Comment[GEAAccession]	E-GEAD-966
MAGE-TAB Version	1.1
Investigation Title	Genome-wide transcriptional profiling of Azorhizobium caulinodans ORS571 (CBX88)
Experiment Description	A 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. It revealed that the sizes of the genome and symbiotic island were 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 bacteroids isolated from stem nodules. The transcriptional profiling showed that the genes involved in sulfur uptake and metabolism, acetone metabolism, and biosynthesis of exopolysaccharide were highly expressed in bacteroids as compared to free-living cells. Some mutants having Tn5 transposon within these up-regulated genes were obtained as nodule-deficient mutants in our previous study. A transcriptomic analysis was also performed on free-living cells in a minimal media supplemented with a flavonoid, naringenin, which is one of the most efficient inducers of A. caulinodans nod genes. Only 18 genes were up-regulated by addition of naringenin, suggesting that regulatory mechanism responding to flavonoid could be simple in A. caulinodans. The combination of the genome-wide transcriptional profiling and our previous genome-wide mutagenesis study has revealed new aspects of nodule formation and maintenance.
Experimental Design	growth condition design
Experimental Factor Name	sample_name
Experimental Factor Type	sample_name
Person Last Name	Aono	Tsukada	Akiba	Liu	Lee	Toyazaki	Oyaizu
Person First Name	Toshihiro	Shuhei	Noriko	Chi-Te	Kyung-bum	Hiroki	Hiroshi
Person Affiliation	The University of Tokyo						
Person Roles	submitter						
Public Release Date	2025-01-31
PubMed ID	19542345
Protocol Name	P-GEAD-4018	P-GEAD-4019	P-GEAD-4020	P-GEAD-4021	P-GEAD-4022	P-GEAD-4023	P-GEAD-4024	P-GEAD-4025
Protocol Type	sample collection protocol	nucleic acid extraction protocol	nucleic acid labeling protocol	nucleic acid hybridization to array protocol	array scanning and feature extraction protocol	normalization data transformation protocol	growth protocol	treatment protocol
Protocol Description	The AzcNxa520550F whole genome array (designed by Affymetrix, USA) contains 7096 probe pair sets (45 for target preparation controls, 4725 for ORFs, 2326 for intergenic regions tiled in both directions) corresponding to 4,703 ORFs and 1,163 intergenic regions of A. caulinodans ORS571, based on the genome sequence. The nucleotide sequence of the entire genome of A. caulinodans ORS571 is available in the DDbJ/EMbL/Genbank databases under the accession number AP009384. Probe length was 25-mer. For all ORFs, 16 probe pairs were chosen. For the intergenic sequences, probes were selected at the density of 16?24 bp to fill the remaining space on the chip. As target preparation controls, the poly-A (dap, lys, phe, thr, trp) and hybridization (biob, bioC, bioD, cre) were included in a chip., S. rostrata seeds were treated with concentrated sulfuric acid for 1 hour, rinsed with sterile water, and soaked in sterile water on trays. The trays were placed for 3 days at 37??C under dark conditions. After germination, S. rostrata were grown for 2 weeks before bacterial inoculation at 35??C under a 24 h light regime at an intensity of 50,000 lux. bacterial cultures grown overnight were inoculated onto the stems. At 7 days post inoculation, the stem nodules on the second stem internode of plants were harvested, immediately frozen in liquid nitrogen, and stored at ?80??C.	Total RNA was isolated from cultured and bacteroid cells using ISOGEN (Nippon Gene, Japan) according to the manufacture's instruction.	