Exploration of intraclonal adaptation mechanisms of Pseudomonas brassicacearum facing cadmium toxicity

Abstract

SummaryPseudomonas 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 mM; 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.

Phenotypic Switching in Pseudomonas brassicacearum Involves GacS- and GacA-Dependent Rsm Small RNAs

“…Previous studies have shown that phenotypic switching from phase I to phase II in P. brassicacearum involves downregulated transcription of lipA (lipase), phlACBD (DAPG biosynthesis), and hcnABC (hydrogen cyanide biosynthesis) (12,31). Using lipA, phlD, and hcnB as molecular markers, we confirmed by RT-PCR that the transcriptional expression of these genes was low in the gacS-negative variant V1 by comparison with the wild type (Fig.…”

“…This phenomenon also occurs in the rhizosphere of Arabidopsis thaliana (2). To identify the regulatory components controlling phenotypic switching in strain NFM421, we initiated a transcriptomic analysis using homemade microarrays (31). The most remarkable changes differentiating the variants from the wild type were seen in transcripts related to biofilm formation as type IV pili and secondary metabolism as 2,4-diacetylphloroglucinol (DAPG) (see Table S2 in the supplemental material).…”

“…Transcriptome analysis of the wild-type strain and variant V1 grown in TSB 1/10 medium during 24 h was performed using homemade DNA microarrays as previously described (31). Experiments were performed in triplicate.…”

Assessing the Toxic Effects of Nickel, Cadmium and EDTA on Growth of the Plant Growth-Promoting Rhizobacterium Pseudomonas brassicacearum

“…Independent of the actual chemical speciation of Cd 2+ and Ni 2+ in the mineral media used for this study, the defined and comparable conditions should thus supply significant information about the interactive influence of Ca 2+ and Mg 2+ at different concentrations as well as of EDTA on growth of P. brassicacearum and on the inhibitory effect caused by Cd 2+ and Ni 2+ . Since the toxic effect of Cd 2+ on P. brassicacearum has been characterised as a bacteriostatic inhibition of growth, but not as a bacteriocidal inactivation (Pages et al 2007), the respective maximum specific growth rate μ max [per hour] of this organism is used as an indicator for the magnitude of toxicity caused by Cd 2+ , Ni 2+ and EDTA.…”

“…In a previous study with cells of P. brassicacearum strain NFM421, Pagès et al reported a maximum tolerable Cd 2+ concentration of 25 μmol L −1 when using a tenfold diluted TSB/10 medium for the cultivations (Pages et al 2007). In the present study, however, the synthetic mineral 4C medium was used to specifically investigate the influence of Cd 2+ and Ni 2+ on growth of P. brassicacearum (type strain CFBP 11706) under various defined conditions.…”