Comparative Genome-Wide Transcriptional Profiling of Azorhizobium caulinodans ORS571 Grown under Free-Living and Symbiotic Conditions

Abstract

The whole-genome sequence of the endosymbiotic bacterium Azorhizobium caulinodans ORS571, which forms nitrogen-fixing nodules on the stems and roots of Sesbania rostrata, was recently determined. The sizes of the genome and symbiosis island are 5.4 Mb and 86.7 kb, respectively, and these sizes are the smallest among the sequenced rhizobia. In the present study, a whole-genome microarray of A. caulinodans was constructed, and transcriptomic analyses were performed on free-living cells grown in rich and minimal media and in bacteroids isolated from stem nodules. Transcriptional profiling showed that the genes involved in sulfur uptake and metabolism, acetone metabolism, and the biosynthesis of exopolysaccharide were highly expressed in bacteroids compared to the expression levels in free-living cells. Some mutants having Tn5 transposons within these genes with increased expression were obtained as nodule-deficient mutants in our previous study. A transcriptomic analysis was also performed on free-living cells grown in minimal medium supplemented with a flavonoid, naringenin, which is one of the most efficient inducers of A. caulinodans nod genes. Only 18 genes exhibited increased expression by the addition of naringenin, suggesting that the regulatory mechanism responding to the flavonoid could be simple in A. caulinodans. The combination of our genome-wide transcriptional profiling and our previous genome-wide mutagenesis study has revealed new aspects of nodule formation and maintenance.The symbiosis between rhizobia and legumes results in the formation of nitrogen-fixing nodules. The symbiotic interaction begins with the induction of bacterial nod genes by flavonoids secreted from the plant roots (8). The nod genes encode proteins that synthesize the nodulation (Nod) factor, which initiates many developmental changes, such as root hair curling and root cell division required for the formation of the nodule primordium in the host plant early during the nodulation process (8,24,50). Bacteria are entrapped in the curled root hair and subsequently infect the root hair through infection threads made of the plant cell wall. Upon release from the infection threads, bacteria invade the plant cell cytoplasm, where they differentiate into bacteroids and provide ammonium to the host plant by reducing atmospheric dinitrogen in exchange for carbon and amino acid compounds (16,49,53). It has been deduced that multiple stages exist in the establishment of nitrogen-fixing symbiosis. To identify novel genes involved in various stages of symbiosis, transcriptomic studies based on complete genome sequences were performed using Sinorhizobium (1, 2, 5, 9), Mesorhizobium (71), and Bradyrhizobium (10,42,54).Azorhizobium caulinodans ORS571 is a microsymbiont of Sesbania rostrata (18)(19)(20). Nitrogen-fixing nodules are formed by A. caulinodans on the stems as well as on the roots of S. rostrata. Stem nodules occur at the site of adventitious root primordia located on the stems after crack-entry invasion by A. caulinodans (70). During...

phrR -Like Gene praR of Azorhizobium caulinodans ORS571 Is Essential for Symbiosis with Sesbania rostrata and Is Involved in Expression of reb Genes

“…The labeled cDNA was mixed with 3.3 l of control oligo(B2) (3 nM; Affymetrix) and 100 l of 2ϫ hybridization mix (Affymetrix) in a volume of 200 l and used in the subsequent hybridization. Hybridization, washing, staining, and scanning were performed as previously described (48). To evaluate the differentially expressed genes, the weighted average difference (WAD) method (25,26) was applied to the log 2 signal.…”

“…Furthermore, we constructed a whole-genome microarray of A. caulinodans and performed transcriptional profiling experiments with A. caulinodans in the free-living and symbiotic states (48). By the screening of Tn5 mutants, we identified 86 genes that were required for A. caulinodans to establish symbiosis (43).…”

“…By the screening of Tn5 mutants, we identified 86 genes that were required for A. caulinodans to establish symbiosis (43). Among these genes, 17 were more highly expressed in bacteroids than in free-living bacteria (48). One of the genes (locus tag on the genome, AZC_0013) was homologous to the phrR gene encoding a putative transcription factor of Sinorhizobium medicae WSM419 (36).…”

TonB-Dependent Receptors in Nitrogen-Fixing Nodulating Bacteria

“…In contrast, no putative TBDRs for thiamine or phytate were identified. This is reasonable because phytate is not a source of phosphorus in the habitats of rhizobacteria (26), and rhizobacteria have the ability to synthesize thiamine (28,36,49). The heme-specific and siderophore-specific TBDRs in rhizobacteria are likely to be responsible for the uptake of heme and iron under free-living conditions, rather than during symbiosis, as their expression is downregulated in bacteroids.…”

Involvement of the Azorhizobial Chromosome Partition Gene ( parA ) in the Onset of Bacteroid Differentiation during Sesbania rostrata Stem Nodule Development

“…In a previous study, we identified many genes that are involved in the developmental process of nodulation through a large-scale in vivo screening of Tn5-inserted mutants of A. caulinodans ORS571 (68). In addition, the genome-wide transcriptional profiles of A. caulinodans ORS571 in free-living and symbiotic states have been established by microarray analyses (71).…”