BW25113 GhoS for RNase activity
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ABSTRACT:
ORGANISM(S): Escherichia coli
SUBMITTER: Thomas Wood
PROVIDER: E-GEOD-36779 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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Project description:Persisters are a subpopulation of metabolically-dormant cells in biofilms that are resistant to antibiotics; hence, understanding persister cell formation is important for controlling bacterial infections. Previously we discerned that MqsR and MqsA of Escherichia coli are a toxin/antitoxin pair that influence persister cell production via their regulation of Hha, CspD, and HokA. Here, to gain more insights into the origin of persisters, we used protein engineering to increase the toxicity of toxin MqsR by reasoning it would be easier to understand the effect of this toxin if it were more toxic. We found that two mutations (K3N and N31Y) increase the toxicity four fold and increase persistence 73 fold compared to native MqsR by making the protein less labile. A whole transcriptome study revealed that the MqsR variant represses acid resistance genes (gadABCEWX and hdeABD), multidrug resistance genes (mdtEF), and osmotic resistance genes (osmEY). Corroborating these microarray results, deletion of rpoS as well as the genes that the master stress response regulator RpoS controls, gadB, gadX, mdtF, and osmY, increased persister formation dramatically to the extent that nearly the whole population became persistent. Therefore, the more toxic MqsR increases persistence by decreasing the ability of the cell to respond to antibiotic stress through its RpoS-based regulation of acid resistance, multidrug resistance, and osmotic resistance systems. For the whole-transcriptome study of BW25113 mqsR/pBS(Kan)-mqsR 2-1 versus BW25113 mqsR/pBS(Kan)-mqsR, planktonic cells were grown to a turbidity of 0.5 at 600 nm in LB medium with 1 mM IPTG at 37 M-BM-0C, adjusted the turbidity to 1, and exposed to 20 M-NM-<g/mL ampicillin with 1 mM IPTG for 1 h. Cells were isolated by centrifuging at 0M-BM-0C, and RNALaterM-BM-. buffer (Ambion, Cat# AM7021) was added to stabilize RNA during the RNA preparation steps. Total RNA was isolated from cell pellets with Qiagen RNeasy mini Kit (Cat# 74104) using a bead beater. cDNA was synthesized and fragmented to obtain 50-200 base cDNA fragments. The fragmented cDNA was labelled with Biotin-ddUTP, and hybridization was performed at 45M-BM-0C with 60 rpm for 16 hours. The probe array was washed, stained using Affymetrix Genechip Fluidics Station 450 and the software GenomeChipOperating Software (GCOS)M-bM-^@M-^], and scanned using Affymetrix Genechip scanner GCS3000 7G system and the software GenomeChipOperating Software (GCOS). Data were processed with MAS 5.0 Expression Analysis Default Setting. Genes were identified as differentially expressed if the expression ratio was higher than the standard deviation: 4.0 fold (enriched and differentially degraded) cutoff for the DNA microarrays (standard deviation 1.3 fold), and if the p-value for comparing two chips was less than 0.05.
2012-04-02 | E-GEOD-31054 | biostudies-arrayexpress
Project description:MqsR/MqsA is a well-characterized toxin/antitoxin system with several regulatory roles. Deletion of mqsRA reduced growth with deoxycholate stress. Here we performed a microarray to determine the expression profile with and without mqsRA under deoxycholate stress to determine transcriptional regulation. Escherichia coli K-12 BW25113 with and without mqsRA was exposed to deoxycholate stress.
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2014-05-13 | E-GEOD-54271 | biostudies-arrayexpress
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2014-09-15 | E-GEOD-47427 | biostudies-arrayexpress
Project description:gene expression profiles by overexpressing Hha from pCA24N-hha using 2 mM IPTG induction in BW25113 wild type suspension cells in LB medium at 37C relative to BW25113 carrying the empty vector plasmid in the same test conditions. Experiment Overall Design: Strains: E. coli K12 BW25113/pCA24N, BW25113/pCA24N-hha Experiment Overall Design: Growth conditions: grow culture in LB medium at 37C to OD600 0.1 and add 2 mM IPTG to induce, and grow to OD600 0.5 to collect cells. RNA was extraction, and gene expression profiles were evaluated.
