We determined the complete genome sequence of Streptomyces griseus IFO 13350, a soil bacterium producing an antituberculosis agent, streptomycin, which is the first aminoglycoside antibiotic, discovered more than 60 years ago. The linear chromosome consists of 8,545,929 base pairs (bp), with an average G+C content of 72.2%, predicting 7,138 open reading frames, six rRNA operons (16S-23S-5S), and 66 tRNA genes. It contains extremely long terminal inverted repeats (TIRs) of 132,910 bp each. The te ...[more]
Project description:Ferric uptake regulator (Fur) is the major regulator of iron acquisition in Escherichia coli and other bacteria. In the present study, the role of Fur in anaerobic S. enterica serovar Typhimurium was determined by transcriptome analysis, reporter assays, and enzymatic assays. In anaerobic Δfur, 298 genes were differentially expressed. In general, Fur repressed genes required for iron acquisition/storage, metabolism, electron transport, oxidative/nitrosative stress, and modulators of virulence. There were 73 genes whose expression required Fur, eleven of which contain a putative Fur box 5‟ of the gene. Transcription of sodA was >9-fold higher in Δfur but there was no corresponding increase in the activity of SodA. Anaerobic cultures of the fur mutant and the WT were used to inoculate two sets of three independent flasks each containing 150 ml of anoxic LB-MOPS-X. The three independent cultures were grown to an optical density at 600 nm (OD600) of 0.25 to 0.35, pooled, and treated with RNAlater (QIAGEN, Valencia, CA) to fix the cells and preserve the quality of the RNA. Total RNA was extracted with the RNeasy RNA extraction kit (QIAGEN), and the samples were treated with RNase-free DNase (Invitrogen, Carlsbad, CA). The absence of contaminating DNA and the quality of the RNA was confirmed by PCR amplification of known genes and by using agarose gel electrophoresis. Aliquots of the RNA samples were kept at –80°C for use in the microarray and quantitative real-time reverse transcription-PCR (qRT-PCR) studies. The SuperScript Indirect cDNA labeling system (Invitrogen) was used to synthesize the cDNA for the hybridizations. Each experiment consisted of two hybridizations, on two slides, and was carried out in Corning Hybridization Chambers at 42°C overnight. Dye swapping was performed to avoid dye-associated effects on cDNA synthesis. The slides were washed at increasing stringencies (2x SSC [1x SSC is 0.15 M NaCl plus 0.015 M sodium citrate], 0.1% sodium dodecyl sulfate [SDS], 42°C; 0.1% SSC, 0.1% SDS, room temperature; 0.1% SSC, room temperature). Following hybridization, the microarrays were scanned for the Cy3 and Cy5 fluorescent signals with a ScanArray 4000 microarray scanner from GSI Lumonics (Watertown, MA). The intensity of every spot was codified as the sum of the intensities of all the pixels within a circle positioned over the spot itself and the background as the sum of the intensities of an identical number of pixels in the immediate surroundings of the circled spot. Cy3 and Cy5 values for each spot were normalized over the total intensity for each dye to account for differences in total intensity between the two scanned images. The consistency of the data obtained from the microarray analysis was evaluated by a pair-wise comparison, calculated with a two-tailed Student's t test and analyzed by the MEAN and TTEST procedures of SAS-STAT statistical software (SAS Institute, Cary, NC) . The signal intensity at each spot from the fur mutant and the WT were background subtracted, normalized, and used to calculate the ratio of gene expression between the two strains. All replicas were combined, and the median expression ratios and standard deviations were calculated for open reading frames (ORFs) showing 2.5-fold change.
Project description:Transcriptomic analysis of the cells grown on biphenyl on a filter placed on a minimal agar medium and those grown on biphenyl to mid-exponential phase in a liquid medium 3x dual channel slides.
Project description:Here we report the first transcriptomic analysis of a Gram-positive bacterium to desiccation. Filtered RHA1 cells incubated at either low relative humidity (20%), as an air drying treatment, or high relative humidity (100%), as a control, were transcriptionally profiled over a comprehensive time series. Keywords: stress response time course Three biological replicates from both the desiccation and control experiments were analyzed using two-colour microarrays. Over a time series for each experiment, Cy-labelled cDNA from treated and time-zero cells were hybridized, with a dye swap for one of the replicates.
Project description:The overall gene expression of a CvfA- mutant of Streptococcus pyogenes grown in C medium was compared to that of the wild type (HSC5spc). Stationary phase cells (8 hrs post-inoculation) were used because the gene expression of the CvfA- mutant was most different from that of the wild type at stationary phase, while the difference of gene expression between the CvfA- mutant and the wild type was negligible at exponential phase. Two biological replicates (two different RNA sets extracted from the CvfA- mutant and the wild type at different day were used): 399773,399887,399888 vs. 399889,399890,399891 Technical replicates (Microarrays were hybridized with the same RNA with the same dye): 399773 and 399887; 399889 and 399890 Dye swap (Microarrays were hybridized with the same RNA but Cy3 and Cy5 dyes were swapped): 399773, 399887 vs. 399888; 399889, 399890 vs. 399891