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RNAseq of Staphylococcus aureus wildtype and two-Cys-type TetR repressor GbaA deletion mutant under nonstress condition

ABSTRACT: RNAseq of Staphylococcus aureus wildtype and two-Cys-type TetR repressor GbaA deletion mutant under nonstress condition

PROVIDER: PRJEB47210 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	ERR6565880_1.fastq.gz	Fastqsanger.gz
	ERR6565880_2.fastq.gz	Fastqsanger.gz
	ERR6565881_1.fastq.gz	Fastqsanger.gz
	ERR6565881_2.fastq.gz	Fastqsanger.gz
	ERR6565882_1.fastq.gz	Fastqsanger.gz

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RNAseq of Staphylococcus aureus wildtype and two-Cys-type TetR repressor GbaA deletion mutant under nonstress condition

Project description:To investigate the changes in gene expression of Staphylococcus aureus COL and the gbaA deletion mutant under nonstress condition, strains were cultivated in RPMI1640 medium (Lonza) under vigorous agitation at 37°C in triplicate until cells have reached an optical density at 500 nm of 0.5. S. aureus cells were harvested and disrupted in 3 mM EDTA/ 200 mM NaCl lysis buffer with a Precellys24 Ribolyzer. RNA isolation was performed using the phenol-chloroform-isoamylalcohol approach. After precipitation with 3 M sodium acetate and isopropanol, the total RNA was washed with cold ethanol and the pellet was solved in sterile water. Initially RNA quality was checked by Trinean Xpose (Gentbrugge,Belgium) and Agilent RNA Nano 6000 kit on Agilent 2100 Bioanalyzer (Agilent Technologies, Böblingen, Germany). Samples contaminated with DNA were treated with DNase (Qiagen), cleaned as described above and rechecked by Xpose and Agilent Bioanalyzer. Finally RNA was free of DNA with an RNA Integrity Number (RIN) > 9 and rRNA Ratio [23s / 16s] > 1.5. Ribo-Zero rRNA Removal Kit (Bacteria) from Illumina (San Diego, CA, USA) was used to remove the ribosomal RNA molecules from the isolated total RNA. Removal of rRNA was checked by Agilent RNA Pico 6000 kit on Agilent 2100 Bioanalyzer (Agilent Technologies, Böblingen, Germany). RNA was free of detectable rRNA. TruSeq Stranded mRNA Library Prep Kit from Illumina (San Diego, CA, USA) was used to prepare cDNA libraries. The resulting cDNAs were sequenced paired end on an Illumina HiSeq 1500 system (San Diego, CA, USA) using 70 bp read length.

2021-10-25 | E-MTAB-10887 | biostudies-arrayexpress

The lack of the TetR-like repressor gene BCG_2177c (Rv2160A) may help mycobacteria overcome intracellular redox stress and survive longer inside macrophages when surrounded by a lipid environment

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The two-Cys-type TetR repressor GbaA confers resistance under disulfide and electrophile stress in Staphylococcus aureus.

Project description:Staphylococcus aureus has to cope with oxidative and electrophile stress during host-pathogen interactions. The TetR-family repressor GbaA was shown to sense electrophiles, such as N-ethylmaleimide (NEM) via monothiol mechanisms of the two conserved Cys55 or Cys104 residues in vitro. In this study, we further investigated the regulation and function of the GbaA repressor and its Cys residues in S. aureus COL. The GbaA-controlled gbaAB-SACOL2595-97 and SACOL2592-nmrA-2590 operons were shown to respond only weakly 3-10-fold to oxidants, electrophiles or antibiotics in S. aureus COL, but are 57-734-fold derepressed in the gbaA deletion mutant, indicating that the physiological inducer is still unknown. Moreover, the gbaA mutant remained responsive to disulfide and electrophile stress, pointing to additional redox control mechanisms of both operons. Thiol-stress induction of the GbaA regulon was strongly diminished in both single Cys mutants, supporting that both Cys residues are required for redox-sensing in vivo. While GbaA and the single Cys mutants are reversible oxidized under diamide and allicin stress, these thiol switches did not affect the DNA binding activity. The repressor activity of GbaA could be only partially inhibited with NEM in vitro. Survival assays revealed that the gbaA mutant confers resistance under diamide, allicin, NEM and methylglyoxal stress, which was mediated by the SACOL2592-90 operon encoding for a putative glyoxalase and oxidoreductase. Altogether, our results support that the GbaA repressor functions in the defense against oxidative and electrophile stress in S. aureus. GbaA represents a 2-Cys-type redox sensor, which requires another redox-sensing regulator and an unknown thiol-reactive ligand for full derepression of the GbaA regulon genes.

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