Project description:In the present study we have determined the global gene expression and biomolecular composition in an Escherichia coli model strain exposed to ten adverse conditions (sodium chloride, ethanol, glycerol, two acids (hydrochloric acid and acetic acid), sodium hydroxide, heat (46°C) and cold (15°C) as well as ethidium bromide and the disinfectant benzalkonium chloride). The large variation in responses and few common genes illustrates the adaptation potential of E. coli and its ability to survive and colonize a wide range of environments. Keywords: gene expression study, stress response The strain used in this study was the genome-sequenced strain E. coli K-12 MG1655 (1) and the following concentrations/conditions were used; CH3COOH (pH 5.9 ± 0.05); HCl (pH 4.75 ± 0.05); NaOH (pH 9.6 ± 0.05); EtOH (5%); NaCl (4.5%); Glycerol (15%); BC (7 and 9 mg/ml); EtBr (150 mg/ml); 46°C; 15°C. The cultures were prepared by inoculating one colony from tryptone soya agar (TSA) (Oxoid) (overnight growth at 37°C) to 5 ml TSB and incubating overnight at 37°C, with shaking (200 rpm). This culture was initially diluted 1:10 in medium and used to inoculate room-temperature TSB for each of the different stress factors (40 ml total volume) to a final concentration of approx. 1X10^7 CFU/ml (1:100 dilution of overnight culture). For cold stress experiment the medium was chilled to 15°C prior to inoculation. The cultures were incubated at 37°C unless otherwise stated (Table 1), shaking at 200 rpm and samples for global gene expression and biomolecular analyses were collected during exponential growth at a cell density of approx. 1X10^7 CFU/ml. All the stress conditions, including the control, were inoculated with the same overnight culture and started at the same time point. The experiment was performed 3 times at different days and with freshly prepared solutions, resulting in 3 biological replicates. Total RNA was extracted from 1 ml cells using the RNeasy Protect Bacteria Mini Prep kit (Qiagen) as recommended by the manufacturer including the "on-column" DNase treatment. The concentration and purity of the total RNA was analyzed using NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Inc.) and the RNA 600 Nano LabChip system (Agilent Technologies). Total RNA (5 ug) and 0.5 ul test/reference Lucidea spike controls RNA (Amersham Biosciences) were reverse transcribed by using random hexamers in the presence of aminoallyl-dUTP (2 aa-dUTP (Ambion): 3 dTTP (Invitrogen)) as described in standard operating procedure no. M007 (The Institute for Genomic Research [TIGR] protocol). The removal of unincorporated aa-dUTP and free amines was performed according to the TIGR protocol using the Qiagen QIAquick PCR purification kit. After drying in a Speed vac, the samples were stored at -20°C. Coupling aminoallyl-labeled cDNA to Cy3/Cy5 mono reactive dyes (Amersham Biosciences) was done according to the TIGR protocol. The E. coli microarrays consisted of E. coli oligos (Operon) printed on Corning Ultra GAPS slides as described previously (2, 6, 7). Microarray slides were treated with a pre-soak solution (Promega/Corning) before incubation in a pre-hybridization solution (5XSSC [1XSSC is 0.15 M NaCl plus 0.015 M sodium citrate], 0.1% dodecyl sulfate [SDS], 1 mg/ml bovine serum albumin) at 42°C for 30-60 minutes. After pre-hybridization, the slides were washed briefly in distilled water, followed by a brief wash in isopropanol. The dried Cy3- and Cy5-labeled cDNA was resuspended in 55 ul hybridization solution (30% formamide, 5XSSC, 0.1%SDS and 0.1 mg/ml Salmon Sperm DNA), denatured and applied to the prehybridized microarray underneath a 25- by 60-mm mSeries LifterSlip coverslip (Erie Scientific Company). The slides were hybridized at 42°C overnight (~16 h). After hybridization the slides were washed twice in 2XSSC buffer with 0.1% SDS at 42°C for 5 minutes, followed by two washes in 0.1XSSC buffer with 0.1% SDS for 10 min at room temperature and four washes in 0.1X SSC buffer for 1 minute at room temperature. The slides were dried by centrifugation (90xg for 12 min). The reference used in the microarray experiments was pooled RNA from all the growth conditions including the control. Slides were scanned with the ScanArray Express 1.0 scanner (Packard BioScience) following the manufacturers guidelines. The fluorescent spot intensities were quantified using ImaGene 5.6.1 (BioDiscovery Inc.) software. Background subtraction and normalization (LOWESS (3, 5)) was performed in GeneSpring 7 (Silicon Genetics). (A lowess curve was fit to the log-intensity versus log-ratio plot. 20% of the data was used to calculate the lowess fit at each point. The curve was used to adjust the control value for each measurement. If the control channel was lower than 10 then the value was set to 10). Only data from spots representing E. coli K-12 MG1655 genes were analyzed in our studies. All analyses were based on 3 biological replicates with the exception of the second biological replicate from conditions HCl and NaOH. These two observations were removed due to bad hybridization giving a total of 34 array hybridizations (observations). Genes not present in any of the 34 observations were filtered out, resulting in the analysis of 4279 out of 4289 MG1655 genes on the array. Missing values (data points) were replaced by using the KNNimpute procedure (4) (k=10) on log2 transformed data. 1. Blattner, F. R., G. Plunkett, 3rd, C. A. Bloch, N. T. Perna, V. Burland, M. Riley, J. Collado-Vides, J. D. Glasner, C. K. Rode, G. F. Mayhew, J. Gregor, N. W. Davis, H. A. Kirkpatrick, M. A. Goeden, D. J. Rose, B. Mau, and Y. Shao. 1997. The complete genome sequence of Escherichia coli K-12. Science 277:1453-74. 2. Constantinidou, C., J. L. Hobman, L. Griffiths, M. D. Patel, C. W. Penn, J. A. Cole, and T. W. Overton. 2006. A reassessment of the FNR regulon and transcriptomic analysis of the effects of nitrate, nitrite, NarXL, and NarQP as Escherichia coli K12 adapts from aerobic to anaerobic growth. J Biol Chem 281:4802-15. 3. Quackenbush, J. 2002. Microarray data normalization and transformation. Nat Genet 32 Suppl:496-501. 4. Troyanskaya, O., M. Cantor, G. Sherlock, P. Brown, T. Hastie, R. Tibshirani, D. Botstein, and R. B. Altman. 2001. Missing value estimation methods for DNA microarrays. Bioinformatics 17:520-5. 5. Yang, Y. H., S. Dudoit, P. Luu, D. M. Lin, V. Peng, J. Ngai, and T. P. Speed. 2002. Normalization for cDNA microarray data: a robust composite method addressing single and multiple slide systematic variation. Nucleic Acids Res 30:e15. 6. Zhang, L., R. R. Chaudhuri, C. Constantinidou, J. L. Hobman, M. D. Patel, A. C. Jones, D. Sarti, A. J. Roe, I. Vlisidou, R. K. Shaw, F. Falciani, M. P. Stevens, D. L. Gally, S. Knutton, G. Frankel, C. W. Penn, and M. J. Pallen. 2004. Regulators encoded in the Escherichia coli type III secretion system 2 gene cluster influence expression of genes within the locus for enterocyte effacement in enterohemorrhagic E. coli O157:H7. Infect Immun 72:7282-93. 7. Zheng, D., C. Constantinidou, J. L. Hobman, and S. D. Minchin. 2004. Identification of the CRP regulon using in vitro and in vivo transcriptional profiling. Nucleic Acids Res 32:5874-93.
2010-05-18 | E-GEOD-11041 | biostudies-arrayexpress
