Defining the regulons of Pseudomonas aeruginosa transcriptional regulators
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ABSTRACT:
PROVIDER: PRJNA685406 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR13255630_1.fastq.gz | Fastqsanger.gz | |||
| SRR13255630_2.fastq.gz | Fastqsanger.gz | |||
| SRR13255631_1.fastq.gz | Fastqsanger.gz | |||
| SRR13255631_2.fastq.gz | Fastqsanger.gz | |||
| SRR13255632_1.fastq.gz | Fastqsanger.gz |
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Project description:The anaerobic metabolism of the opportunistic pathogen Pseudomonas aeruginosa is important for growth and survival during persistent infections. The two Fnr-type transcription factors Anr and Dnr regulate different parts of the underlying network. Both are proposed to bind to a non-distinguishable DNA sequence named Anr box. The aim of this study was the identification of genes induced under anaerobic conditions in the P. aeruginosa wild type and identification of genes under control of the Anr or Dnr regulators.
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Project description:The Pseudomonas aeruginosa quorum-sensing (QS) systems contribute to bacterial homeostasis and pathogenicity. Although many regulators have been characterized to control the production of virulence factors and QS signaling molecules, its detailed regulatory mechanisms still remain elusive. Here, we performed chromatin immunoprecipitation followed by high-throughput DNA sequencing (ChIP-seq) on 10 key QS regulators. The direct regulation of these genes by corresponding regulator has been confirmed by Electrophoretic mobility shift assays (EMSAs) and quantitative real-time polymerase chain reactions (qRT-PCR). Binding motifs are found by using MEME suite and verified by footprint assays in vitro. Collectively, this work provides new cues to better understand the detailed regulatory networks of QS systems. ChIP-seq of 10 QS regulators in Pseudomonas aeruginosa
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Project description:The anaerobic metabolism of the opportunistic pathogen Pseudomonas aeruginosa is important for growth and survival during persistent infections. The two Fnr-type transcription factors Anr and Dnr regulate different parts of the underlying network. Both are proposed to bind to a non-distinguishable DNA sequence named Anr box. The aim of this study was the identification of genes induced under anaerobic conditions in the P. aeruginosa wild type and identification of genes under control of the Anr or Dnr regulators. We performed three comparisons to identify genes induced under anaerobic denitrifying conditions in the P. aeruginosa wild type strain and genes which are under control of the Anr or Dnr regulators under these anaerobic conditions. Since the anr and dnr mutant strains do not grow under anaerobic denitrifying conditions, we applied anaerobic shift experiments. Pseudomonas aeruginosa was grown in a modified AB minimal medium, containing 25 µM FeSO4, 20 mM glucose and 50 mM NaNO3. The 200 ml aerobic cultures were grown in 1 l Erlenmeyer flasks at 37 oC and 300 rpm. The aerobic culture was grown to an OD578 of 0.3. For the aerobic culture, cells were harvested at this point. For the anaerobic shift experiments 130 ml of the respective aerobic culture were transferred to a 135 ml sealed serum flask. Control experiments verified that oxygen tension decreased within 3 - 5 min below the detection limit of an oxygen electrode. The cells were harvested after incubation for additional 2h under anaerobic conditions. Within these 2h incubation period no growth of the wild type, the anr mutant or the dnr mutant strain was observed. First comparison: Identification of genes induced or repressed under anaerobic conditions in the P. aeruginosa wild type PAO1. Here we compared the transcriptome profile of P. aeruginosa PAO1 grown under aerobic conditions up to an OD578 of 0.3 with the transcriptome profile of the PAO1 strain, which was first grown under aerobic conditions up to an OD578 of 0.3 and than shifted to anaerobic conditions by transfer to a sealed serum flask and further incubated for two hours under anaerobic conditions. Second comparison: Identification of genes regulated differently in the anr mutant strain PAO6261. Here we compared the transcriptome profile of the P. aeruginosa wild type PAO1 with the transcriptome profile of the P. aeruginosa anr mutant strain PAO6261. Both strains were harvested after 2h incubation under anaerobic conditions. Third comparison: Identification of genes regulated differently in the dnr mutant strain RM536. Here we compared the transcriptome profile of the P. aeruginosa wild type PAO1 with the transcriptome profile of the P. aeruginosa dnr mutant strain RM536. Both strains were harvested after 2h incubation under anaerobic conditions.
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Project description:Regulatory networks including virulence-related transcriptional factors (TFs) determine bacterial pathogenicity in response to different environmental cues. Pseudomonas aeruginosa, a Gram-negative opportunistic pathogen of humans, recruits numerous TFs in quorum sensing (QS) system, type III secretion system (T3SS) and Type VI secretion system (T6SS) to mediate the pathogenicity. Although many virulence-related TFs have been illustrated individually, very little is known about their crosstalks and regulatory network. Here, based on chromatin immunoprecipitation coupled with high-throughput sequencing (ChIP-seq) and transcriptome profiling (RNA-seq), we primarily focused on understanding the crosstalks of 20 virulence-related TFs, which led to construction of a virulence regulatory network named PAGnet (Pseudomonas aeruginosa Genomic integrated regulatory network), including 82 crosstalk targets. The PAGnet uncovered the intricate mechanism of virulence regulation and revealed master regulators in QS, T3SS and T6SS pathways. In particular, GacA and ExsA showed novel functions in QS and nitrogen metabolism. In addition, an online PAGnet platform was provided to analyze these TFs and more virulence factors. Taken together, the present study revealed the function-specific crosstalks of virulence regulatory network, which might provide new strategies for treating infections in P. aeruginosa in the future.
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