Disruption of Transporters Affiliated with Enantio-Pyochelin Biosynthesis Gene Cluster of Pseudomonas protegens Pf-5 Has Pleiotropic Effects

Abstract

Pseudomonas protegens Pf-5 (formerly Pseudomonas fluorescens) is a biocontrol bacterium that produces the siderophore enantio-pyochelin under conditions of iron starvation in a process that is often accompanied by the secretion of its biosynthesis intermediates, salicylic acid and dihydroaeruginoic acid. In this study, we investigated whether several transporters that are encoded by genes within or adjacent to the enantio-pyochelin biosynthetic cluster, serve as efflux systems for enantio-pyochelin and/or its intermediates. In addition, we determined whether these transporters have broad substrates range specificity using a Phenotype Microarray system. Intriguingly, knockouts of the pchH and fetF transporter genes resulted in mutant strains that secrete higher levels of enantio-pyochelin as well as its intermediates salicylic acid and dihydroaeruginoic acid. Analyses of these mutants did not indicate significant change in transcription of biosynthetic genes involved in enantio-pyochelin production. In contrast, the deletion mutant of PFL_3504 resulted in reduced transcription of the biosynthetic genes as well as decreased dihydroaeruginoic acid concentrations in the culture supernatant, which could either point to regulation of gene expression by the transporter or its role in dihydroaeruginoic acid transport. Disruption of each of the transporters resulted in altered stress and/or chemical resistance profile of Pf-5, which may reflect that these transporters could have specificity for rather a broad range of substrates.

Fig 1. Pathway for biosynthesis of salicylic acid (SA), Dihydroaeruginoic acid (DHA) and Enantio-pyochelin (E-Pch), based on the information provided in Maspoli et al., 2014 [30].

Fig 2. Concentrations of E-Pch, DHA and SA in culture supernatants of P. protegens strains determined by HPLC.

Levels of E-Pch presented are combination of both E-Pch diastereoisomers. Error bars represent standard deviations between three biological replicates. Strains tested were P. protegens Pf-5 (wildtype) and mutants of Pf-5 having deletions in putative transporter genes (ΔpchH, ΔfetF, and Δ3504) as well as mutants complemented (labeled as ‘compl.’) with the corresponding gene in the plasmid vector pBBR1-MCS2. The label ‘WT (pBBR1-MCS2)’ indicates wild-type strain with control plasmid.

Fig 3. Transcriptional profiling of genes located around the enantio-pyochelin biosynthetic gene cluster of P. protegens Pf-5.

Transcript levels were determined by qRT-PCR and the fold change (log₂) of each transcript is shown for strains ΔpchH, ΔfetF and Δ3504 relative to WT Pf-5. qRT-PCR on the truncated genes was performed on non-truncated gene regions. Error bars represent standard deviations between replicates. Coloured arrows depict the corresponding gene organization with the putative operons of the E-Pch biosynthetic gene cluster.