Growth Temperature: -6°C +/- 0.1°C Optical Density (OD600) at Harvest: 0.1024 Growth Rate: 0.0002
Growth protocol
Growth Media and Procedure: For all experiments, single colonies of Psychrobacter arcticus 273-4 were used to inoculate primary cultures in marine broth (MB) which grew to OD600 ~ 0.8 at 4°C. One liter of MB consisted of 3% (w/v) sea salts (Sigma-Aldrich, St. Louis, MO), 5 g/l tryptone (BD, Sparks, MD) and 1 g/l yeast extract (Difco). A 1% (v/v) inoculum of this starter culture was transferred to 7 ml of acetate medium. One liter acetate medium contained 75 g l-1 sea salts (Sigma-Aldrich), 50 mM MOPS buffer (Sigma-Aldrich), 20 mM sodium acetate (Baker), 5 mM NH4Cl (Baker), pH 7.0 with NaOH, 1 mM K2HPO4 (Baker), 1X Wolfe’s Vitamins, and 1X trace minerals mix. Wolfe’s Vitamins (1000X) and trace minerals solutions were made according to M1 defined medium from Kostka and Nealson (31). P. arcticus 273-4 was acclimatized to the acetate medium through two passages to OD600 ~ 0.8 at 4°C with shaking at 150 rpm before inoculation into experimental samples. A 5 ml inoculum from the final starter culture of P. arcticus in acetate medium was introduced to 500 ml acetate medium in 300 cm2 untreated and vent-capped Falcon tissue culture flask. Cultures were allowed to grow without shaking at -6°C, 0°C, 17°C or 22°C until they reached an OD600 between 0.09 and 0.25 (mid-exponential phase at all temperatures).
Extracted molecule
total RNA
Extraction protocol
When cultures reached mid- exponential phase, 25 ml of culture was preserved with 2 volumes of RNAprotect Bacteria Reagent (Qiagen, Valencia, CA) which had been chilled to the growth temperature of the culture. Cells in RNAprotect were incubated at room temperature for 5 min then pelleted by centrifugation in a Sorvall RC-5B centrifuge at 4°C. Cells were resuspended in 1 ml room temperature RNAprotect and pelleted again in an Eppendorf 5417R centrifuge at 4°C. Supernatant was decanted and the cell pellet was frozen at -80°C. RNA extractions were performed according to the RNEasy mini kit (Qiagen) with some modifications. Specificially, lysozyme digestion was carried out for 30 min. Following cell lysis using buffer RLT (Qiagen), 1 μl each of a two-fold dilution series of SpotReport mRNAs (Stratagene) ranging from 1 ng to 1 pg of RNA were added to the lysate as controls for RNA degradation during sample preparation. Purified RNAs were analysed by gel electrophoresis in a 1X FA gel containing 0.67% formaldehyde and 1.2% agarose.
Label
Cy5
Label protocol
Three 5 μg aliquots of total RNA were denatured with 6 μg random hexamers (Invitrogen, Carlsbad, CA) in a 17.5 μl volume for 10 min at 70°C and snap-cooled for 5 min on ice. Denatured total RNA mixtures were reverse transcribed to amino-allyl labeled cDNAs by combining the total RNA mixture with 6 μl 5X First Strand Synthesis Buffer, 3 μl 0.1 M dithiothreitol, 1 μl RNaseOUT (Invitrogen), 1.2 μl 25X dNTPs (3 aa-dUTP:2 dTTP), and 2 μl Superscript II reverse transcriptase (Invitrogen). Amino-allyl dUTP was obtained from Ambion (Austin, TX). Reactions were incubated at 42°C overnight and stopped by addition of 10 μl 0.5 M EDTA. RNA was hydrolyzed by incubation with 10 μl 1 M NaOH at 65°C for 15 min. RNA hydrolysis was neutralized with 10 μl 1 M HCl. Amino-allyl labeled cDNAs were purified according to the amino-allyl labeling protocol published by The Institute for Genomic Research (TIGR) (Hegde et al 2000).
