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

Abstract

The alphaproteobacterium 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-homoserinelactone 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 an NGR234-⌬traI and an NGR234-⌬ngrI mutant and their genome-wide transcriptome analysis. A high-resolution RNA sequencing (RNA-seq) analysis of early-stationary-phase cultures in the NGR234-⌬traI background suggested that up to 316 genes were differentially expressed in the NGR234-⌬traI mutant versus the parent strain. Similarly, in the background of NGR234-⌬ngrI 466 differentially regulated genes were identified. Accordingly, a common set of 186 genes was regulated by the TraI/R and NgrI/R regulon. Coregulated genes included 42 flagellar biosynthesis genes and 22 genes linked to exopolysaccharide (EPS) biosynthesis. Among the genes and open reading frames (ORFs) that were differentially regulated in NGR234-⌬traI were those linked to replication of the pNGR234a symbiotic plasmid and cytochrome c oxidases. Biotin and pyrroloquinoline quinone biosynthesis genes were differentially expressed in the NGR234-⌬ngrI mutant as well as the entire cluster of 21 genes linked to assembly of the NGR234 type III secretion system (T3SS-II). Further, we also discovered that genes responsible for rhizopine catabolism in NGR234 were strongly repressed in the presence of high levels of N-acyl-homoserine-lactones. Together with nodulation assays, the RNA-seq-based findings suggested that quorum sensing (QS)-dependent gene regulation appears to be of higher relevance during nonsymbiotic growth rather than for life within root nodules.

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

“…Both genes encode lactonases in NGR234, and we have previously shown that they are involved in AI degradation (21). RNA-seq data and previous studies in our laboratory suggested that these genes are expressed independently of the presence of AIs (21,44). Interestingly, the expression levels of both QQ genes were highly heterogeneous in fresh and 48-h-old cultures on a singlecell level.…”

“…Our recent RNA-seq data indicate that the two QQ genes and the virB cluster encoded on pNGR234b are transcribed in an AI-independent manner and the gspD gene in an AI-dependent manner (44). This observation is interesting, since it implies that the heterogeneous regulation of genes is a more common phenomenon in NGR234 and does not depend only on the presence of AI.…”

“…Experiments with NGR234 cells harboring pBBR1MCS-5 with the inserted PgspD::rfp fusion suggested that this gene also is heterogeneously expressed during growth in laboratory cultures. gspD is the first gene of the gsp (general secretion pathway, type II) gene cluster in NGR234, and previous studies indicated that this gene is regulated in a QS-dependent manner (44). For the gspD promoter fusion, the highest levels of phenotypic heterogeneity were observed during the exponential growth phase.…”

“…To test whether the pNGR234b-borne T4SS pilus is hetero-or homogeneously expressed on a single-cell level, we chose the promoter region in front of the first gene (NGR_b10240, hypothetical protein) of the T4SS operon. NGR_b10240 and the T4SS cluster most likely are regulated in an AI-independent manner and are transcribed from a single promoter located in the 5= direction of NGR_b10240 (44). During growth in TY medium, almost equal amounts of cells were in a virB-ON and a virB-OFF mode in the exponential growth phase.…”

“…Therefore, the presence of at least five loci involved in AI degradation suggests a complex regulatory circuit in NGR234 that strictly controls the internal AI concentration. Interestingly, RNA sequencing (RNA-seq) data from our laboratory suggested an AI-independent regulation of these QQ genes (44).…”