2008-10-20 | E-GEOD-10116 | biostudies-arrayexpress
Project description:This Series involves two studies: 1) The gene expression of E. coli K-12 BW25113 ompA mutant strain vs. wild type strain glasswool biofilm cells and E. coli K-12 BW25113 ompA mutant vs. wild type polystyrene biofilm cells. 2) The gene expression of E. coli BW25113 ompA/pCA24N_ompA vs. ompA/pCA24N suspension cells. Strains: E. coli K-12 BW25113 wild type, ompA mutant Medium: LB Cell type: Biofilm cells grown on glasswool and polystyrene surfaces Time: 15 h Temperature: 37C Strains: BW25113 ompA/pCA24N_ompA and ompA/pCA24N Medium: LB Time: 7 h Temperature: 37C Cell type: suspension cells, induced by 0.1 mM IPTG
2010-11-03 | E-GEOD-14064 | biostudies-arrayexpress
Project description:It has been proposed that the gastro-intestinal tract environment containing high levels of neuroendocrine hormones is important for gut-derived P. aeruginosa infections. In this study, we report that the hormone norepinephrine increases P. aeruginosa PA14 growth, virulence factor production, invasion of HCT-8 epithelial cells, and swimming motility in a concentration-dependent manner. Transcriptome analysis of P. aeruginosa exposed to 500 µM, but not 50 µM, norepinephrine for 7 h showed that genes involved in the regulation of the virulence determinants pyocyanin, elastase, and Pseudomonas quinolone signal (PQS, 2-heptyl-3-hydroxy-4-quinolone) were up-regulated. The production of rhamnolipids, which are also important in P. aeruginosa infections, was significantly decreased on semi-solid surfaces, but not in planktonic cultures, upon exposure to norepinephrine. Swarming motility, a phenotype that is directly influenced by rhamnolipids, was also decreased upon 500 µM norepinephrine exposure. The increase in the transcriptional activation of lasR, but not that of rhlR, suggest that the effects of norepinephrine are mediated primarily through the las quorum sensing pathway. Together, our data strongly suggests that norepinephrine can play an important role in gut-derived infections by increasing the pathogenicity of P. aeruginosa PA14. For growth rate measurements, a single colony of PA14 was grown overnight in Luria Bertini (LB) broth, and re-inoculated in serum-RPMI medium in the presence of 50 µM and 500 µM NE to an initial turbidity of ~ 0.05 at 600 nm. The turbidity of the cultures at 600 nm was monitored every hour and growth rate of the exponentially growing cultures was calculated. Growth curves were obtained in triplicate using three independent cultures, and the statistical significance of specific growth rate was determined using the unpaired Student’s t-test. Total RNA isolation and microarray analysis PA14 was grown overnight in LB to turbidity at 600 nm of ~5.0 and diluted in 100 mL of serum-RPMI medium to an initial turbidity at 600 nm of 0.05. Different concentrations of NE (50 µM or 500 µM) were added and the cultures grown for 7 hours till late exponential phase (turbidity at 600 nm of ~0.5, 1.1 and 1.5 with 0 µM, 50 µM and 500 µM NE, respectively). Cell pellets were prepared and RNA was isolated as described previously (Ren et al. 2004a). The P. aeruginosa Genome Array (Affymetrix, P/N 510596) containing 5,500 of the 5,570 open reading frames of P. aeruginosa PA01, was used to analyze changes in the PA14 transcriptome. cDNA synthesis, fragmentation and hybridizations were as described previously (Gonzalez Barrios et al. 2006). Hybridization was performed for 16 h at 50°C, and the total cell intensity was scaled to an average value of 500. The probe array images were inspected for any image artifact. Background values, noise values and scaling factors of all the arrays were examined and were comparable. The intensities of polyadenosine RNA control were used to monitor the labelling process. For each binary microarray comparison of differential genes expression, if the gene with the larger transcription rate did not have a consistent transcription rate based on the 13 probe pairs (P-value less than 0.05), these genes were discarded. A gene was considered differentially expressed when the P-value for comparing two chips was lower than 0.05 (to assure that the change in gene expression was statistically significant and that false positives arise less than 5%), and the expression ratio (between cells treated with NE and control) was greater than 4.0 for the 50 µM NE array and 2.0 for 500 µM NE array (based on the standard deviation of fold-change values) (Ren et al. 2004b). Gene functions and locus tag numbers were obtained from the Pseudomonas Genome Database (http://www.pseudomonas.com/download.jsp).