Growth Temperature: 22°C +/- 0.5°C Optical Density (OD600) at Harvest: 0.1551 Growth Rate: 0.014 per h
Growth protocol
Growth Media and Procedure: For all experiments, single colonies of Psychrobacter arcticus 273-4 were used to inoculate primary cultures in marine broth (MB) which grew to OD600 ~ 0.8 at 4°C. One liter of MB consisted of 3% (w/v) sea salts (Sigma-Aldrich, St. Louis, MO), 5 g/l tryptone (BD, Sparks, MD) and 1 g/l yeast extract (Difco). A 1% (v/v) inoculum of this starter culture was transferred to 7 ml of acetate medium. One liter acetate medium contained 75 g l-1 sea salts (Sigma-Aldrich), 50 mM MOPS buffer (Sigma-Aldrich), 20 mM sodium acetate (Baker), 5 mM NH4Cl (Baker), pH 7.0 with NaOH, 1 mM K2HPO4 (Baker), 1X Wolfe’s Vitamins, and 1X trace minerals mix. Wolfe’s Vitamins (1000X) and trace minerals solutions were made according to M1 defined medium from Kostka and Nealson (31). P. arcticus 273-4 was acclimatized to the acetate medium through two passages to OD600 ~ 0.8 at 4°C with shaking at 150 rpm before inoculation into experimental samples. A 5 ml inoculum from the final starter culture of P. arcticus in acetate medium was introduced to 500 ml acetate medium in 300 cm2 untreated and vent-capped Falcon tissue culture flask. Cultures were allowed to grow without shaking at -6°C, 0°C, 17°C or 22°C until they reached an OD600 between 0.09 and 0.25 (mid-exponential phase at all temperatures).
Extracted molecule
total RNA
Extraction protocol
When cultures reached mid- exponential phase, 25 ml of culture was preserved with 2 volumes of RNAprotect Bacteria Reagent (Qiagen, Valencia, CA) which had been chilled to the growth temperature of the culture. Cells in RNAprotect were incubated at room temperature for 5 min then pelleted by centrifugation in a Sorvall RC-5B centrifuge at 4°C. Cells were resuspended in 1 ml room temperature RNAprotect and pelleted again in an Eppendorf 5417R centrifuge at 4°C. Supernatant was decanted and the cell pellet was frozen at -80°C. RNA extractions were performed according to the RNEasy mini kit (Qiagen) with some modifications. Specificially, lysozyme digestion was carried out for 30 min. Following cell lysis using buffer RLT (Qiagen), 1 μl each of a two-fold dilution series of SpotReport mRNAs (Stratagene) ranging from 1 ng to 1 pg of RNA were added to the lysate as controls for RNA degradation during sample preparation. Purified RNAs were analysed by gel electrophoresis in a 1X FA gel containing 0.67% formaldehyde and 1.2% agarose.
Label
Cy3
Label protocol
Three 5 μg aliquots of total RNA were denatured with 6 μg random hexamers (Invitrogen, Carlsbad, CA) in a 17.5 μl volume for 10 min at 70°C and snap-cooled for 5 min on ice. Denatured total RNA mixtures were reverse transcribed to amino-allyl labeled cDNAs by combining the total RNA mixture with 6 μl 5X First Strand Synthesis Buffer, 3 μl 0.1 M dithiothreitol, 1 μl RNaseOUT (Invitrogen), 1.2 μl 25X dNTPs (3 aa-dUTP:2 dTTP), and 2 μl Superscript II reverse transcriptase (Invitrogen). Amino-allyl dUTP was obtained from Ambion (Austin, TX). Reactions were incubated at 42°C overnight and stopped by addition of 10 μl 0.5 M EDTA. RNA was hydrolyzed by incubation with 10 μl 1 M NaOH at 65°C for 15 min. RNA hydrolysis was neutralized with 10 μl 1 M HCl. Amino-allyl labeled cDNAs were purified according to the amino-allyl labeling protocol published by The Institute for Genomic Research (TIGR) (Hegde et al 2000).
