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Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Probing the RyhB-2 regulon in Salmonella Typhimurium

ABSTRACT: Typically, the expression of sRNAs is activated in response to environmental stimuli in order to regulate gene expression through post-transcriptional mechanisms. In the present work we show that the Salmonella Typhimurium paralog sRNAs RyhB-1 and RyhB-2 are induced in response to the nitrosating agent S-nitrosoglutathione (GSNO). Inactivation of these sRNAs decreased S. Typhimurium resistance to GSNO and increased the levels of nitrosylated proteins. These results prompted us to evaluate a possible role of these sRNAs in nitrosative stress resistance. RNA profiling was used as a screening to identify novel RyhB-1 and RyhB-2 regulated targets, and a subset of genes was filtered based on their potential role in the response to nitrosative stress. To confirm our observation, expression of the candidate targets was analyzed by quantitative RT-PCR in a wild type, single and double mutant strains (M-NM-^TryhB1, M-NM-^TryhB2 and M-NM-^TryhB1 M-NM-^TryhB2) treated with GSNO. In response to GSNO RyhB-1 and RyhB-2 negatively regulate the expression of the genes cyoABC (cytochrome o oxidase complex), cydB (cytochrome d oxidase complex), cybC (cytochrome b-562), and positively regulate the nirBCD operon (nitrite reductase system). Together, these results suggest that RyhB-1 and RyhB-2 finely tune the expression of genes coding for cytochrome and the nitrate reductase system, allowing the cell to cope with GSNO-induced stress. Total RNA was harvested from two biological replicates of wt S. Typhimurium overexpressing the sRNA RyhB-2 grown in LB medium, in order to identify RhyB-2 targets that might be implicated in reactive nitrogen stress resistance.

ORGANISM(S): Salmonella enterica subsp. enterica serovar Typhimurium str. LT2

SUBMITTER: Michael McClelland

PROVIDER: E-GEOD-58110 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Probing the RyhB-2 regulon in Salmonella Typhimurium

Project description:Typically, the expression of sRNAs is activated in response to environmental stimuli in order to regulate gene expression through post-transcriptional mechanisms. In the present work we show that the Salmonella Typhimurium paralog sRNAs RyhB-1 and RyhB-2 are induced in response to the nitrosating agent S-nitrosoglutathione (GSNO). Inactivation of these sRNAs decreased S. Typhimurium resistance to GSNO and increased the levels of nitrosylated proteins. These results prompted us to evaluate a possible role of these sRNAs in nitrosative stress resistance. RNA profiling was used as a screening to identify novel RyhB-1 and RyhB-2 regulated targets, and a subset of genes was filtered based on their potential role in the response to nitrosative stress. To confirm our observation, expression of the candidate targets was analyzed by quantitative RT-PCR in a wild type, single and double mutant strains (ΔryhB1, ΔryhB2 and ΔryhB1 ΔryhB2) treated with GSNO. In response to GSNO RyhB-1 and RyhB-2 negatively regulate the expression of the genes cyoABC (cytochrome o oxidase complex), cydB (cytochrome d oxidase complex), cybC (cytochrome b-562), and positively regulate the nirBCD operon (nitrite reductase system). Together, these results suggest that RyhB-1 and RyhB-2 finely tune the expression of genes coding for cytochrome and the nitrate reductase system, allowing the cell to cope with GSNO-induced stress.

2014-06-10 | GSE58110 | GEO

Transcriptome response to nitrosative stress in Rhodobacter sphaeroides 2.4.1

Project description:DNA microarray analysis was employed to investigate the transcriptome response to nitrosative stress in a non-denitrifying facultative photosynthetic bacterium Rhodobacter sphaeroides 2.4.1. We focused on the role played by a nitric oxide-response transcriptional regulator NnrR in the response. The transcriptome profiles of R. sphaeroides 2.4.1 and its nnrR mutant before and after exposure to nitrosating agents S-nitrosoglutathione (GSNO) or sodium nitroprusside (SNP) under semiaerobic conditions were analyzed. R. sphaeroides 2.4.1 and its nnrR mutant were cultivated in glass bottles under semiaerobic growth conditions. When the optical density at 600 nm reached approximately 0.15-0.2, 1 mM GSNO or SNP was added to the medium. RNA was isolated from a 50 ml aliquot of the culture prior to the addition of GSNO or SNP and at 15 min after the addition. The experiment was performed in duplicate independent cultures.

2012-11-11 | E-GEOD-33641 | biostudies-arrayexpress

Response of Porphyromonas gingivalis to nitrosative stress

Project description:Transcriptional profiling of P. gingivalis cells comparing cells grown in anaerobic conditions to cells grown in anaerobic conditions and exposed to nitrite and to GSNO. Two-condition experiment, anaerobically vs. anaerobically exposed to nitrosative stress cells. Biological replicates: 1 independently grown and harvested bacterial cells for each nitrosative exposure tested. Four technical replicates for each experiment.

