Tiling microarray-based identification of small regulatory RNAs in the group A Streptococcus (GAS)
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ABSTRACT:
ORGANISM(S): Streptococcus pyogenes
PROVIDER: GSE17789 | GEO | 2009/08/25
SECONDARY ACCESSION(S): PRJNA123287
REPOSITORIES: GEO
Similar Datasets
Project description:Small regulatory RNAs (sRNAs) represent a major mechanism of virulence regulation in several pathogens. To date, only three sRNAs have been described in GAS in any detail. To identify whether sRNAs are common within the GAS genome, we created a custom Affymetrix tiling microarray in which all intergenic regions (>49 bp) of the MGAS2221 genome were tiled at high density. Triplicate cultures of strain MGAS2221 in THY broth were grown to the exponential phase (O.D.600 of 0.5) of growth and RNA was isolated, converted to cDNA, fragmented, labeled, and hybridized to our custom array (one array per RNA sample [ hence three arrays total]). Our data identified the presence of 40 candidate sRNAs within the MGAS2221 genome. Three cultures of MGAS2221 were grown in THY broth to the exponential (O.D. 0.5) phase of growth. Two volumes of RNA protect were added to the samples, the samples incubated at room temperature for 5 minutes, and the bacteria collected through centrifugation. Total RNA was isolated via a mechanical disruption method, converted to cDNA, fragmented, labeled, and hybridized to our Affymetrix microarray. Data was analyzed using tiling analysis software to combine the data from the three arrays and generate signal intensities. The supplementary files 'GSE17789_2221sRNA_signal.bar' and 'GSE17789_2221sRNA_pvalue.bar' contain the signals and p-values, respectively, for Samples GSM444010-GSM444012. The BAR files are generated by TAS software using PM/MM signal intensities. Signal intensity data is reported in a linear scale; the p-value is reported in -10log10 scale. Probe analysis used a bandwidth of 40. Interval analysis used a threshold of 0, a maximum gap of 20, and a minimum run of 60.
2010-05-06 | E-GEOD-17789 | biostudies-arrayexpress
Project description:The rivR gene encodes a putative transcriptional regulator, while the rivX gene encodes a putative small regulatory RNA. To determine the RivRX regulon during exponential phase growth in THY broth we compared parental strain MGAS2221 with isogenic rivRX mutant strain 2221?rivRX. GAS strains were grown in THY broth to mid-exponential phase, corresponding to an O.D.600 of 0.5. A 4.5ml aliquot of each GAS culture was added to 9ml of RNAprotect (Qiagen Inc.). Following addition of RNAprotect, samples were incubated at room temperature for 5 mins before centrifuging. Supernatant was removed and the pellets quick frozen in liquid nitrogen and stored at -80 °C until ready to use. RNA isolation, cDNA synthesis, and use of our custom Affymetrix microarray was as previously described (Perez et al., 2009)
2012-12-01 | E-GEOD-36646 | biostudies-arrayexpress
Project description:PEL is a small regulatory RNA that is present within the S. pyogenes genome. In some strains of S. pyogenes, the PEL sRNA regulates many of the known virulence factors produced by this pathogen. A genome-wide analysis of PEL-regulated genes had not been performed previously so we analyzed this by comparing the parental M1T1 isolate MGAS2221 with an isogenic PEL mutant. Comparing gene transcript levels between these two strains at the exponential and stationary phases of growth, we identified that PEL has no regulatory function in MGAS2221. Three cultures of each GAS strain were grown in THY broth to exponential (O.D. 0.5) and stationary (O.D. 1.7) phases of growth. Two volumes of RNA protect were added to samples recovered at these O.D.'s, the samples incubated at room temperature for 5 minutes, and the bacteria collected through centrifugation. Total RNA was isolated via a mechanical disruption method, converted to cDNA, fragmented, labeled, and hybridized to our Affymetrix microarray. Estimates of gene expression were calculated using GCOS software v1.4.
