RNA-seq in 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

The α-proteobacterium Sinorhizobium fredii NGR234 has an exceptionally wide host range as it forms nitrogen-fixing nodules with more legumes than any other known microsymbiont. Within its 6.9 Mbp genome it encodes two N-acyl-homoserine-lactone synthase genes (i.e. traI and ngrI) involved in the biosynthesis of two distinct autoinducer I-type molecules. Here we report on the construction of a NGR234-ΔtraI and a NGR234-ΔngrI mutant and the genome wide RNA-seq-based transcriptome analysis in the parent strain and in the two constructed mutants. A high-resolution RNA-seq analysis of early stationary phase cultures in the background of NGR234-ΔtraI suggested that up to 316 genes (4.9% of all predicted genes) were differentially expressed in the mutant vs. the parent strain. Similarly, in the background of NGR234-ΔngrI 466 differentially regulated genes (7.3% of all predicted genes) were identified. A considerable overlap in the gene expression pattern was uncovered between both mutants resulting in a common set of 186 genes which were almost identically regulated. Co-regulated genes that were linked to the ngrI- and the traI-dependent autoinducer regulatory circuits included 53 flagella biosynthesis genes and genes linked to EPS succinoglycan biosynthesis. Among the genes and ORFs that were differentially regulated in NGR234-ΔtraI were those linked to replication of the pNGR234a symbiotic plasmid and cytochrome c-related genes. In the NGR234-ΔngrI mutant biotin and pyrroloquinoline quinone (PQQ) biosynthesis genes were differentially expressed as well as the entire cluster of 21 genes linked to assembly of the NGR234 type three secretion system II (T3SS-II). We also discovered that genes responsible for octopine catabolism in NGR234 are strongly repressed in the presence of high levels of N-acyl-homoserine-lactones (AHLs). Surprisingly, only few truly symbiosis-related genes were identified that appeared to be quorum sensing regulated. Together with nodulation assays, the RNAseq-based findings suggested that QS-dependent gene regulation appears to be of higher relevance during non-symbiotic growth rather than for life within root nodules. In total 12 samples were analyzed (six different treatments with two independent samples), treatment A: NGR234 wild type strain in stationary phase, treatment B: NGR234 traI-mutant strain in stationary phase, treatment C: NGR234 ngrI-mutant strain in stationary phase, treatment D: NGR234 wild type strain in exponential phase, treatment E: NGR234 wild type supplemented with 0.05 µM AHLs in exponential phase, treatment F: NGR234 wild type supplemented with 50 µM AHLs in exponential phase