When Genome-Based Approach Meets the “Old but Good”: Revealing Genes Involved in the Antibacterial Activity of Pseudomonas sp. P482 against Soft Rot Pathogens

Abstract

Dickeya solani and Pectobacterium carotovorum subsp. brasiliense are recently established species of bacterial plant pathogens causing black leg and soft rot of many vegetables and ornamental plants. Pseudomonas sp. strain P482 inhibits the growth of these pathogens, a desired trait considering the limited measures to combat these diseases. In this study, we determined the genetic background of the antibacterial activity of P482, and established the phylogenetic position of this strain. Pseudomonas sp. P482 was classified as Pseudomonas donghuensis. Genome mining revealed that the P482 genome does not contain genes determining the synthesis of known antimicrobials. However, the ClusterFinder algorithm, designed to detect atypical or novel classes of secondary metabolite gene clusters, predicted 18 such clusters in the genome. Screening of a Tn5 mutant library yielded an antimicrobial negative transposon mutant. The transposon insertion was located in a gene encoding an HpcH/HpaI aldolase/citrate lyase family protein. This gene is located in a hypothetical cluster predicted by the ClusterFinder, together with the downstream homologs of four nfs genes, that confer production of a non-fluorescent siderophore by P. donghuensis HYST. Site-directed inactivation of the HpcH/HpaI aldolase gene, the adjacent short chain dehydrogenase gene, as well as a homolog of an essential nfs cluster gene, all abolished the antimicrobial activity of the P482, suggesting their involvement in a common biosynthesis pathway. However, none of the mutants showed a decreased siderophore yield, neither was the antimicrobial activity of the wild type P482 compromised by high iron bioavailability. A genomic region comprising the nfs cluster and three upstream genes is involved in the antibacterial activity of P. donghuensis P482 against D. solani and P. carotovorum subsp. brasiliense. The genes studied are unique to the two known P. donghuensis strains. This study illustrates that mining of microbial genomes is a powerful approach for predictingthe presence of novel secondary-metabolite encoding genes especially when coupled with transposon mutagenesis.

7‐hydroxytropolone is the main metabolite responsible for the fungal antagonism of Pseudomonas donghuensis strain SVBP6

“…S1). This genomic region has been recently associated with the antibacterial activity of P. donghuensis strain P482 and with iron scavenging in P. donghuensis strains HYS (Krzyzanowska et al, 2016;Chen et al, 2018). Moreover, in the latter strain, the homologous gene cluster was found to be responsible for the production of 7HT (Jiang et al, 2016;Chen et al, 2018).…”

“…Remarkably, the hallmark phenotype of each of the three strains originated in different continents is linked to the same genomic locus (Fig. 1) (Krzyzanowska et al, 2016;Chen et al, 2018), thus suggesting that the biosynthesis of tropolonoid compounds is indispensable for the antibacterial activity of strain P482 (Krzyzanowska et al, 2016), iron scavenging in strain HYS (Jiang et al, 2016;Chen et al, 2018) and the antifungal activity in strain SVBP6.…”

Pseudomonas species isolated via high-throughput screening significantly protect cotton plants against verticillium wilt

“…P. donghuensis HYS and P. donghuensis 22G5 conservatively produce a novel siderophore, 7-HT. 7-HT was first reported as a siderophore in P. donghuensis HYS in 2016 (Jiang et al 2016 ), and the gene cluster responsible for its production has been well established (Chen et al 2018 ; Krzyżanowska et al 2016 ). Recently, 7-HT was found to be the main metabolite responsible for the fungal antagonism of P. donghuensis SVBP6 by testing its antagonism activities against Macrophomina phaseolina , Fusarium graminearum , Fusarium semitectum and Bacillus subtilis (Muzio et al 2020 ).…”

A Complex Mechanism Involving LysR and TetR/AcrR That Regulates Iron Scavenger Biosynthesis in Pseudomonas donghuensis HYS

“…brasiliense strains. Strains with mutations of gene 4709 from strain P482 (denoted orf8 in P. donghuensis HYS), along with mutations of other genes located in the same cluster, were deficient in antagonism against pathogens (30).…”