2009-08-25 | E-GEOD-17767 | biostudies-arrayexpress
Project description:GAS strains were grown in THY broth to early exponential phase and RNA extracted. cDNA was generated and the expression profiles were determined using the RMLgenechip. Comparisons between the sample groups allow the identification of genes differentially expressed between strains. This experiment compared pre- and post- mouse passaged GAS strains. Keywords: GAS comparison
2006-02-01 | GSE3899 | GEO
Project description:The fasX gene encodes a small regulatory RNA in the group A Streptococcus (GAS). To determine the FasX regulon during GAS exposure to human plasma we compared parental strain MGAS2221 with isogenic fasX mutant strain 2221ΔFasX. Gene expression was analyzed 0, 15, and 60 minutes post-plasma exposure. GAS strains were grown in THY broth to mid-exponential phase, corresponding to an O.D.600 of 0.5. Two 16ml aliquots of each GAS culture were pelleted by centrifugation and the supernatant removed. Each GAS cell pellet was resuspended in 16ml of pre-warmed human plasma. Thus, four GAS-plasma suspensions were created, two each for each of the GAS strains, with one of the two using plasma from person A, and the other two using plasma from person B. Aliquots were recovered from each of the four GAS-plasma suspensions at T = 0, 15, and 60 mins. For the first two time-points 4.5ml of GAS-plasma suspension were added to 9ml of RNAprotect (Qiagen Inc.). For the final time-point, 4ml was added to 8ml of RNAprotect. Following addition of RNAprotect, samples were incubated at room temperature for 5 mins before centrifuging. Supernatant was removed and the pellets quick frozen in liquid nitrogen and stored at -80 °C until ready to use. RNA isolation, cDNA synthesis, and use of our custom Affymetrix microarray was as previously described (Perez et al., 2009)
2012-07-31 | E-GEOD-36645 | biostudies-arrayexpress
Project description:Two covR mutant derivatives of parental strain MGAS2221 were recovered from mice experimentally infected with MGAS2221 and shown to differ in terms of the number and concentration of secreted proteins. One of the covR mutant strains had a secretion phenotype identical to a covS mutant strain, while the other had a secretion phenotype identical to a constructed covR mutant strain. To further investigate the potential differences between the two covR mutant strains we performed expression microarray analysis. Single cultures of each of the four GAS strains tested were grown in THY broth to early exponential phase (O.D. 0.2). Two volumes of RNA protect were added, the samples incubated at room temperature for 5 minutes, and the bacteria collected through centrifugation. Total RNA was isolated via a mechaniscal disruption method, converted to cDNA, fragmented, labeled, and hybridized to our Affymetrix microarray. Estimates of gene expression were calculated using GCOS software v1.4. Data represent probes for serotype M1.
2009-06-05 | E-GEOD-16436 | biostudies-arrayexpress
Project description:We used RNA-Seq to assess how the presence/absence of the RD2 pathogenicity island influences global gene expression in the parental serotype M1 GAS isolate MGAS2221 and the parental serotype M49 isolate NZ131
2021-03-12 | GSE167343 | GEO
Project description:GAS strains were grown in THY broth to early exponential phase and RNA extracted. Targets from two biological replicates of each were generated and the expression profiles were determined using the RMLgenechip. Comparisons between the sample groups allow the identification of genes differentially expressed between strains. Keywords: repeat
2006-02-01 | GSE3900 | GEO
Project description:Small non-coding RNAs (sRNAs) have attracted attention as a new class of gene regulators in eukaryotes and in bacteria. In this study, we identified novel sRNAs of the human pathogen Streptococcus pyogenes M49 (GAS M49). A temporal expression profile of potential sRNAs was determined for (GAS M49). Cells were grown in chemical defined medium (CDM), Todd-Hewitt broth (Invitrogen) supplemented with 0.5% yeast extract (THY) or Brain-Heart-Infusion (BHI) complex medium using tiling arrays representing the intergenic regions of the GAS M49 genome. We identified 55 putative sRNAs in GAS M49 that were expressed during growth. Of those, 35 sRNAs were novel, whereas 20 sRNAs were known. Already described sRNAs included molecules belonging to one of the common non-coding RNA families covered by the rfam collection and streptococcal sRNAs that have been detected in previous studies. Comparison to a recently published bioinformatics screen showed a low overlap between putative sRNA genes. This is in accordance with the results from other studies, which underlines the fact that a comprehensive analysis of sRNAs expressed by a given organism requires the complementary use of different methods and the investigation of several environmental conditions. Despite a high conservation of sRNA genes within streptococci, the expression of sRNAs is rather strain specific. Identification of sncRNA candidates transcribed by S. pyogenes was undertaken with bacteria from cultures grown to exponential (OD600: 0.4) and stationary (OD600: 1.2) phase of growth and each for three growth media. For samples from bacteria grown in CDM medium four biological replicates were included, for samples from bacteria grown in THY and BHI medium two biological replicates were included.
2011-08-05 | E-GEOD-31228 | biostudies-arrayexpress
Project description:Small non-coding RNAs (sRNAs) have attracted attention as a new class of gene regulators in eukaryotes and in bacteria. In this study, we identified novel sRNAs of the human pathogen Streptococcus pyogenes M49 (GAS M49). A temporal expression profile of potential sRNAs was determined for (GAS M49). Cells were grown in chemical defined medium (CDM), Todd-Hewitt broth (Invitrogen) supplemented with 0.5% yeast extract (THY) or Brain-Heart-Infusion (BHI) complex medium using tiling arrays representing the intergenic regions of the GAS M49 genome. We identified 55 putative sRNAs in GAS M49 that were expressed during growth. Of those, 35 sRNAs were novel, whereas 20 sRNAs were known. Already described sRNAs included molecules belonging to one of the common non-coding RNA families covered by the rfam collection and streptococcal sRNAs that have been detected in previous studies. Comparison to a recently published bioinformatics screen showed a low overlap between putative sRNA genes. This is in accordance with the results from other studies, which underlines the fact that a comprehensive analysis of sRNAs expressed by a given organism requires the complementary use of different methods and the investigation of several environmental conditions. Despite a high conservation of sRNA genes within streptococci, the expression of sRNAs is rather strain specific.
2011-08-06 | GSE31228 | GEO