Transcriptome analysis of Rhizobium etli’s free-living and symbiotic non-growing state.
Ontology highlight
ABSTRACT:
ORGANISM(S): Rhizobium etli CFN 42
SUBMITTER: Kristof Engelen
PROVIDER: E-GEOD-25094 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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Project description:89 small non-coding RNAs (ncRNAs) were identified in the soil-dwelling alpha-proteobacterium Rhizobium etli by comparing an extensive compilation of ncRNA predictions to intergenic expression data of a whole-genome tiling array. The differential expression levels of some of these ncRNAs during free-living growth and during interaction with the eukaryotic host plant may indicate a role in adaptation to changing environmental conditions. In order to study expression in the free-living state, wild-type R. etli CFN42 was grown at 30ËC in acid minimal salts medium supplied with 10 mM NH4Cl and 10 mM succinate while monitoring the optical density (OD) of the culture. Samples were taken at OD600 = 0.3, 0.7 and 6 hours after reaching the maximum OD, representing early/late exponential and stationary phase, respectively. In order to study gene expression during host-associated growth, common bean plants (Phaseolus vulgaris cv. Limburgse vroege) were cultivated and inoculated as described previously. Nodules were harvested 2 and 3 weeks after inoculation and the bacteroids were purified by differential centrifugation.
2010-06-19 | E-GEOD-18580 | biostudies-arrayexpress
Project description:The impact on gene expression by the alarmone (p)ppGpp during growth and non-growth was determined by comparing the transcriptome of R. etli CFN42 wild type and rel mutant. This allowed us to better understand the pleiotropic stress phenotype of the rel mutant as well as identifying specific (p)ppGpp-dependent stress regulators. In order to study expression, R. etli CFN42 wild type and rel mutant was grown at 30˚C in acid minimal salts medium supplied with 10 mM NH4Cl and 10 mM succinate while monitoring the optical density (OD) of the culture. Samples were taken at OD600 = 0.3, 0.7 and 6 hours after reaching the maximum OD, representing early/late exponential and stationary phase, respectively.
2010-09-15 | E-GEOD-23961 | biostudies-arrayexpress
Project description:A core component of the M-NM-1-proteobacterial general stress response is the extracytoplasmic function (ECF) sigma factor EcfG, exclusively present in this taxonomic class. Half of the completed M-NM-1-proteobacterial genome sequences contain two or more copies of genes encoding M-OM-^CEcfG-like sigma factors, with the primary copy typically located adjacent to genes coding for a cognate anti-sigma factor (NepR) and two-component response regulator (PhyR). So far, the widespread occurrence of additional, non-canonical M-OM-^CEcfG copies has not satisfactorily been explained. This work explores the hierarchical relation between Rhizobium etli M-OM-^CEcfG1 and M-OM-^CEcfG2, canonical and non-canonical M-OM-^CEcfG proteins, respectively. Contrary to reports in other species, we find that M-OM-^CEcfG1 and M-OM-^CEcfG2 act in parallel, as nodes of a complex regulatory network, rather than in series, as elements of a linear regulatory cascade. We demonstrate that both sigma factors control unique yet also shared target genes, corroborating phenotypic evidence. M-OM-^CEcfG1 drives expression of rpoH2, explaining the increased heat sensitivity of an ecfG1 mutant, while katG is under control of M-OM-^CEcfG2, accounting for reduced oxidative stress resistance of an ecfG2 mutant. We also identify non-coding RNA genes as novel M-OM-^CEcfG targets. We propose a modified model for general stress response regulation in R. etli, in which M-OM-^CEcfG1 and M-OM-^CEcfG2 function largely independently. Based on a phylogenetic analysis and considering the prevalence of M-NM-1-proteobacterial genomes with multiple M-OM-^CEcfG copies, this model may also be applicable to numerous other species. In this study, we investigate to which degree R. etli EcfG1 and EcfG2 regulation is interdependent. Furthermore, we demonstrate that both sigma factors control distinct regulons and pinpoint unique EcfG2 target genes. We also identify non-coding RNAs (ncRNAs) as novel targets of EcfG1 and EcfG2 and show that expression of at least one of these ncRNAs is under direct EcfG control. The presence of ncRNAs in the regulon of an EcfG type sigma factor has not been described previously and adds an extra level of complexity to the regulation of the general stress response in Alpha-proteobacteria. Moreover, considering the widespread existence of Alpha-proteobacterial genomes with multiple EcfG copies, the here presented results not only lead to a better understanding of the general stress response in R. etli, but also contribute to a conceptual paradigm for general stress response regulation by multiple EcfG proteins in Alpha-proteobacteria. Comparative transcriptome analyses were carried out on four samples: the parental strain (WT) and mutants in either sigma factor gene ecfG1, sigma factor gene ecfG2, and both genes combined.
