strain: ATCC 3502 genotype/variation: Wild Type temperature: 37 °C
Growth protocol
TPGY, anaerobic, 37 °C, up to OD600=1.0 (5-7 hours), then subjected to temperature downshift to 15 °C and thereafter incubated at 15 °C
Extracted molecule
total RNA
Extraction protocol
Five-ml samples from three replicate C. botulinum ATCC 3502 wild type or cbo365 KO-mutant cultures were collected at mid-logarithmic growth phase at 37 °C and 1 h after temperature downshift to 15 °C. The samples were collected into sterile plastic tubes containing ice-cold ethanol-phenol (9:1) solution (Sigma Aldrich, St. Louis, MO, USA), mixed thoroughly, and incubated on ice for 30 min. Cells were harvested by centrifugation (4 °C, 8000 x G) for 5 min. The cell pellets were immediately frozen to -70 °C until RNA extraction. The cell pellets were thawed on ice for 5 min and used for RNA extraction with the RNeasy Midi Kit (Qiagen, Hilden, Germany) according to manufacturer’s instructions. The cells were lysed with a solution containing 25 mg/ml lysozyme (Sigma Aldrich, St. Louis, MO, USA) and 250 IU/ml mutanolysin (Sigma Aldrich) in Tris-EDTA buffer (pH 8.0, Fluka BioChemica, Buchs, Switzerland) and agitated at 37 °C for 30 min. The final elution volume of RNAse free water was 300 µl. To confirm efficient removal of all genomic DNA, an additional DNase treatment was carried out using the DNA-free Kit (Ambion, Austin, TX, USA) according to manufacturer’s instructions. The RNA yield and purity (A260/A280) were checked using the NanoDrop ND-1000 device (NanoDrop Technologies, Wilmington, DE, USA). The RNA purity ratio was >2.0 for all samples. Integrity of RNA was confirmed using a miniatyrized gel electrophoresis in the Agilent Bioanalyzer (Agilent Technologies, Waldbronn, Germany). The RNA integrity number was >9.2 for all RNA samples.
Label
Cy3
Label protocol
A total of 2 µg of each RNA sample was reverse-transcribed into cDNA and simultaneously labeled with fluorescent dyes. In brief, each 30-µl labeling reaction contained 0.2 µg/µl of random hexamers (Invitrogen), 0.01 M DTT (Invitrogen), 1.3 U/µl ribonuclease inhibitor (Invitrogen), 0.5 µM dATP, dTTP and dGTP, 0.2 µM dCTP, 1.7 nmol of Cy-3 or Cy-5 labeled dCTP (GE Healthcare, Pittsburgh, PA), 13 U/µl of SuperScript III reverse transcriptase (Invitrogen), and appropriate buffer (1 x First Strand Buffer, Invitrogen) and was incubated at 46 °C for 3 h. RNA hydrolysis and reaction inactivation were performed by addition of 10 µl of 0.1 M NaOH and 0.5 mM EDTA and incubation at 70 °C for 15 min. The reactions were subsequently neutralized by addition of 10 µl of 0.1 M HCl. The cDNA was purified with QIAquick PCR Purification Kit (Qiagen), with final elution volume of 40 µl. The cDNA concentration of each sample was measured with NanoDrop.
strain: ATCC 3502 genotype/variation: CBO0365 KO mutant temperature: 37 °C
Growth protocol
TPGY, anaerobic, 37 °C, up to OD600=1.0 (5-7 hours), then subjected to temperature downshift to 15 °C and thereafter incubated at 15 °C
Extracted molecule
total RNA
Extraction protocol
Five-ml samples from three replicate C. botulinum ATCC 3502 wild type or cbo365 KO-mutant cultures were collected at mid-logarithmic growth phase at 37 °C and 1 h after temperature downshift to 15 °C. The samples were collected into sterile plastic tubes containing ice-cold ethanol-phenol (9:1) solution (Sigma Aldrich, St. Louis, MO, USA), mixed thoroughly, and incubated on ice for 30 min. Cells were harvested by centrifugation (4 °C, 8000 x G) for 5 min. The cell pellets were immediately frozen to -70 °C until RNA extraction. The cell pellets were thawed on ice for 5 min and used for RNA extraction with the RNeasy Midi Kit (Qiagen, Hilden, Germany) according to manufacturer’s instructions. The cells were lysed with a solution containing 25 mg/ml lysozyme (Sigma Aldrich, St. Louis, MO, USA) and 250 IU/ml mutanolysin (Sigma Aldrich) in Tris-EDTA buffer (pH 8.0, Fluka BioChemica, Buchs, Switzerland) and agitated at 37 °C for 30 min. The final elution volume of RNAse free water was 300 µl. To confirm efficient removal of all genomic DNA, an additional DNase treatment was carried out using the DNA-free Kit (Ambion, Austin, TX, USA) according to manufacturer’s instructions. The RNA yield and purity (A260/A280) were checked using the NanoDrop ND-1000 device (NanoDrop Technologies, Wilmington, DE, USA). The RNA purity ratio was >2.0 for all samples. Integrity of RNA was confirmed using a miniatyrized gel electrophoresis in the Agilent Bioanalyzer (Agilent Technologies, Waldbronn, Germany). The RNA integrity number was >9.2 for all RNA samples.
Label
Cy5
Label protocol
A total of 2 µg of each RNA sample was reverse-transcribed into cDNA and simultaneously labeled with fluorescent dyes. In brief, each 30-µl labeling reaction contained 0.2 µg/µl of random hexamers (Invitrogen), 0.01 M DTT (Invitrogen), 1.3 U/µl ribonuclease inhibitor (Invitrogen), 0.5 µM dATP, dTTP and dGTP, 0.2 µM dCTP, 1.7 nmol of Cy-3 or Cy-5 labeled dCTP (GE Healthcare, Pittsburgh, PA), 13 U/µl of SuperScript III reverse transcriptase (Invitrogen), and appropriate buffer (1 x First Strand Buffer, Invitrogen) and was incubated at 46 °C for 3 h. RNA hydrolysis and reaction inactivation were performed by addition of 10 µl of 0.1 M NaOH and 0.5 mM EDTA and incubation at 70 °C for 15 min. The reactions were subsequently neutralized by addition of 10 µl of 0.1 M HCl. The cDNA was purified with QIAquick PCR Purification Kit (Qiagen), with final elution volume of 40 µl. The cDNA concentration of each sample was measured with NanoDrop.
Hybridization protocol
Agilent (Agilent, Santa Clara, USA) 8x15K CGH protocol, 65°C for 18-20 h.
Scan protocol
GenePix 4200 AL (Axon Instruments) using pixel resolution of 5 µm, line average 2
Data processing
Image analysis with GenePix Pro 6.0, bad spots were manually flagged (flag-value -100). Data preprocessing in R with limma package: background subtraction with normexp and offset 50. Log-ratio was calculated between KO-mutant and WT and normalized with loess.
The cold-induced two-component system CBO0366/CBO0365 regulates metabolic pathways with novel roles in cold tolerance of Group I Clostridium botulinum ATCC 3502
