Dataset Information

GNPS Soil Pseudomonas - GAC signal identification

ABSTRACT: Three different strains of PGPR pseudomonads and their respective Tn5 mutants. Metabolomic extraction in dichloromethane.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Pseudomonas Chlororaphis (ncbitaxon:587753) Pseudomonas Donghuensis (ncbitaxon:1163398) Pseudomonas Fluorescens Cha0 (ncbitaxon:1124983)

SUBMITTER: Daniel Petras

PROVIDER: MSV000089821 | GNPS | Wed Jul 06 05:26:00 BST 2022

REPOSITORIES: GNPS

ACCESS DATA

Dataset's files

Source:

			Action	DRS
		Other

Items per page:

1 - 1 of 1

Similar Datasets

Project description:Purpose: Recently we have characterize 43 clinical isolates of Pseudomonas aeruginosa overproducing the efflux pump MexEF-OprN. About 45% of these strains did not display mutations in any of the regulators known to control mexEF-oprN expression. Here we identified a novel regulator of the efflux operon mexEF-oprN that we named CmrA by characterizing in vitro selected mutants from the strain PA14. Methods: MexEF-OprN overproducing spontaneous mutants were selected in vitro using the reference strain PA14 in presence of chloramphenicol. Selected mutants (PJ mutants) were characterized using different criteria such as antibiotic susceptibility and production of virulence traits. Gene expression was evaluated by RT-qPCR. Whole genome sequencing of selected mutants (PJ mutants) was performed using IonTorrent technology. Knock-out mutants were constructed by overlapping PCR and homologous recombination. Finally, a transcriptomic analysis by RNAseq was performed to identify the genes depending on CmrA. Results: Characterization of the 4 selected PJ mutants showed that these strains have resistance and virulence profiles slightly differentent from those typically observed in MexEF-OprN overproducing strains (mexS- NfxC strains). The cmrA+ strains (here called NfxC2) showed moderate resistance to MexEF-OprN antibiotic substrates (ciprofloxacin, chloramphenicol and trimethoprim from 8- to 32-fold compared to PA14) and showed a less compromised capacity to produce some virulence traits (rhamnolipids, elastase, biofilm and pyocyanin) as well as the swarming motility. Whole genome sequencing of PJ mutants led to the identification of SNPs in the gene PA14_38040 (PA14_RS15465 after 2015 genome annotation uptdate). The gene PA14_RS15465 codes for an unknown transcriptional activator and was named cmrA for chlormaphenicol resistance activator. Aminoacid substitutions in CmrA found in PJ mutants were shown to constantly activate this regulator. Further characterization by gene inactivation showed that cmrA is responsible for the activation of the expression of the mexEF-oprN operon in PJ mutants. The locus cmrA was characterized and the complete sequence, including regulatory sequeces, was submitted to GenBank (accession number KX274690). Further analysis showed that the CmrA-activating pathway was dependent on mexS and mexT, both genes are known to affect mexEF-oprN expression. Finally, the transcriptomic analysis of the PJ01 mutant (compared to PA14 and to PJ01∆mexT) revealed that CmrA significally activate the expression of 11 genes most of them coding for enzymes involved in redox regulation. Conclusions: The discovery of cmrA opens the posibility to give an explanation to those clinical strains overproducing the efflux pump MexEF-OprN for unknown reasons. Our data shows that this regulator can activate the efflux pump in a way that the strain is capable to tolerate the antibiotics substrates of the pump and to mantain its capacity to produce some virulence traits. CmrA seems to have a role in the activation of regulatory pathways involved in redox homeostasis as shown by the transcriptomic analysis. The fact that the MexEF-OprN pump is activated at the same time that a redox system let us think that this pump could be related to intracellular detoxification of molecules altering the intracellular homeostasis as it has been previously proposed. We are currently analyzing the transcriptome of CmrA to describe the whole activation pathway of this regulator.

Dataset Information

GNPS Soil Pseudomonas - GAC signal identification

Dataset's files

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets