rpm, 4 °C) and total RNA was isolated from the aqueous phase using the High Pure RNA Isolation Kit (Roche Molecular Biochemicals, Mannheim, Germany), according the manufacturer’s instructions.
Label
Cy3
Label protocol
Incorporation of amino allyl-modified dUTPs during cDNA synthesis allowed Cy3/Cy5 labeling with the CyScribe Post labeling Kit (Amersham Biosciences, Piscataway, NJ, USA), according to the supplier’s instructions. All intermediate and final purifications of either labeled or unlabeled cDNA were performed with a NucleoSpin Extract II Kit (Clontech Laboratories, Mountain View, CA, USA), according to manufacturer’s instructions except when purifying unlabeled cDNA, where 80 % ethanol was used in a second washing step and 0.1 M sodium bicarbonate, pH 9.0, was used as the elution buffer.
At exponential growth phase (OD600 = 0.4) diacetyl was added at a concentration of 5 mM. After 30 min, the equivalent of 10 OD600 units of volume (volume in mL multiplied by OD600) was taken. Cells were collected by centrifugation in 50 mL Greiner tubes in an Eppendorf 5810R centrifuge (Eppendorf AG, Hamburg, Germany), during 10 min centrifugation, speed 10,000 rpm, at 4 °C.
Growth protocol
The bacterial strain Lactococcus lactis was grown as standing cultures at 30 °C in M17 broth (DifcoTM BD, NJ, USA) supplemented with glucose (GM17; Sigma-Aldrich, MO, USA) at a concentration of 0.5 % (w/v).
Extracted molecule
total RNA
Extraction protocol
For RNA isolation, 0.5 g of glass beads (~ 100 μm in diameter), 50 μL of 10% sodium dodecylsulfate (SDS; Sigma-Aldrich, MO, USA), 500 μL of premixed phenol:chloroform:isoamylalcohol (25:24:1) and 175 μL of macaloid suspension were added to the thawed cells in the screw cap tube. Cells were disrupted using 2 cycles of 45 s of bead beating with a 1 min interval on ice. The cell lysate was cleared by centrifugation in an Eppendorf 5417R centrifuge (Eppendorf AG, Hamburg, Germany) during 10 min centrifugation, speed 10000 rpm, at 4 °C. Next, the upper phase was extracted with 500 μL of chloroform: isoamylalcohol (24:1). The two phases were resolved by centrifugation (10 min, 10000 rpm, 4 °C) and total RNA was isolated from the aqueous phase using the High Pure RNA Isolation Kit (Roche Molecular Biochemicals, Mannheim, Germany), according the manufacturer’s instructions.
Label
Cy5
Label protocol
Incorporation of amino allyl-modified dUTPs during cDNA synthesis allowed Cy3/Cy5 labeling with the CyScribe Post labeling Kit (Amersham Biosciences, Piscataway, NJ, USA), according to the supplier’s instructions. All intermediate and final purifications of either labeled or unlabeled cDNA were performed with a NucleoSpin Extract II Kit (Clontech Laboratories, Mountain View, CA, USA), according to manufacturer’s instructions except when purifying unlabeled cDNA, where 80 % ethanol was used in a second washing step and 0.1 M sodium bicarbonate, pH 9.0, was used as the elution buffer.
Hybridization protocol
Hybridization to the probes spotted on the L. lactis MG1363 mixed amplicon and oligonucleotide DNA microarray slides, covering 2308 of the 2435 predicted ORF’s, was done using the Slidehyb 1 hybridization buffer (Ambion Biosystems, Foster City, CA, USA), during 16 h at 45 °C. After hybridization, slides were washed for 5 min in 2X SSC (150 mM NaCl and 15 mM trisodium citrate) with 0.5 % SDS, 2 times for 5 min in 1X SSC with 0.25 % SDS and 5 min in 1X SSC with 0.1 % SDS. All washing steps were performed at 30 °C with preheated buffers. The washing buffers were removed via centrifugation (Eppendorf 5810R, 2 min, 2000 rpm).
Scan protocol
The DNA microarray slides were scanned using a GenePix Autoloader 4200AL confocal laser scanner (Molecular Devices Corporation, Sunnyvale, CA, USA).
Description
Biological replicate 3 of 3: Cy3 induced, Cy5 control
Data processing
The resulting images were analyzed using the ArrayPro Analyzer 4.5 software (Media Cybernetics, Silver Spring, MD, USA). Signal intensities were quantified for each spot on the DNA microarray slides after subtracting the background intensities, which were determined for each spot by reading the signals in the regions that separated diagonal spots.
