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Phenotype Enhancement Screen of a Regulatory spx Mutant Unveils a Role for the ytpQ Gene in the Control of Iron Homeostasis.

ABSTRACT: Spx is a global regulator of genes that are induced by disulfide stress in Bacillus subtilis. Most of the Spx-regulated genes (SRGs) are of unknown function, but many encode products conserved in low %GC Gram positive bacteria. Using a gene-disruption library of B. subtilis genomic mutations, the SRGs were screened for phenotypes related to Spx-controlled activities, such as growth in minimal medium and sensitivity to methylglyoxal, but nearly all of the SRG mutations showed little if any phenotype. To uncover SRG function, the mutations were rescreened in an spx mutant background to determine which mutant SRG allele would enhance the spx mutant phenotype. One of the SRGs, ytpQ, was the site of a mutation that, when combined with an spx null mutation, elevated the severity of the Spx mutant phenotype, as shown by reduced growth in a minimal medium and by hypersensitivity to methylglyoxal. Proteomic and transcriptomic data indicated that the ytpQ mutation caused the derepression of the Fur and PerR regulons, as well as heightened LexA-controlled gene expression. Our study suggests that the ytpQ gene, encoding a conserved DUF1444 protein, functions directly or indirectly in repairing or stabilizing Fe-bearing proteins, which are sensitive to thiol reactive agents and, therefore, likely influenced by Spx-controlled genes.

ORGANISM(S): Bacillus subtilis Bacillus subtilis subsp. subtilis str. 168

PROVIDER: GSE28872 | GEO | 2011/10/25

SECONDARY ACCESSION(S): PRJNA140593

REPOSITORIES: GEO

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Project description:Abstract of associated manuscript: The Bacillus subtilis extracytoplasmic function (ECF) sigma(M) factor is activated by cell envelope stress elicited by antibiotics, and by acid, heat, ethanol and superoxide stresses. Here, we have used several complementary approaches to identify genes controlled by sigma(M). In many cases, expression is only partially dependent on sigma(M) because of both overlapping promoter recognition with other ECF sigma factors and the presence of additional promoter elements. Genes regulated by sigma(M) have a characteristic pattern of induction in response to cell envelope-acting antibiotics as evidenced by hierarchical clustering analysis. sigma(M) also contributes to the expression of the Spx transcription factor and thereby indirectly regulates genes of the Spx regulon. Cell envelope stress responses also include regulons controlled by sigma(W), sigma(B) and several two-component regulatory systems (e.g. LiaRS, YycFG, BceRS). Activation of the sigma(M) regulon increases expression of proteins functioning in transcriptional control, cell wall synthesis and shape determination, cell division, DNA damage monitoring, recombinational repair and detoxification. WT (-van) vs. WT (+van), sigM (-van) vs. sigM (+van), WT (-van) vs. sigM (-van), WT (+van) vs. sigM (+van), WT (-van) vs. spx (-van), WT (+van) vs. spx (+van). Each experiment was conducted at least twice using two independent total RNA preparations. For vancomycin untreated and treated experiments, untreated samples were labeled with Alexa Fluor 555 and treated samples with Alexa Fluor 647. For WT vs. mutant experiments, wild type was labeled with Alexa Fluor 555 and mutants with Alexa Fluor 647. For dye swap experiment, wild-type was labeled with Alexa Fluor 647 and mutant with Alexa Fluor 555. Bacillus subtilis CU1065, WT (-van) vs. WT (+van), sigM (-van) vs. sigM (+van), WT (-van) vs. sigM (-van), WT (+van) vs. sigM (+van), WT (-van) vs. spx (-van), WT (+van) vs. spx (+van)

Project description:More than 97% of the mammalian genome is non-protein coding, and repetitive elements account for more than 50% of noncoding space. However, the functional importance of many non-coding RNAs generated by these elements and their connection with pathologic processes remains elusive. We have previously shown that B2 RNAs, a class of non-coding RNAs that belong to the B2 family of SINE repeats, mediate the transcriptional activation of stress response genes (SRGs) upon application of a stimulus. Notably, B2 RNAs bind RNA Polymerase II (RNA Pol II) and suppress SRG transcription during pro-stimulation state. Upon application of a stimulus, B2 RNAs are processed into fragments and degraded, which in turn releases RNA Pol II from suppression and upregulates SRGs. Here, we demonstrate a novel role for B2 RNAs in transcriptome response to amyloid beta toxicity and pathology in mouse hippocampus. In healthy hippocampi, activation of SRGs is followed by a transient upregulation of pro-apoptotic factors, such as p53 and miRNA-34c, which target SRGs creating a negative feedback loop that facilitates return to the pro-stimulation state. Using an integrative RNA genomics approach, we show that in mouse hippocampi with amyloid pathology and in an in vitro cell culture model of amyloid beta toxicity, this regulatory loop is dysfunctional due to increased levels of B2 RNA processing, constitutively elevated SRG expression and high p53 levels. Evidence indicates that Hsf1, a master regulator of stress response, mediates B2 RNA processing in cells, and is upregulated during amyloid toxicity accelerating the processing of SINE RNAs and SRG hyper-activation. This data attributes a role to SINE RNA processing in a pathological process as well as a new function to Hsf1 that is independent of its transcription factor activity. Our study reveals that in mouse, SINE RNAs constitute a novel pathway deregulated in amyloid beta pathology, with potential implications for similar cases in the human brain, such as Alzheimer’s disease (AD).

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Phenotype Enhancement Screen of a Regulatory spx Mutant Unveils a Role for the ytpQ Gene in the Control of Iron Homeostasis.

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