Pleiotropic effects of a rel mutation on stress survival of Rhizobium etli CNPAF512

The rel gene of Rhizobium etli (relRet), the nodulating endosymbiont of the common bean plant, determines the cellular level of the alarmone (p)ppGpp and was previously shown to affect free-living growth and symbiosis. Here, we demonstrate its role in cellular adaptation and survival in response to various stresses.Journal ArticleResearch Support, Non-U.S. Gov'tinfo:eu-repo/semantics/publishe

Genome-wide detection of predicted non-coding RNAs in Rhizobium etli expressed during free-living and host-associated growth using a high-resolution tiling array

Non-coding RNAs (ncRNAs) play a crucial role in the intricate regulation of bacterial gene expression, allowing bacteria to quickly adapt to changing environments. In the past few years, a growing number of regulatory RNA elements have been predicted by computational methods, mostly in well-studied gamma-proteobacteria but lately in several alpha-proteobacteria as well. Here, we have compared an extensive compilation of these non-coding RNA predictions to intergenic expression data of a whole-genome high-resolution tiling array in the soil-dwelling alpha-proteobacterium Rhizobium etli.Journal ArticleResearch Support, Non-U.S. Gov'tinfo:eu-repo/semantics/publishe

Stress response regulators identified through genome-wide transcriptome analysis of the (p)ppGpp-dependent response in Rhizobium etli

Background: The alarmone (p) ppGpp mediates a global reprogramming of gene expression upon nutrient limitation and other stresses to cope with these unfavorable conditions. Synthesis of (p) ppGpp is, in most bacteria, controlled by RelA/SpoT (Rsh) proteins. The role of (p) ppGpp has been characterized primarily in Escherichia coli and several Gram-positive bacteria. Here, we report the first in-depth analysis of the (p) ppGpp-regulon in an alpha-proteobacterium using a high-resolution tiling array to better understand the pleiotropic stress phenotype of a relA/rsh mutant. Results: We compared gene expression of the Rhizobium etli wild type and rsh (previously rel) mutant during exponential and stationary phase, identifying numerous (p) ppGpp targets, including small non-coding RNAs. The majority of the 834 (p) ppGpp-dependent genes were detected during stationary phase. Unexpectedly, 223 genes were expressed (p) ppGpp-dependently during early exponential phase, indicating the hitherto unrecognized importance of (p) ppGpp during active growth. Furthermore, we identified two (p) ppGpp-dependent key regulators for survival during heat and oxidative stress and one regulator putatively involved in metabolic adaptation, namely extracytoplasmic function sigma factor EcfG2/PF00052, transcription factor CH00371, and serine protein kinase PrkA. Conclusions: The regulatory role of (p) ppGpp in R. etli stress adaptation is far-reaching in redirecting gene expression during all growth phases. Genome-wide transcriptome analysis of a strain deficient in a global regulator, and exhibiting a pleiotropic phenotype, enables the identification of more specific regulators that control genes associated with a subset of stress phenotypes. This work is an important step toward a full understanding of the regulatory network underlying stress responses in alpha-proteobacteria

Transferring an optimized TAP-toolbox for the isolation of protein complexes to a portfolio of rice tissues

Proteins are the cell's functional entities. Rather than operating independently, they interact with other proteins. Capturing in vivo protein complexes is therefore crucial to gain understanding of the function of a protein in a cellular context. Affinity purification coupled to mass spectrometry has proven to yield a wealth of information about protein complex constitutions for a broad range of organisms. For Oryza sativa, the technique has been initiated in callus and shoots, but has not been optimized ever since. We translated an optimized tandem affinity purification (TAP) approach from Arabidopsis thaliana toward Oryza sativa, and demonstrate its applicability in a variety of rice tissues. A list of non-specific and false positive interactors is presented, based on re-occurrence over more than 170 independent experiments, to filter bona fide interactors. We demonstrate the sensitivity of our approach by isolating the complexes for the rice ANAPHASE PROMOTING COMPLEX SUBUNIT 10 (APC10) and CYCLIN-DEPENDENT KINASE D (CDKD) proteins from the proliferation zone of the emerging fourth leaf. Next to APC10 and CDKD, we tested several additional baits in the different rice tissues and reproducibly retrieved at least one interactor for 81.4 % of the baits screened for in callus tissue and T1 seedlings. By transferring an optimized TAP tag combined with state-of-the-art mass spectrometry, our TAP protocol enables the discovery of interactors for low abundance proteins in rice and opens the possibility to capture complex dynamics by comparing tissues at different stages of a developing rice organ

C21orf57 is a human homologue of bacterial YbeY proteins

The product of the human C21orf57 (huYBEY) gene is predicted to be a homologue of the highly conserved YbeY proteins found in nearly all bacteria. We show that, like its bacterial and chloroplast counterparts, the HuYbeY protein is an RNase and that it retains sufficient function in common with bacterial YbeY proteins to partially suppress numerous aspects of the complex phenotype of an Escherichia coli ΔybeY mutant. Expression of HuYbeY in Saccharomyces cerevisiae, which lacks a YbeY homologue, results in a severe growth phenotype. This observation suggests that the function of HuYbeY in human cells is likely regulated through specific interactions with partner proteins similarly to the way YbeY is regulated in bacteria.National Institutes of Health (U.S.) (Grant GM31010)National Institutes of Health (U.S.) (Grant GM17151

High-resolution transcriptomics and functional genomics of Rhizobium etli during growth and non-growth

