Transcriptomic analysis of Rhizobium leguminosarum biovar viciae in symbiosis with host plants Pisum sativum and Vicia cracca

Abstract

Rhizobium leguminosarum bv. viciae forms nitrogen-fixing nodules on several legumes, including pea (Pisum sativum) and vetch (Vicia cracca), and has been widely used as a model to study nodule biochemistry. To understand the complex biochemical and developmental changes undergone by R. leguminosarum bv. viciae during bacteroid development, microarray experiments were first performed with cultured bacteria grown on a variety of carbon substrates (glucose, pyruvate, succinate, inositol, acetate, and acetoacetate) and then compared to bacteroids. Bacteroid metabolism is essentially that of dicarboxylate-grown cells (i.e., induction of dicarboxylate transport, gluconeogenesis and alanine synthesis, and repression of sugar utilization). The decarboxylating arm of the tricarboxylic acid cycle is highly induced, as is gamma-aminobutyrate metabolism, particularly in bacteroids from early (7-day) nodules. To investigate bacteroid development, gene expression in bacteroids was analyzed at 7, 15, and 21 days postinoculation of peas. This revealed that bacterial rRNA isolated from pea, but not vetch, is extensively processed in mature bacteroids. In early development (7 days), there were large changes in the expression of regulators, exported and cell surface molecules, multidrug exporters, and heat and cold shock proteins. fix genes were induced early but continued to increase in mature bacteroids, while nif genes were induced strongly in older bacteroids. Mutation of 37 genes that were strongly upregulated in mature bacteroids revealed that none were essential for nitrogen fixation. However, screening of 3,072 mini-Tn5 mutants on peas revealed previously uncharacterized genes essential for nitrogen fixation. These encoded a potential magnesium transporter, an AAA domain protein, and proteins involved in cytochrome synthesis.

FIG. 1.

Comparison of RNA structures in free-living bacteria and mature bacteroids. (A) Free-living R. leguminosarum bv. viciae 3841. (B) Twenty-eight-day pea bacteroids of 3841. In free-living cells (A), there is some processing of 23S rRNA, as shown by the large 1.3-kb doublet; however, a 23S rRNA peak is clearly visible. In the bacteroid sample (B), there is almost complete processing of 23S rRNA into smaller fragments. A Bio-Rad Experion microfluidic analyzer was used to determine the RNA structure.

FIG. 2.

Expression of gfp-mut3.1 gene fusions to inositol-regulated genes (iolC and idhA) in R. leguminosarum bv. viciae 3841 and iolR mutant backgrounds, grown with either glucose or inositol as the sole carbon source. The error bars indicate standard error of the means.

FIG. 3.

Hierarchical gene tree of expression from 13 experiments over the course of bacteroid development on peas (7, 15, and 21 days p.i. of 7 day-old plants and 28 days p.i. of seeds). The hierarchical tree was generated with the gene tree clustering algorithm in GeneSpring 7.2. using Pearson correlation and average linkage of the 386 genes that were ≥3-fold upregulated in nodule bacteria. Red indicates highly expressed genes, yellow intermediate, and blue low. Nine clusters can be distinguished and are indicated.

FIG. 4.

Classification based on Riley codes of genes differentially regulated (≥3-fold) during bacteroid development. Genes expressed only in early/intermediate bacteroids (7 days p.i.) are represented by black bars, and genes expressed only in mature bacteroids (≥15 days p.i.) are represented by unfilled bars. Genes expressed throughout bacteroid development are represented by gray bars. (A) Upregulated genes. (B) Downregulated genes. The functional classifications were derived from annotations available via RhizoDB.