Pantoea ananatis, a plant pathogenic bacterium, inflicts significant economic losses to the agricultural and forestry industries. It is ubiquitous and capable of surviving in a diverse range of environmental conditions. The mechanism underlying host infection and colonization by this pathogen is poorly understood. The genome sequence of P. ananatis led to the discovery of putative pathogenicity determinants such as quorum sensing. In this study, a PCR-mediated protocol that makes use of the lambda () red genes was used to knockout the genes for the three quorum sensing systems in P. ananatis LMG 2665T. The mutant strain was named EanΔI/R,RhlΔI/R,ΔLuxS. Growth assays conducted using this mutant and the wild-type strain showed that the mutations did not affect its growth in liquid broth. This mutant was used to determine the role of quorum sensing in the virulence of P. ananatis. Virulence assays conducted showed that quorum sensing is required for virulence in P. ananatis.
To elucidate the role of individual quorum sensing systems in the virulence of P. ananatis, mutants lacking one system were constructed following the Red-mediated PCR protocol. The mutant strains were complemented by cloning the wild-type genes for the respective quorum sensing systems into the broad-host-range plasmid pBR1MCS-5. The mutant strains were named EanΔI/R, RhlΔI/R and ΔLuxS based on the quorum sensing genes that were mutated. The complemented strains were named EanΔI/R::EanI/R, RhlΔI/R::RhlI/R and ΔLuxS::LuxS, respectively. In vitro growth studies showed that the genetically modified P. ananatis strains were not impaired in their growth.
The P. ananatis quorum sensing mutant strains and complemented mutant strains were used to determine the functional role of each quorum sensing system in P. ananatis. Characterization of the quorum sensing mutant strains revealed that the three quorum sensing systems are required for virulence of P. ananatis in onion seedlings. The virulence assays conducted showed that the LuxS quorum sensing system is the most crucial system for virulence in P. ananatis. Furthermore, in vitro studies of quorum sensing regulation of specific phenotypes of P. ananatis showed that quorum sensing governs biofilm formation and exopolysaccharide production. The phenotypes that were impaired in the quorum sensing mutant strains were restored to wild-type levels by genetic complementation. This study also showed that swarming and twitching motility, as well as rhamnolipid production are not influenced by quorum sensing in P. ananatis. The dependence of specific phenotypes on quorum sensing indicates the significance of the functional role of quorum sensing genes in the virulence of P. ananatis.