Identification of novel regulators of the necrosis-inducing protein (Nip) and carbapenem antibiotic production in the phytopathogen Pectobacterium

Abstract

Pectobacterium is a Gram-negative phytopathogen that causes soft rot and blackleg disease of potatoes. It produces plant cell wall degrading enzymes (PCWDEs) and is capable of swimming and swarming motility. Some strains also produce a simple β-lactam carbapenem antibiotic, 1-carbapen-2-em-3-carboxylic acid. This carbapenem cluster is cryptic in several strains of Pectobacterium but can be activated by introduction of its cognate regulator gene, carR, if provided in trans. Pectobacterium is also known to produce a necrosis-inducing protein, Nip, which triggers immunity-associated defence mechanisms. Prior studies identified Hor, RpoS and RsmA as potential regulators of carbapenem. Three regulators of Nip have also been described: the small non-coding RNA rsmB and flagellar proteins FhlKL. Both are under quorum sensing control. In this study random transposon mutagenesis was used to identify novel regulators of Nip and carbapenem antibiotic production. A mutant library was constructed for Pectobacterium carotovorum subsp. carotovorum strain CP1000 by exploiting a nip::phoA translational fusion screen. These included mutants with insertions in genes fadA and modB_1, in addition to pstA_2 and phoU which are part of the pstSCAB-phoU operon for phosphate uptake; expA which encodes the response regulator of the ExpS/ExpA two-component system and orf03204 - of unknown function. The orf03204 gene was part of a 10.5 kb likely transcript located within a novel 50.9 kb complete prophage, suggesting that orf03204, or a polar impact, contributes to the regulation of Nip through lysogenic conversion. Pectobacterium carotovorum strain SCRI 198 contains the complete carbapenem gene cluster but is cryptic for carbapenem production; antibiotic activity in this wild type strain was not seen under laboratory growth conditions. A second mutant library was screened for activated producers of the carbapenem antibiotic. A mutant from this screen carried an insertion within the intergenic region (IGR) of rho. The rho-IGR mutation circumvented the crypticity, allowing carbapenem production, but also caused pleiotropic effects on cellulase production, flagellar-mediated swimming and swarming motility. A quantitative proteomic analysis was performed comparing the rho-IGR mutant and a carR bypass mutant of the wild type SCRI 198, to further understand the regulatory role of Rho.