Evidence of Autoinducer-Dependent and -Independent Heterogeneous Gene Expression in Sinorhizobium fredii NGR234

Abstract

Populations of genetically identical Sinorhizobium fredii NGR234 cells differ significantly in their expression profiles of autoinducer (AI)-dependent and AI-independent genes. Promoter fusions of the NGR234 AI synthase genes traI and ngrI showed high levels of phenotypic heterogeneity during growth in TY medium on a single-cell level. However, adding very high concentrations of N-(3-oxooctanoyl-)-L-homoserine lactone resulted in a more homogeneous expression profile. Similarly, the lack of internally synthesized AIs in the background of the NGR234-⌬traI or the NGR234-⌬ngrI mutant resulted in a highly homogenous expression of the corresponding promoter fusions in the population. Expression studies with reporter fusions of the promoter regions of the quorum-quenching genes dlhR and qsdR1 and the type IV pilus gene cluster located on pNGR234b suggested that factors other than AI molecules affect NGR234 phenotypic heterogeneity. Further studies with root exudates and developing Arabidopsis thaliana seedlings provide the first evidence that plant root exudates have strong effects on the heterogeneity of AI synthase and quorum-quenching genes in NGR234. Therefore, plant-released octopine appears to play a key role in modulation of heterogeneous gene expression.

Phenotypic Heterogeneity, a Phenomenon That May Explain Why Quorum Sensing Does Not Always Result in Truly Homogenous Cell Behavior

“…The upregulation of the AI systems in the background of the AI synthase deletion mutants was in part verified by population-wide transcriptome data (31). Not only the AI themselves but also the addition of crude plant root exudates or octopine as a plant-released molecule caused a more homogenous expression of the AI systems (30). Part of this study was confirmed by tests with the close relative Sinorhizobium meliloti using a sinI reporter gene fusion (32).…”

“…Part of this study was confirmed by tests with the close relative Sinorhizobium meliloti using a sinI reporter gene fusion (32). Further investigations in S. fredii NGR234 indicated that quorum quenching genes and a type IV pilus gene cluster showed phenotypic heterogeneity that was independent of the influence of cell-cell communication signals (30).…”

“…In batch cultures, the two AI systems, TraI/R and NgrI/R, are heterogeneously expressed (Fig. 1A and B) (30). Further work from our laboratory thereby implies that some cells turn on the traI gene and others the ngrI gene; however, only a few cells turn on both genes at the same time (Fig.…”

“…In contrast to the heterogeneous behavior of P. syringae and X. campestris, the heterogeneous behavior of the two S. fredii AI systems can be quenched by either the external addition of 50 M AI or the deletion of the corresponding AI synthase gene (30). The upregulation of the AI systems in the background of the AI synthase deletion mutants was in part verified by population-wide transcriptome data (31).…”

RNA Sequencing Analysis of the Broad-Host-Range Strain Sinorhizobium fredii NGR234 Identifies a Large Set of Genes Linked to Quorum Sensing-Dependent Regulation in the Background of a traI and ngrI Deletion Mutant

“…While in general it is assumed that a homogeneous expression of these genes occurred throughout an isogenic population, there is now initial evidence that this may not be the natural situation. Instead, a fraction of the NGR234 genes are most likely heterogeneously expressed on a single-cell level (98). In the light of these novel findings, future work will now have to elucidate the complex pattern of gene expression and regulation on a single-cell level in this fascinating microorganism.…”