Project Details
Projekt
Molecular switches and networks involved in regulation of phenotypic heterogeneity in Sinorhizobium fredii NGR234
Applicant
Professor Dr. Wolfgang Streit
Subject Area
Microbial Ecology and Applied Microbiology
Term
from 2012 to 2019
Project identifier
Deutsche Forschungsgemeinschaft (DFG) - Project number 217398597
Phenotypic heterogeneity and bistability are widespread and common phenomena in bacteria, however the molecular switches that are involved remain poorly understood. In the first funding period we have analyzed and described the basic principles of phenotypic heterogeneous gene expression in the Gram-negative and plant-associated model organism Sinorhizobium fredii NGR234. Thereby, we demonstrated that the autoinducer (AI) synthase genes traI and ngrI are heterogeneously expressed in laboratory cultures. We further provided evidence that the switching depended to a large extend on the available concentration of the synthesized AI molecules. Heterogeneous gene expression was analyzed in the background of the parent strain and newly constructed knockout mutants. Interestingly, heterogeneous expression was not only observed for AI-dependent genes but it was also observed for genes which were not subject to quorum sensing (QS) regulation such as the quorum quenching (QQ) genes dlhR and qsdR1. Furthermore we have shown that phenotypic heterogeneity was affected by plant-produced compounds such as octopine and that in close distance to plant roots heterogeneous expression is obviously only of minor importance. This observation was made independent from the plant species. Because of these findings we hypothesize that beside the well-known QS circuits other regulons are subject to phenotypic heterogeneous regulation in NGR234 and that other signal molecules than the AI molecules are involved in this phenomenon.To now further analyze the regulatory circuits involved in switching between heterogeneous and homogeneous gene expression in NGR234, in the second funding period of this project we will exploit the molecular mechanisms involved in this phenomenon. We will focus on the two QS-associated traI- and ngrI AI-synthase genes as well as on the QS-independently regulated genes dlhR and qsdR1. We will analyze the regulatory networks linked to these genes and we will mainly employ different strategies to identify the key players involved in sensing and switching from heterogeneous to homogeneous gene expression. Within this framework we hypothesize that in NGR234 other important regulators affect phenotypic heterogeneity such as the ExpR, TraM, QseC and several of the LuxR orphans encoded in this organism. We will construct knockouts of these regulators and assess the phenotypes these mutations have with respect to phenotypic heterogeneity. In parallel approaches we will use high-resolution RNA sequencing strategies to identify genes that are involved in the switching process. Further, we will be using a random mutagenesis approach and bioinformatics tools together with chromatin immunoprecipitation combined with DNA sequencing (ChIP-seq) approaches to identify promoter sequences that are regulated. Thereby, we will build up a detailed map of the regulatory network that assists the cell to switch between heterogeneous and homogeneous gene expression.
DFG Programme
Priority Programmes
