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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  HYS T . 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.

frontiers in microbiology

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