RNA Seq of passages of a small colony variant P. aeruginosa clinical isolate
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ABSTRACT:
PROVIDER: PRJNA622542 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR11465562.fastq.gz | Fastqsanger.gz | |||
| SRR11465563.fastq.gz | Fastqsanger.gz | |||
| SRR11465564.fastq.gz | Fastqsanger.gz | |||
| SRR11465565.fastq.gz | Fastqsanger.gz | |||
| SRR11465566.fastq.gz | Fastqsanger.gz |
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Project description:Purpose : The goal of this study was to use RNA Seq to explore the correlation of gene expression of a collection of clinical P. aeruginosa isolates to various phenotypes, such as colony morphology, c-di-GMP production and biofilm formation Methods : mRNA profiles were generated for Pseudomonas aerugionsa clinical samples by deep sequencing.Ribosomal RNA was removed by using the RiboZero Bacteria kit (Illumina). cDNA libraries were synthesized using the SMARTScribe Reverse Transcriptase (Takara) followed by a PCR enrichment using the AccuPrime HiFi Taq polymerase (Invitrogen). Enzymatic reactions were carried out in the presence of SUPERase·In™ RNase Inhibitor (Invitrogen); RNACleanXP beads (Agencourt) were used for all RNA purification steps. Quality checks were performed before, during and after cDNA library preparation with the RNA Nano Kit and an Agilent Bioanalyzer 2100 (Agilent Technologies). Libraries were sequenced on an Illumina NovaSeq 6000 (paired-end mode; 2 x 50 bp) and mRNA reads were trimmed using the tool ‘cutadapt’ (version 3.5) with customized settings and mapped to the NC_008463.1 (PA14) reference genome from NCBI using ‘bowtie2’ (version 2.3.5.1) with the settings “--very-sensitive-local; --no-mixed; --fr; --no-unal”.
2022-11-05 | GSE147921 | GEO
Project description:In this study we have profiled a clinical tobramycin resistant P. aeruginosa strain that exhibited a small colony variant (SCV) phenotype. Both, the resistance and the colony morphology phenotypes were lost upon passaging the isolate under rich medium conditions. Transcriptional and mutational profiling revealed that the SCV harbored activating mutations in the two two-component systems AmgRS and PmrAB. Introduction of these mutations singularly into the type strain PA14 conferred tobramycin and colistin resistance, respectively. However, their combined introduction had an additive effect on the tobramycin resistance phenotype. Activation of the AmgRS system slightly reduced the colony size of the PA14 wild-type, whereas the simultaneous overexpression of gacA, the response regulator of the GacSA two component system, further reduced colony size. In conclusion, we uncovered combinatorial influences of two-component systems on clinically relevant phenotypes, such as resistance and the expression of the SCV phenotype. Our results clearly demonstrate that combined activation of P. aeruginosa two-component systems exhibit pleiotropic effects with unforeseen consequences.
2017-08-22 | GSE99729 | GEO
Project description:Genomic inversion drives small colony variant formation and increased virulence in P. aeruginosa
| PRJEB12456 | ENA
Project description:Transcriptional profiling of the small colony variant (SCV) S. aureus isolate (JKD6229) compared to the parent isolate with a normal phenotype (JKD6210). Both isolates were from a patient with persistent S. aureus infection, and the SCV strain arose during failed antibiotic therapy.
2010-08-13 | GSE20957 | GEO
Project description:Transcriptional profiling of the small colony variant (SCV) S. aureus isolate (JKD6229) compared to the parent isolate with a normal phenotype (JKD6210). Both isolates were from a patient with persistent S. aureus infection, and the SCV strain arose during failed antibiotic therapy. Two condition experiment JKD6229 vs JKD6210. 4 biological replicates, one replicate per slide.
2010-08-13 | E-GEOD-20957 | biostudies-arrayexpress
Project description:Background Small colony variants (SCVs) are slow-growing bacteria, which often show increased resistance to antibiotics and cause latent or recurrent infections. It is therefore important to understand the mechanisms at the basis of this phenotypic switch. Methodology/Principal findings One SCV (termed PAO-SCV) was isolated, showing high resistance to gentamicin and to the cephalosporine cefotaxime. PAO-SCV was prone to reversion as evidenced by emergence of large colonies with a frequency of 10-5 on media without antibiotics while it was stably maintained in presence of gentamicin. PAO-SCV showed a delayed growth, defective motility, and strongly reduced levels of the quorum sensing Pseudomonas quinolone signal (PQS). Whole genome expression analysis further suggested a multi-layered antibiotic resistance mechanism, including simultaneous over-expression of two drug efflux pumps (MexAB-OprM, MexXY-OprM), the LPS modification operon arnBCADTEF, and the PhoP-PhoQ two-component system. Conversely, the genes for the synthesis of PQS were strongly down-regulated in PAOSCV. Finally, genomic analysis revealed the presence of mutations in phoP and phoQ genes as well as in the mexZ gene encoding a repressor of the mexXY and mexABoprM genes. Only one mutation occurred only in REV, at nucleotide 1020 of the tufA gene, a paralog of tufB, both encoding the elongation factor Tu, causing a change of the rarely used aspartic acid codon GAU to the more common GAC, possibly causing an increase of tufA mRNA translation. High expression of phoP and phoQ was confirmed for the SCV variant while the revertant showed expression levels reduced to wild-type levels. Conclusions By combining data coming from phenotypic, gene expression and proteome analysis, we could demonstrate that resistance to aminoglycosides in one SCV mutant is multifactorial including overexpression of efflux mechanisms, LPS modification and is accompanied by a drastic down-regulation of the Pseudomonas quinolone signal quorum sensing system. We used microarrays to study changes in gene expression during early and late stationary phase of SCV and WT strains. SCV and WT cultures were grown in triplicate until early (24h) and late (48h) stationary phase. RNA was extracted, labelled and hybridised on Affymetrix P. aeruginosa expression microarrays.
