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Pseudomonas putida strain:MnB1

ABSTRACT: RNA-seq of Pseudomonas putida

PROVIDER: PRJNA770004 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR16286430_1.fastq.gz	Fastqsanger.gz
	SRR16286430_2.fastq.gz	Fastqsanger.gz
	SRR16286431_1.fastq.gz	Fastqsanger.gz
	SRR16286431_2.fastq.gz	Fastqsanger.gz
	SRR16286432_1.fastq.gz	Fastqsanger.gz

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Characterization of DkaiC mutants of Pseudomonas putida KT2440/2 and Pseudomonas protegens CHA0

Project description:KaiC is the central cog of the circadian clock in Cyanobacteria. Close homologs of this protein are widespread among bacteria not known to have a circadian physiology. The function, interaction network, and mechanism of action of these KaiC homologs are still largely unknown. Here, we focus on KaiC homologs found in environmental Pseudomonas species. We characterize experimentally the only KaiC homolog present in Pseudomonas putida KT2440 and Pseudomonas protegens CHA0. Through phenotypic assays and transcriptomics, we show that KaiC is involved in osmotic and oxidative stress resistance in P. putida and in biofilm production in both P. putida and P. protegens.

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RNA-seq of Pseudomonas putida KT2440 and the derived strain P. putida pig21 treated with 1-octanol and PQS (Pseudomonas quinolone signal), causing chemical membrane stress.

Project description:Pseudomonas species have become promising cell factories for the production of natural products due to their inherent robustness. Here, we explored membrane adaptations of Pseudomonas putida KT2440, in particular outer membrane vesicle (OMV) formation in response to 1-octanol, PQS and prodigiosin, causing chemical membrane stress via RNA-seq of mRNA. Pseudomonas putida wild type KT2440 (Nelson et al. 2002) and the derived strains P. putida pig21 were cultivated in biological triplicates under continuous shaking (130 rpm) at 30 °C in 10 mL LB (lysogeny broth) medium (10 g L-1 tryptone, 5 g L-1 yeast extract, 10 g L-1 sodium chloride; Carl Roth®, Karlsruhe, Germany). Antibiotics were added to the culture medium when appropriate to the following final concentrations: 25 µg mL-1 kanamycin, 25 µg mL-1 irgasan, 25 µg mL-1 gentamicin, 50 µg mL-1 tetracycline. For chemical induction of OMV formation, P. putida KT2440 was exposed to 1 mM 1-octanol or 50 µM PQS (Pseudomonas quinolone signal) after reaching the logarithmic growth phase. For transcriptome analysis, cells were cultivated as described above, the cell pellet was harvested after 7 h, adjusted to an optical density (OD700 nm) of 1 and flash frozen . Total RNA was isolated from 3 biological replicates using Quick-RNA Miniprep Plus kit (Zymo Research). The samples were treated with DNase (Zymo Research) and RNA was again purified with an RNA Clean&Concentrator-5 kit (Zymo Research). Ribosomal rRNA was removed with a riboPOOL for bacteria (siTOOLs Biotech GmbH). The purity of RNA and removal of rRNA was then tested with an Agilent RNA Pico 6000 kit and an Agilent 2100 Bioanalyzer (Agilent Technologies). TruSeq Stranded mRNA Sample Preparation guide (Illumina) was then used to construct the cDNA library. The constructed cDNA library was then sequenced with Illumina NextSeq500 high output mode paired end using a read length of 75 bases.

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