The alpha-proteobacterium Rhizobium etli occurs either in a free-living state in the soil or in a nitrogen-fixing symbiosis with its leguminous host plant, Phaseolus vulgaris. During either condition, R. etli resides primarily in a non-growing state. The transition from fast-growing to non-growing conditions is pivotal in the lifecycle of bacteria and essential for successful adaptation to diverse environments. In this work, we examined the molecular basis of adaptation to non-growth of R. etli outside and inside the host plant. The starting point was a genome-wide transcriptome analysis of free-living bacteria during exponential growth and stationary phase as well of nitrogen-fixing bacteroids in root nodules using a custom-designed high-resolution tiling array. This thesis consists of three main parts. In the first part, we identified novel non-coding RNAs (ncRNAs) differentially expressed according to its growth state. In the second part, we characterized for the first time in a alpha-proteobacterium the regulon of the stress alarmone guanosine 3′,5′-bispyrophosphate or (p)ppGpp, and identified three key regulators for stress survival. In the third part, we determined condition-specific genes regulating adaptation in either the free-living or symbiotic non-growing state.ncRNAs play a crucial role in the intricate regulation of bacterial gene expression, allowing bacteria to quickly adapt to changing environments. By comparing an extensive compilation of ncRNA predictions in alpha-proteobacteria to the whole-genome intergenic expression data, we were able to detect 89 candidate ncRNAs corresponding to functionally well-characterized or uncharacterized ncRNAs, and novel ncRNAs that were computationally predicted but had not been experimentally identified earlier. The latter comprise both putative small RNAs and cis-regulatory ncRNAs. A selection of novel ncRNAs was experimentally validated, confirming their existence. Interestingly, transcript levels of numerous ncRNAs varied during free-living growth and during interaction with the host plant, pointing to possible ncRNA-dependent regulation of adaptation to these processes. These results also support the practical value of ncRNA prediction algorithms and significantly expand the list of ncRNAs encoded in the intergenic regions of R. etli and, by extension, of alpha-proteobacteria. Moreover, we show tiling arrays to be suitable tools for studying intergenic ncRNA transcription profiles across the genome.(p)ppGpp mediates the global reprogramming of gene expression upon nutrient limitation and other stresses to cope with these unfavourable growth conditions. Synthesis of (p)ppGpp is in most bacteria controlled by Rel proteins. An in-depth analysis of the (p)ppGpp regulon in R. etli allowed us to better understand the pleiotropic stress phenotype of a rel mutant. We compared gene expression ofR. etli wild type and rel mutant during exponential and stationary phase,identifying numerous (p)ppGpp targets, including ncRNAs. The majority of the (p)ppGpp-dependent genes were detected during stationary phase. Unexpectedly, various genes were expressed in a (p)ppGpp-dependent manner also during growth, indicating the hitherto largely unrecognized importance of (p)ppGpp during active growth. Furthermore, we identified two (p)ppGpp-dependent key regulators for survival during heat and oxidative stress and one regulator putatively involved in metabolic adaptation, i.e. an extracytoplasmic function sigma factor, a DNA-binding transcription factor, and a serine protein kinase. Hence, a genome-wide transcriptome analysis of a strain deficient in a global regulator, and exhibiting a pleiotropic phenotype, enables the identification of more specific regulators that control genes associated with a subset of stress phenotypes. The comparison of the transcriptome of mature nitrogen-fixing bacteroids in nodules and free-living stationary phase cells using exponentially growing bacteria as a reference allowed us to identify commonly regulated genes in both non-growing states, as well as condition-specific genes. The transcriptome of the symbiotic state shows how bacteroids adjust their metabolism and operate under stress. The transcriptome of stationary phase cells demonstrates the increase in their potential to use different carbon sources and how free-living cells use diverse regulators, including ncRNAs and alternative sigma factors, to reprogram global gene expression. Our data highlight the similarity in gene repression between both states, but also a clear difference in gene induction. Hence, R. etli adjusts to non-growth primarily by shutting down expression of a common core of genes, while activation of condition-specific sets of genes results in adaptation to the particular challenges posed by non-growth in either the free-living or symbiotic state.List of abbreviations ix Samenvatting xi Summary xv 1. The rhizobia-legume symbiosis 1 1.1 Initial signal exchange 2 1.2 Infection process 6 1.3 Symbiosis 7 2 The (p)ppGpp regulon, variations on a theme 1 2.1 Metabolism of (p)ppGpp 12 2.2 Regulatory function of (p)ppGpp 15 2.3 Regulon of (p)ppGpp 17 2.4 Conclusion 49 3 ncRNA detection in R. etli 53 3.1 Summary 53 3.2 Background 55 3.3 Results and Discussion 58 3.4 Conclusions 71 3.5 Materials and Methods 73 3.6 Additional files 79 4 The (p)ppGpp-response in R. etli 81 4.1 Summary 81 4.2 Background 82 4.3 Results and Discussion 86 4.4 Conclusions 117 4.5 Materials and Methods 120 4.6 Additional files 127 5 The non-growing state of R. etli 129 5.1 Summary 129 5.2 Background 130 5.3 Results and Discussion 132 5.4 Conclusions 169 5.5 Materials and Methods 170 5.6 Additional files 172 6 General conclusions and perspectives 175 Appendix A List of publications 185 Appendix B List of candidate ncRNAs 187 Appendix C The (p)ppGpp regulon 191nrpages: 260status: publishe

Alarmone-dependent regulators in Rhizobium etli.

Journal Articleinfo:eu-repo/semantics/publishe

Unveiling the intergenic transcriptome across Rhizobium etli's lifecycle.

Journal Articleinfo:eu-repo/semantics/publishe