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Using RNA Seq to define the regulon of the Pseudomonas aeruginosa transcription factor Anr in low-oxygen colony biofilms.

ABSTRACT: 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.

ORGANISM(S): Pseudomonas aeruginosa Pseudomonas aeruginosa PAO1

PROVIDER: GSE68534 | GEO | 2015/06/29

SECONDARY ACCESSION(S): PRJNA283002

REPOSITORIES: GEO

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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 ÂµM 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.

Project description:Bacteria growing in biofilms are physiologically heterogeneous, due in part to their adaptation to local environmental conditions. Here, we characterized the local transcriptional responses of Pseudomonas aeruginosa growing in biofilms by using microarray analysis of isolated biofilm subpopulations. The results demonstrated that cells at the top of the biofilms had high mRNA abundances for genes involved in general metabolic functions, while mRNAs for these housekeeping genes were low in cells at the bottom of the biofilms. Selective GFP labeling showed that cells at the top of the biofilm were actively dividing. However, the dividing cells had high mRNAs levels for genes regulated by the hypoxia induced regulator, Anr. Slow-growing cells deep in the biofilms had little expression of Anr-regulated genes and may have experienced long-termanoxia. Transcripts for ribosomal proteins were primarily associated with the metabolically active cell fraction, while ribosomal RNAs were abundant throughout the biofilms, indicating that ribosomes are stably maintained even in slowly growing cells. Consistent with these results was the identification of mRNAs for ribosome hibernation factors (rmf and PA4463) at the bottom of the biofilms. A P. aeruginosa M-bM-^HM-^Frmf strain had increased uptake of the membrane integrity stain, propidium iodide. Using selective GFP labeling and cell sorting, we showed that the dividing cells were more susceptible to tobramycin and ciprofloxacin than the dormant subpopulation. The results demonstrate that in thick P. aeruginosa biofilms, cells are physiologically distinct spatially, with cells deep in the biofilm in a viable but antibiotic-tolerant slow-growth state. 52-hour Pseudomonas aeruginosa TSA colony biofilms were cryoembedded, thin sectioned, and laser dissected (LCM) to obtain samples from the top and bottom 50 M-BM-5m of the biofilms. 9 sections per biofilm were pooled. RNA was extracted with the RNeasy Micro kit, Turbo DNase treated, poly(A) tailed, and amplified using the Quantitect WTA kit. After clean up, the resulting product was fragmented and end labeled before hybridization.

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Using RNA Seq to define the regulon of the Pseudomonas aeruginosa transcription factor Anr in low-oxygen colony biofilms.

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