2012-10-05 | E-GEOD-38220 | biostudies-arrayexpress

Respiratory backpressure determines normoxic distribution of terminal oxygen reduction pathways

Project description:Cytochrome bd (CydAB), a high-affinity oxidase that performs terminal oxygen reduction in cellular respiration, is used by various bacteria to grow in hypoxic environments, form nonreplicating persister cells, or resist antimicrobial killing. It is thought that oxygen concentration solely determines the usage of CydAB in bacteria, with the tradeoff being that CydAB-containing respiratory chains generate less ATP than those containing low-affinity proton-pumping cytochrome oxidases, as CydAB does not pump protons when generating a proton motive force (PMF). However, CydAB has roles in acid, nitrosative and oxidative stress that suggest the existence of oxygen-independent CydAB usage. Here, we show that an obligate aerobe, the soil actinomycete Mycobacterium smegmatis, uses CydAB to normoxically maintain a homeostatic balance of the PMF, involving an oxygen-independent interaction with the classical hypoxia regulator (DosR). We show that overpotentials of the PMF apply feedback inhibition on the proton-pumping, low-affinity, cytochrome c oxidase supercomplex bcc-aa3, and that CydAB can immediately escape this inhibition and promote a PMF-responsive programme.

2023-02-28 | GSE167408 | GEO

Project description:Transcriptional profiling of Salmonella enterica sv Typhimurium under NO stress and comparison of profiles between wild type and lpdA mutant cultures. We investigated the bacteriostatic nature of NO·. S. Typhmurium 14028s is prototrophic for all amino acids but cannot synthesize Methionine or Lysine during nitrosative stress. This MK auxotrophy results from reduced availability of succinyl-CoA, a consequence of NO· targeting lipoamide-dependent LpdA activity. LpdA is an essential component of the pyruvate and α-ketoglutarate dehydrogenase complexes. Additional effects of NO· on gene regulation prevent compensatory pathways of succinyl-CoA production. By microarray analysis, more than 50% of the transcriptional response of S. Typhimurium to nitrosative stress is attributable to LpdA inhibition.

2011-06-04 | GSE29735 | GEO

Transcriptome response to nitrosative stress in Rhodobacter sphaeroides 2.4.1

2012-11-11 | GSE33641 | GEO

RNA-Seq samples of E. coli K12 MG1655, deletion of ryhB sRNA gene, 2,2’-Dipyridy

Project description:Bacteria adapt to environmental changes by remodeling their gene expression programs. The change in expression of specific genes is due to the combined effects of various regulators, among them small RNAs (sRNAs). Here we present a straightforward RNA-seq-based approach to infer the contribution of a sRNA to the total change in gene expression levels of specific genes in response to shifts in environmental conditions, demonstrated here for the sRNA RyhB in cells growing under iron limitation stress. We show that the contribution of RyhB to changes in expression levels of specific genes upon iron limitation varies between the genes, from relatively mild contribution to the expression change of some genes to ~100% effect on the expression change of other genes. Interestingly, the contribution of RyhB is high for genes undergoing in total mild expression changes and relatively low for genes undergoing in total high expression changes, implying that for almost all affected genes RyhB renders small changes in expression upon iron limitation. Determining the contribution of a sRNA to the total changes in expression levels of genes in response to an environmental change is important for understanding the integration of regulation by sRNAs in the cellular networks regulating cellular physiology.

2022-12-09 | E-MTAB-10502 | biostudies-arrayexpress

2011-06-04 | E-GEOD-29735 | biostudies-arrayexpress

Evidence for a key role of cytochrome bo3 oxidase in respiratory energy metabolism of Gluconobacter oxydans

Project description:The obligatory aerobic acetic acid bacterium Gluconobacter oxydans oxidizes a variety of substrates in the periplasm by membrane-bound dehydrogenases, which transfer the reducing equivalents to ubiquinone. Two quinol oxidases, cytochrome bo3 and cytochrome bd, then catalyze transfer of the electrons from ubiquinol to molecular oxygen. In this study, mutants lacking either of these terminal oxidases were characterized. Deletion of the cydAB genes for cytochrome bd had no obvious influence on growth, whereas the lack of the cyoBACD genes for cytochrome bo3 severely reduced the growth rate and the cell yield. Using a respiration activity monitoring system and adjusting different levels of oxygen availability, hints for a low oxygen affinity of cytochrome bd oxidase were obtained, which were supported by measurements of oxygen consumption in a respirometer. The H+/O ratio of the M-NM-^TcyoBACD mutant with mannitol as substrate was 0.56 M-BM-1 0.11 and more than 50% lower than that of the reference strain (1.26 M-BM-1 0.06) and the delta-cydAB mutant (1.31 M-BM-1 0.16), indicating that cytochrome bo3 oxidase is the main component for proton extrusion via the respiratory chain. Plasmid-based overexpression of cyoBACD led to increased growth rates and growth yields, both in the wild type and the delta-cyoBACD mutant, suggesting that cytochrome bo3 might be the rate-limiting factor of the respiratory chain. The three transcriptome comparisons of G. oxydans M-NM-^TuppM-NM-^TcyoBACD vs. G.oxydans M-NM-^Tupp were repeated independently three times in biological replicates resulting in 3 hybridizations as termed by sample 1 to 3.

2013-07-26 | E-GEOD-47782 | biostudies-arrayexpress

MS2-affinity purification coupled with RNA sequencing (MAPS) reveals RyhB sRNA targetome

Project description:Despite the overwhelming information about sRNAs, one of the biggest challenges in the sRNA field is characterizing sRNA targetomes. Thus, we develop a novel method to identify RNAs that interact with a specific sRNA, regardless of the type of regulation (positive or negative) or targets (mRNA, tRNA, sRNA). This method is called MAPS: MS2 affinity purification coupled with RNA sequencing. As proof of principle, we identified RNAs bound to RyhB, a well-characterized E. coli sRNA. Identification of RNAs co-purified with MS2-RyhB in a rne131 ?ryhB strain. RyhB (without MS2) was used as control

2015-04-16 | E-GEOD-66519 | biostudies-arrayexpress

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