Dataset Information

Genomic comparison of seed rhizobia

ABSTRACT: The proteomic analysis was performed to look for proteins abundant in rhizobial seed strains and to compare with those from nodules. Despite that strain in comparison belong to the same species; we observed that abundant proteins were different. They were identified and analyzed by function and metabolic participation. In general, seed strains had abundant proteins from coenxyme and cofactor synthesis and nodule strains from other central processes.

INSTRUMENT(S): autoflex

ORGANISM(S): Sinorhizobium Americanum Sinorhizobium Americanum Ccgm7 Rhizobium Sp. Ciat652 Rhizobium Leguminosarum Bv. Phaseoli Ccgm1

TISSUE(S): Prokaryotic Cell, Growth Phase Culture

SUBMITTER: Magdalena Hernandez Ortiz

LAB HEAD: Jaime Mora

PROVIDER: PXD004702 | Pride | 2018-10-27

REPOSITORIES: Pride

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Publications

Genomic studies of nitrogen-fixing rhizobial strains from Phaseolus vulgaris seeds and nodules.

Peralta Humberto H Aguilar Alejandro A Díaz Rafael R Mora Yolanda Y Martínez-Batallar Gabriel G Salazar Emmanuel E Vargas-Lagunas Carmen C Martínez Esperanza E Encarnación Sergio S Girard Lourdes L Mora Jaime J

BMC genomics 20160906

<h4>Background</h4>Rhizobia are soil bacteria that establish symbiotic relationships with legumes and fix nitrogen in root nodules. We recently reported that several nitrogen-fixing rhizobial strains, belonging to Rhizobium phaseoli, R. trifolii, R. grahamii and Sinorhizobium americanum, were able to colonize Phaseolus vulgaris (common bean) seeds. To gain further insight into the traits that support this ability, we analyzed the genomic sequences and proteomes of R. phaseoli (CCGM1) and S. amer ...[more]

PMID: 27601031

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Project description:Background Regulation of transcription is essential for any organism and Rhizobium etli (a multi-replicon, nitrogen-fixing symbiotic bacterium) is no exception. This bacterium is commonly found in the rhizosphere (free-living) or inside of root-nodules of the common bean (Phaseolus vulgaris) in a symbiotic relationship. Abiotic stresses, such as high soil temperatures and salinity, compromise the genetic stability of R. etli and therefore its symbiotic interaction with P. vulgaris. However, it is still unclear which genes are up- or down-regulated to cope with these stress conditions. The aim of this study was to identify the genes and non-coding RNAs (ncRNAs) that are differentially expressed under heat and saline shock, as well as the promoter regions of the up-regulated loci. Results Analysing the heat and saline shock responses of R. etli CE3 through RNA-Seq, we identified 756 and 392 differentially expressed genes, respectively, and 106 were up-regulated under both conditions. Notably, the set of genes over-expressed under either condition was preferentially encoded on plasmids, although this observation was more significant for the heat shock response. In contrast, during either saline shock or heat shock, the down-regulated genes were principally chromosomally encoded. Our functional analysis shows that genes encoding chaperone proteins were up-regulated during the heat shock response, whereas genes involved in the metabolism of compatible solutes were up-regulated following saline shock. Furthermore, we identified thirteen and nine ncRNAs that were differentially expressed under heat and saline shock, respectively, as well as eleven ncRNAs that had not been previously identified. Finally, using an in silico analysis, we studied the promoter motifs in all of the non-coding regions associated with the genes and ncRNAs up-regulated under both conditions. Conclusions Our data suggest that the replicon contribution is different for different stress responses and that the heat shock response is more complex than the saline shock response. In general, this work exemplifies how strategies that not only consider differentially regulated genes but also regulatory elements of the stress response provide a more comprehensive view of bacterial gene regulation. mRNA of nine independent cultures of wild type strain Rhizobium etli CE3 were sequenced using Illumina GAIIx. Our parameters were: all cultures were taken from exponential phase, at 30M-BM-0C for 30min in control condition; 42M-BM-0C for 30min for the culture subject to heat -shock, and 30min at 30M-BM-0C in supplementary PY medium with 80mM NaCl for the saline shock condition.

Project description:DNA sequence and local chromatin landscape act jointly to determine transcription factor (TF) binding intensity profiles. To disentangle these influences, we developed an experimental approach, called protein/DNA binding and high-throughput sequencing (PB-seq), that allows the binding energy landscape to be characterized genome-wide in the absence of chromatin. We applied our methods to the Drosophila Heat Shock Factor (HSF), which inducibly binds a target DNA sequence element (HSE) following heat shock stress. PB-seq involves incubating sheared naked genomic DNA with recombinant HSF, partitioning the HSF-bound and HSF-free DNA, and then detecting HSF-bound DNA by high throughput sequencing. We compared PB-seq binding profiles with ones observed in vivo by ChIP-seq, and developed statistical models to predict the observed departures from idealized binding patterns based on covariates describing the local chromatin environment. We found that DNase I hypersensitivity and tetra-acetylation of H4 were the most influential covariates in predicting changes in HSF binding affinity. We also investigated the extent to which DNA accessibility, as measured by digital DNase I footprinting data, could be predicted from MNase-seq data and the ChIP-chip profiles for many histone modifications and TFs, and found GAGA element associated factor (GAF), tetra-acetylation of H4, and H4K16 acetylation to be the most predictive covariates. Lastly, we generated an unbiased model of HSF binding sequences, which revealed distinct biophysical properties of the HSF/HSE interaction and a previously unrecognized substructure within the HSE. These findings provide new insights into the interplay between the genomic sequence and the chromatin landscape in determining transcription factor binding intensity. We performed an in vitro binding experiment with purified HSF and naked, sheared genomic Drosophila S2 DNA (PB-seq), to derive an accurate set of potential HSF binding sites in the Drosophila genome. HSF-bound DNA was specifically eluted and detected by high throughput sequencing. Drosophila HSF was N-terminally tagged with glutathione s-transferase and a tobacco etch virus (TEV) protease cleavage site. The C-terminus of the recombinant HSF was fused to the 3xFLAG epitope. Recombinant HSF was purified from E. coli with glutathione resin as previously described (PMID: 20078429) , with the following modifications: HSF-3xFLAG elution was achieved by addition of 6xHistidine tagged TEV protease and TEV protease was cleared from the HSF preparation using a Nickel-NTA column. We incubated 600pM HSF and 2500ng genomic DNA (sonicated to 100-600bp fragment size as previously described in PMID: 20844575) in 1500μl final volume of 1xHSF binding buffer and let it come to equilibrium for an hour at room temperature. We added 20μl ANTI-FLAG M2 affinity gel for 10 minutes, washed 8 times with 1xHSF binding buffer to remove unbound DNA; 3xFLAG peptide was added to a final concentration of 200ng/μl to specifically elute HSF and HSF-bound DNA. The mock IP was done in the absence of recombinant HSF.

			Action	DRS
	CCGM1vsCIAT652.TIF	Other
	CCGM7vsCFNEI73.TIF	Other
	R1.dat	Other
	R1.dat-pride.pride.mgf.gz	Mgf
	R1.dat-pride.pride.mztab.gz	Mztab