Hybridization protocol
Amino-allyl labeled cDNAs were resuspended in 4.5 μl 0.1 M Na2CO3, pH 9.0 for 10 min at room temperature and combined with 4.5 μl Cy3 or Cy5 NHS-ester in DMSO (GE Biosciences, Piscataway, NJ). Dye incorporation reactions were incubated at 25°C for 1.5 h. Equipicomolar nucleotide amounts of dye-labeled cDNA samples were hybridized such that neither Cy3 nor Cy5 exceeded 550 pmoles. cDNAs were dried in a SpeedVac for 1.5 h. Hybridization mix was prepared by resuspending cDNA pellets in 50 μl 5X SSC, 25% formamide, 0.1% SDS and 0.1 mg/ml Salmon Testes DNA (Sigma-Aldrich). Glass slide arrays were prehybridized by incubation in 5X SSC, 0.1% SDS, 0.1 mg ml-1 BSA for 60 min at 49°C. Slides were then washed twice in room temperature 0.1X SSC for 5 min. Slides were washed twice for 30 sec in sterile water. Prehybridized slides were then immediately dried by centrifugation for 3 min at 1600 rpm in a Sorvall RT 6000D centrifuge with an H1000B hanging basket rotor. Lifterslips (22 x 60 mm, Erie Scientific, Portsmouth, NH) were washed in sterile water followed by 100% ethanol and dried under filtered forced air. Hybridization mix was preheated to 95°C for 10 min, then pipeted onto the slide. Hybridization cassettes were sealed and incubated in a 49°C water bath shaking at 30 rpm for 20 h.
Scan protocol
Slides were scanned using a Genepix 4000B scanner (Molecular Devices, Sunnyvale, CA). Images were analyzed using Genepix 6.0 software and results were analyzed for quality using the marray and arrayQuality packages in R 2.3.0 (http://www.r-project.org/). Only slides passing the quality control analysis were included in the biological analysis.
Description
Here we report that P. arcticus 273-4 exhibits two states of growth metabolism over temperature, a fast growth state at Topt and a resource efficiency state at temperature < 4°C. While numerous discoveries are reported, three hypotheses were the focus of this transcriptome analysis: i) transcriptomes at low temperature would be more similar to each other than to the transcriptomes at optimal growth temperatures, ii) genes for cold acclimation proteins such as RNA and protein chaperones, fatty acid desaturase and translation factors such as rbfA would be transcribed at higher levels during growth at low temperatures, and iii) acetate metabolism and energy metabolism would be up-regulated at low temperature to compensate for increased energy demand per generation. It was also expected that exchange of isozyme expression of temperature would be detectable in the microarray experiment. The transcriptome results were compared to a small scale proteome analysis and knockout mutants were also generated to test specific hypotheses resulting from the microarray. To the best of our knowledge, this is the first report of transcriptome analysis during growth below 0°C.
Data processing
Statistical significance of the temperature effect on gene expression was computed using R/MAANOVA 1.0 to determine statistical significance and LIMMA 2.7.10 to calculate pairwise comparisons of gene expression. In both cases data were normalized using regional (print-tip) lowess normalization. Normalized data were fit to a mixed-effects ANOVA model with gene, dye and temperature as fixed terms. Slide and biological replicate were random terms. F tests were carried out using the variance shrinking Fs statistic to determine the statistical significance of differential expression with 500 permutations to evaluate the distribution of Fs. Genes were considered differentially expressed if P < 0.01 after Benjamini-Hochberg P-value adjustment for multiple hypothesis testing. The false discovery rate for the statistically significant genes was estimated according to the method of Storey using the qvalue package in R. All pairwise temperature comparison coefficients were estimated using LIMMA.