To synthesize cDNA, 10 microg of total RNA was mixed with 2 microL of the 260 fold diluted poly-A target preparation control containing dap, lys, phe, thr mRNA (Affymetrix, USA), and was reverse-transcribed by SuperScript III (Invitrogen, USA). After digesting the remaining RNA with RNaseH for 20 min at 37degree C, cDNA was collected with MinElute kit (Qiagen, Germany). Fragmentation was performed with DNase I with 1 x One-Phor-All buffer (GE Healthcare, UK). Fragmented cDNA was end-labeled with Terminal Deoxynucleotidyl Transferase (Promega, USA) and GeneChip DNA Labeling Reagent (Affymetrix, USA).	Hybridization was carried out using GeneChip Hybridization, Wash and Stain kit (Affymetrix). 1.0?1.5 microg of labeled cDNA was mixed with control oligo b2 (3 nM, Affymetrix) in a volume of 150 microl hybridization solution. Hybridization was carried out overnight at 50degree C for 16 hours.	Scanning was carried with GeneChip Scanner 3000 (Affymetrix, Inc.)	Image analysis was carried out with the GeneChip Operating Software (GCOS) version 1.4 (Affymetrix)., The target intensity value was set to 500. The scaling factors were between 1.5 and 4.9. Default statistical parameters of GCOS (Alpha1 = 0.04, Alpha2 = 0.06, Tau = 0.015, Gamma1L = 0.001111, Gamma1H = 0.001111, Gamma2L = 0.001333, Gamma2H = 0.001333, perturbation = 1.1) were applied.	Grown at 37degree C in  TY medium (5 g/L tryptone, 3 g/L yeast extract, 0.83 g/L CaCl2?2H2O, pH 6.8) as a rich medium. To isolate RNA from free-living cells. A. caulinodans were grown in 100 ml of media up to an OD600 of 0.5?0.7., Grown at 37degree C in MMO medium (5 g/L sodium lactate, 5 g/L disodium succinate 6H2O, 1.67 g/L K2HPO4, 0.87 g/L KH2PO4, 0.1 g/L MgSO4?7H2O, 0.1 g/L NaCl, 1 g/L (NH4)2SO4, 0.04 g/L CaCl2?2H2O, 2 mg/L biotin, 4 mg/L pantothenic acid, 4 mg/L nicotinic acid, pH6.8) as a minimal medium. To isolate RNA from free-living cells, A. caulinodans were grown in 100 ml of media up to an OD600 of 0.5?0.7., Grown at 37degree C in MMO medium (5 g/L sodium lactate, 5 g/L disodium succinate 6H2O, 1.67 g/L K2HPO4, 0.87 g/L KH2PO4, 0.1 g/L MgSO4?7H2O, 0.1 g/L NaCl, 1 g/L (NH4)2SO4, 0.04 g/L CaCl2?2H2O, 2 mg/L biotin, 4 mg/L pantothenic acid, 4 mg/L nicotinic acid, pH6.8) as a minimal medium.  To analyze the effect of flavonoids, naringenin (20 microM) was added to MMO medium 6 h before harvesting cells. To isolate RNA from free-living cells, A. caulinodans were grown in 100 ml of media up to an OD600 of 0.5?0.7.	Cultured cells were harvested by centrifugation at 8,000 g, 4degree C, for 5 min. The pellets were frozen in liquid nitrogen and stored at  ?80degree C until RNA was isolated., To isolate bacteroids, stem nodules were vigorously homogenized with MMS buffer (40 mM MOPS, 20 mM KOH, 2 mM MgSO4, 0.3 M sucrose, pH 7.0) containing 4% polyvinylpyrrolidone. The resulting homogenate was passed through three layers of miracloth (Calbiochem, USA), and filtrate was centrifuged at 500 g for 5 min to remove plant cell fractions and other debris. Then supernatant was centrifuged at 5,000 g for 10 min, and the pellet containing bacteroids was collected. To remove the green layer containing the contaminated chloroplast on the surface of white pellet, MMS buffer containing 25% polyvinylpyrrolidone was added onto the pellet, and surface of the pellet was washed by vigorous pipetting, then the buffer was removed.. After this washing procedure, the pellet was resuspended in MMS buffer without polyvinylpyrrolidone, and centrifuged at 5,000 g for 2 min, and the supernatant was removed. This procedure was carried out twice. The white pellet was used as a bacteroid sample for RNA isolation.
SDRF File	E-GEAD-966.sdrf.txt
Comment[Number of channel]	single-channel
Comment[Array Design REF]	A-GEAD-171
Comment[AEExperimentType]	transcription profiling by array
Comment[SecondaryAccession]	CBX88
Comment[BioProject]	PRJDB20065
Comment[CIBEX Accept Date]	2010-01-01
Comment[CIBEX Public Release Date]	2009-11-24
Comment[Last Update Date]	2025-01-31