Exploration of intraclonal adaptation mechanisms of Pseudomonas brassicacearum facing cadmium toxicity

Pseudomonas brassicacearum forms phenotypic variants in vitro as well as in planta during root colonization under natural conditions, leading to subpopulations (phase I and II cells) that differ in colony morphology and production of exoenzymes/secondary metabolites. The maximal concentration of cadmium allowing both variants growth was 25 μM; however, phase II cells accumulated fivefold higher Cd than phase I cells, even though both variants showed the same growth rate and kinetics, comprising a long stasis period (50 h). The whole transcriptome analysis of both variants in response to Cd was investigated using the home-made DNA microarrays. This analysis revealed completely different adaptation mechanisms developed by each variant to withstand and grow in the presence of the toxic. A re-organization of the cell wall to limit Cd entrance was noticed for phase I cells, as genes encoding levan exopolymers were downregulated at the expense of an upregulation of genes encoding alginate, and an upregulation of transporters such as cadA, and a downregulation of copper transporters. Phase II cells were unable to prevent Cd entrance and recruited genes under the control of oxyR and soxR regulation to face osmotic and oxidant stresses generated by Cd. Putrescine and spermidine metabolism appeared to play a central role in Cd tolerance. Microarray data were validated by biological analyses such as motility, oxidative stress assay, metabolite profiling with ICR-FT/MS and UPLC, capillary electrophoresis analysis of biogenic amines

Pseudomonas community structure and antagonistic potential in the rhizosphere: insights gained by combining phylogenetic and functional gene-based analyses

The Pseudomonas community structure and antagonistic potential in the rhizospheres of strawberry and oilseed rape (host plants of the fungal phytopathogen Verticillium dahliae) were assessed. The use of a new PCR-DGGE system, designed to target Pseudomonas-specific gacA gene fragments in environmental DNA, circumvented common biases of 16S rRNA gene-based DGGE analyses and proved to be a reliable tool to unravel the diversity of uncultured Pseudomonas in bulk and rhizosphere soils. Pseudomonas-specific gacA fingerprints of total-community (TC) rhizosphere DNA were surprisingly diverse, plant-specific and differed markedly from those of the corresponding bulk soils. By combining multiple culture-dependent and independent surveys, a group of Pseudomonas isolates antagonistic towards V. dahliae was shown to be genotypically conserved, to carry the phlD biosynthetic locus (involved in the biosynthesis of 2,4-diacetylphloroglucinol - 2,4-DAPG), and to correspond to a dominant and highly frequent Pseudomonas population in the rhizosphere of field-grown strawberries planted at three sites in Germany which have different land use histories. This population belongs to the Pseudomonas fluorescens phylogenetic lineage and showed closest relatedness to P. fluorescens strain F113 (97% gacA gene sequence identity in 492-bp sequences), a biocontrol agent and 2,4-DAPG producer. Partial gacA gene sequences derived from isolates, clones of the strawberry rhizosphere and DGGE bands retrieved in this study represent previously undescribed Pseudomonas gacA gene clusters as revealed by phylogenetic analysis