2011-12-06 | E-GEOD-34141 | biostudies-arrayexpress
Project description:Background Small colony variants (SCVs) are slow-growing bacteria, which often show increased resistance to antibiotics and cause latent or recurrent infections. It is therefore important to understand the mechanisms at the basis of this phenotypic switch. Methodology/Principal findings One SCV (termed PAO-SCV) was isolated, showing high resistance to gentamicin and to the cephalosporine cefotaxime. PAO-SCV was prone to reversion as evidenced by emergence of large colonies with a frequency of 10-5 on media without antibiotics while it was stably maintained in presence of gentamicin. PAO-SCV showed a delayed growth, defective motility, and strongly reduced levels of the quorum sensing Pseudomonas quinolone signal (PQS). Whole genome expression analysis further suggested a multi-layered antibiotic resistance mechanism, including simultaneous over-expression of two drug efflux pumps (MexAB-OprM, MexXY-OprM), the LPS modification operon arnBCADTEF, and the PhoP-PhoQ two-component system. Conversely, the genes for the synthesis of PQS were strongly down-regulated in PAOSCV. Finally, genomic analysis revealed the presence of mutations in phoP and phoQ genes as well as in the mexZ gene encoding a repressor of the mexXY and mexABoprM genes. Only one mutation occurred only in REV, at nucleotide 1020 of the tufA gene, a paralog of tufB, both encoding the elongation factor Tu, causing a change of the rarely used aspartic acid codon GAU to the more common GAC, possibly causing an increase of tufA mRNA translation. High expression of phoP and phoQ was confirmed for the SCV variant while the revertant showed expression levels reduced to wild-type levels. Conclusions By combining data coming from phenotypic, gene expression and proteome analysis, we could demonstrate that resistance to aminoglycosides in one SCV mutant is multifactorial including overexpression of efflux mechanisms, LPS modification and is accompanied by a drastic down-regulation of the Pseudomonas quinolone signal quorum sensing system. We used microarrays to study changes in gene expression during early and late stationary phase of SCV and WT strains.
2011-12-06 | GSE34141 | GEO
Project description:Pseudomonas aeruginosa is a gram-negative, pathogenic, bacterium that produces biofilms containing phenotypically distinct subpopulations (Dodson et al., 2022). It was discovered that rugose small colony variants (RSCVs) were isolated with the biofilm subpopulations. RSCVs are hyper-biofilm producing bacterial mutants with increased antibiotic tolerance and are often found in chronic infections. When separating and characterizing the three biofilm cell subpopulations from a single P. aeruginosa PA14 biofilm, we discovered three novel RSCVs (denoted RSCV_1, RSCV_2, and RSCV_3). These RSCVs differed from stationary wild type (WT) PA14, the PA14 biofilm subpopulations, and between the RSCVs themselves. The observed phenotypic changes in the RSCVs included differences in cellular morphology, exopolysaccharide production, biosynthesis of virulence factors, biofilm formation, and antibiotic tolerance. Different molecules were ionized on the cell membranes of RSCVs and stationary phase WT PA14 using matrix assisted laser desorption ionization (MALDI) mass spectrometry. RNA sequencing analysis of stationary phase cells identified a high number of transcripts that were differentially expressed between the RSCVs and WT PA14 as well as between RSCV_1 and RSCV_3. The levels of the intracellular signaling molecule bis-(3′, 5′)-cyclic-dimeric-guanosine monophosphate (cyclic-di-GMP) were higher in all of the RSCVs compared to WT PA14 and significantly lower in RSCV_3 as compared to both RSCV_1 and RSCV_2. Whole-genome DNA sequencing analysis revealed single-nucleotide deletions and single-nucleotide polymorphisms (SNPs) among the RSCVs and between the RSCVs to WT PA14. The detected differences in the RSCVs have significant implications for biofilm production, antibiotic tolerance, and virulence.
2025-10-21 | GSE293895 | GEO
Project description:The iron starvation response of the Staphylococcus aureus hemB small colony variant (SCV)
| PRJNA763127 | ENA
Project description:Purpose : The goal of this study was to use RNA Seq to define the regulon of the transciption factor Anr by comparing global transcriptional profiles of Pseudomonas aeruginosa strain PAO1 and a clinical isolate with their isogenic ?anr mutants, grown in colony biofilms at 1% oxygen. Methods : mRNA profiles were generated for laboratory strain PAO1 and for a clinical isolate J215, as well as for ?anr derivatives of each strain, in duplicate, by deep sequencing. Strains were grown for 12 hours in colony biofilms at 1% O2, 5% CO2 prior to RNA harvest. Ribosomal and transfer RNAs were removed using the MICROBExpress kit (Life Technologies). mRNA reads were trimmed and mapped to the PAO1 NC_002516 reference genome from NCBI using the ClC Genomics Workbench platform and defaut parameters. mRNA profiles of 12 hour colony biofilms were generated for P. aeruginosa strains PAO1 WT, PAO1 ?anr, clinical isolate J215, and J215 ?anr, each in duplicate, by deep sequencing using Illumina HiSeq.
2015-06-29 | E-GEOD-68534 | biostudies-arrayexpress