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2014-01-01 | E-GEOD-52541 | biostudies-arrayexpress
Project description:Abstract A comparative proteomic study at 6 h of growth in minimal medium (MM) and bacteroids at 18 days of symbiosis of Rhizobium etli CFN42 with the Phaseolus vulgaris leguminous plant was performed. A gene ontology classification of proteins in MM and bacteroid, showed 31 and 10 pathways with higher or equal than 30% and 20% of proteins with respect to genome content per pathway, respectively. These pathways were for energy and environmental compound metabolism, contributing to understand how Rhizobium is adapted to the different conditions. Metabolic maps based on orthology of the protein profiles, showed 101 and 74 functional homologous proteins in the MM and bacteroid profiles, respectively, which were grouped in 34 different isoenzymes showing a great impact in metabolism by covering 60 metabolic pathways in MM and symbiosis. Taking advantage of coexpression of transcriptional regulators (TF´s) in the profiles, by selection of genes whose matrices were clustered with matrices of TF´s, TRN´s were deduced by the first time for these metabolic stages. In these clustered TF-MM and clustered TF-bacteroid networks, containing 654 and 246 proteins, including 93 and 46 TFs, respectively, showing valuable information of the TF´s and their regulated genes with high stringency. Isoenzymes were specific for adaptation to the different conditions and a different transcriptional regulation for MM and bacteroid was deduced. The parameters of the TRNs of these expected biological networks and biological networks of E. coli and B. subtilis segregate from the random theoretical networks. These are useful data to design experiments on TF gene–target relationships for bases to construct a TRN.
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Project description:mRNAs are generally assumed to be loaded instantly with ribosomes upon entry into the cytoplasm. To measure ribosome density on nascent mRNA, we developed nascent Ribo-Seq (nRibo-Seq) by combining Ribo-Seq with progressive 4-thiouridine labelling. In mouse macrophages, we experimentally determined, for the first time, the lag between the appearance of nascent RNA and its association with ribosomes, which was calculated to be 20 - 22 min for bulk mRNA, and approximated the time it takes for mRNAs to be fully loaded with ribosomes to be 41 - 44 min. Notably, ribosomal loading time is adapted to gene function as rapid loading was observed with highly regulated genes. The lag and ribosomal loading time correlate positively with ORF size and mRNA half-life, and negatively with tRNA adaptation index. Similar results were obtained in mouse embryonic stem cells, where the lag in ribosome loading was even more pronounced with 35 - 38 min. We validated our measurements after stimulation of macrophages with lipopolysaccharide, where the lag between cytoplasmic and translated mRNA leads to uncoupling between input and ribosome-protected fragments. Uncoupling is stronger for mRNAs with long ORFs or half-lives, a finding we also confirmed at the level of protein production by nascent chain proteomics. As a consequence of the lag in ribosome loading, ribosome density measurements are distorted when performed under conditions where mRNA levels are far from steady state expression, and transcriptional changes affect ribosome density in a passive way. This study uncovers an unexpected and considerable lag in ribosome loading, and provides guidelines for the interpretation of Ribo-Seq data taking passive effects on ribosome density into account.
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Project description:The NifA-RpoN complex is a master regulator of the nitrogen fixation genes in alpha-proteobacteria. Based on the complete Rhizobium etli genome sequence, we constructed the R. etli CFN42 oligonucleotide (70 mer) microarray, and utilized this tool to survey changes in gene expression in R. etli CFN42 wild type compared with NifA CFNX247 mutant strain in symbiosis with Phaseolus vulgaris. As expected, the genes associated with a NifA and RpoN binding sites were downregulated in the NifA mutant strain.
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Project description:Gene expression during stationary phase and symbiosis of R. etli CFN42 was compared to that of exponentially growing cells. This allowed us to better understand how R. etli adapts to a non-growing lifestyle, both the free-living and symbiotic state, as well as to determine to what extent this adaptation is similar in both states.
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