2009-08-27 | E-GEOD-13326 | biostudies-arrayexpress
Project description:E. coli K-12 BW25113 persister cells generated via H202 pre-treatment and deletion of rpoS, relative to BW25113 wild-type stationary phase gene expression. Persister cells were generated following exposure to ampicillin 20 ug/mL. Strains: BW25113 wild-type, pre-treated with H202 10 min, ampicillin 5 h; BW25113 delta rpoS, ampicillin 5 h; BW25113 wild-type OD at 600 nm of 3.0, ampicillin 30 mins. Medium: LB ampicillin 20 ug/mL used to generate persisters; ampicillin 5 ug/mL added to stationary phase BW25113 wild-type
2012-04-02 | E-GEOD-34028 | biostudies-arrayexpress
Project description:Persisters are cells which evade stresses like antibiotics and which are characterized by reduced metabolism and a lack of genetic alterations required to achieve this state. We showed previously that MqsR and MqsA of Escherichia coli are a toxin-antitoxin pair that influence cell physiology (e.g., biofilm formation and motility) via RNase activity as well as through regulation of toxin CspD. Here, we show that deletion of the mqsRA locus decreases persister cell formation and, consistent with this result, overexpression of MqsR increases persister cell formation. Furthermore, toxins Hha, CspD, and HokA increase persister cell formation. In addition, by overproducing MqsR in a series of isogenic mutants, we show that Hha and CspD are necessary for persister cell formation via MqsR overexpression. Surprisingly, Hfq, a small RNA chaperone, decreases persistence. A whole-transcriptome study shows that Hfq induces transport-related genes (oppA, oppB, oppC, oppD, oppF, and dppA), outer membrane protein-related genes (ybfM and ybfN), toxins (hha), and proteases (clpX, clpP, and lon). Taken together, these results indicate that toxins CspD and Hha influence persister cell formation via MqsR and that Hfq plays an important role in the regulation of persister cell formation via regulation of transport or outer membrane proteins. Strains: E. coli BW25113 K-12 hfq deleted mutant vs. wild-type Medium: LB Culture: Planktonic cell grown OD=0.5, adjusted OD=1.0, and then exposed to 100 ug/mL ampicillin for 2 h.
2009-11-07 | E-GEOD-18852 | biostudies-arrayexpress
Project description:To provide more evidence of the specificity of the RNase activity of GhoS, we performed a whole-transcriptome study for the production of GhoS vs. an empty plasmid so that we could investigate all of the cell’s transcripts for cleavage with GhoS, in vivo (i.e., BW25113/pCA24N-ghoS vs. BW25113/pCA24N with 1 mM IPTG induction of ghoS for 90 min). Under these conditions, only 20 genes were found to be repressed by more than 4-fold; there were no induced genes. These GhoS-repressed genes were all involved in the biosynthesis/transport of purines and pyrimidines; among them, pyrI was most highly repressed (-20 fold). These results suggest that GhoS selectively cleaves only a few cellular targets.
2012-08-30 | GSE36779 | GEO