Transcriptomic profiling of haloarchaeal denitrification through RNA-Seq analysis
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ABSTRACT:
ORGANISM(S): Haloferax mediterranei
PROVIDER: GSE245042 | GEO | 2024/04/22
REPOSITORIES: GEO
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Project description:Many proteins and enzymes involved in denitrification in haloarchaea can be inferred to be located between the cytoplasmic membrane and the S-layer, based on the presence of a Tat signal sequence and the orientation of the active site that some of these enzymes have. The membrane fraction of the haloarchaeon Haloferax mediterranei (R-4), grown under anaerobic conditions in the presence of nitrate, was solubilised to identify the respiratory proteins associated or anchored to it. Using Triton X-100, CHAPS and n-Octyl-β-d-glucopyranoside at different concentrations we found the best conditions for isolating membrane proteins in micelles, in which enzymatic activity and stability were maintained. Then, they were subjected to purification using two chromatographic steps followed by the analysis of the eluents by NANO-ESI Chip-HPLC-MS/MS. The results showed that the four main enzymes of denitrification (nitrate, nitrite, nitric oxide and nitrous oxide reductases) in H. mediterranei were identified and they were co-purified thanks to the micelles made with Triton X-100 (20% w/v for membrane solubilisation and 0.2% w/v in the buffers used during purification). In addition, several accessory proteins involved in electron transfer processes during anaerobic respiration as well as proteins supporting ATP synthesis, redox balancing and oxygen sensing were detected. This is the first characterization of anaerobic membrane proteome of haloarchaea under denitrifying conditions using liquid chromatography-mass spectrometry. It provides new information for a better understanding of the anaerobic respiration in haloarchaea.
2020-12-07 | PXD022473 | Pride
Project description:Bradyrhizobium japonicum RegSR regulatory proteins belong to the family of two-component regulatory systems, and orthologs are present in many Proteobacteria where they globally control gene expression mostly in a redox-responsive manner. In this work, we have performed a transcriptional profiling of wild-type and regR mutant cells grown under anoxic denitrifying conditions. The comparative analyses of wild-type and regR strains revealed that almost 620 genes induced in the wild type under denitrifying conditions were regulated (directly or indirectly) by RegR, pointing out the important role of this protein as a global regulator of denitrification. Genes controlled by RegR included nor and nos structural genes encoding nitric oxide and nitrous oxide reductase, respectively, genes encoding electron transport proteins such as cycA (blr7544), or cy2 (bll2388), genes involved in nitric oxide detoxification (blr2806-09), copper homeostasis (copCAB), as well as two regulatory genes (bll3466, bll4130). Purified RegR interacted with the promoters of norC (blr3214), nosR (blr0314), a fixK-like gene (bll3466), and bll4130 which encodes a LysR-type regulator. By using fluorescently labeled oligonucleotide extension (FLOE), we were able to identify two transcriptional start sites located at about 35 (P1) and 22 (P2) bp upstream of the putative translational start codon of norC. P1 matched with the previously mapped 5M-bM-^@M-^Yend of norC mRNA which we demonstrate in this work to be under FixK2 control. P2 is a start site modulated by RegR and specific for anoxic conditions. Moreover, qRT-PCR experiments, expression studies with a norC-lacZ fusion, and heme c-staining analyses revealed that anoxia and nitrate are required for RegR-dependent induction of nor genes, and that this control is independent of the sensor protein RegS. A total of eight Affymetrix GeneChips are included in this study. Per strain (wild-type B. japonicum 110spc4, delta regR mutant 2426) four biological replicates were processed and analyzed. Strains were grown under anoxic conditions in Bergersen minimal medium supplemented with succinate as carbon source and KNO3 as terminal electron acceptor. For comparison, a microarray expression dataset generated with B. japonicum wild-type (Hauser et al., Mol. Genet. Genomics 278:255-271, 2007) and delta regR mutant cells (Lindemann et al., J. Bacteriol. 189:8928-8943, 2007) was used.
2014-09-01 | E-GEOD-56668 | biostudies-arrayexpress
Project description:Bradyrhizobium japonicum RegSR regulatory proteins belong to the family of two-component regulatory systems, and orthologs are present in many Proteobacteria where they globally control gene expression mostly in a redox-responsive manner. In this work, we have performed a transcriptional profiling of wild-type and regR mutant cells grown under anoxic denitrifying conditions. The comparative analyses of wild-type and regR strains revealed that almost 620 genes induced in the wild type under denitrifying conditions were regulated (directly or indirectly) by RegR, pointing out the important role of this protein as a global regulator of denitrification. Genes controlled by RegR included nor and nos structural genes encoding nitric oxide and nitrous oxide reductase, respectively, genes encoding electron transport proteins such as cycA (blr7544), or cy2 (bll2388), genes involved in nitric oxide detoxification (blr2806-09), copper homeostasis (copCAB), as well as two regulatory genes (bll3466, bll4130). Purified RegR interacted with the promoters of norC (blr3214), nosR (blr0314), a fixK-like gene (bll3466), and bll4130 which encodes a LysR-type regulator. By using fluorescently labeled oligonucleotide extension (FLOE), we were able to identify two transcriptional start sites located at about 35 (P1) and 22 (P2) bp upstream of the putative translational start codon of norC. P1 matched with the previously mapped 5’end of norC mRNA which we demonstrate in this work to be under FixK2 control. P2 is a start site modulated by RegR and specific for anoxic conditions. Moreover, qRT-PCR experiments, expression studies with a norC-lacZ fusion, and heme c-staining analyses revealed that anoxia and nitrate are required for RegR-dependent induction of nor genes, and that this control is independent of the sensor protein RegS.
2014-09-01 | GSE56668 | GEO
Project description:Deep Lake is a hypersaline system in Antarctica (68°33’36.8S, 78°11’48.7E) that is so saline it remains liquid at –20°C (DeMaere et al 2013). The lake is dominated by haloarchaea, comprising a low-complexity community that differs greatly to warm-hot latitude hypersaline systems, is hierarchical structured, and supports a high level of intergenera gene exchange. Metaproteomics was performed on biomass that was collected in the austral summer of 2008 by sequential size fractionation (20 – 3 µm, 3 – 0.8 µm, 0.8 – 0.1 µm). The data were integrated to obtain a systems level view of the active host-virus interactions occurring in this novel aquatic Antarctic system. DeMaere MZ, Williams TJ, Allen MA, Brown MV, Gibson JA, Rich J, Lauro FM, Dyall-Smith M, Davenport KW, Woyke T, Kyrpides NC, Tringe SG, Cavicchioli R (2013) High level of intergenera gene exchange shapes the evolution of haloarchaea in an isolated Antarctic lake. Proc Natl Acad Sci USA 110: 16939-16944
2015-07-21 | PXD001436 | Pride
Project description:Beller, H. R., T. E. Letain, A. Chakicherla, S. R. Kane, T. C. Legler, and M. A. Coleman. 2006. Whole-genome transcriptional analysis of chemolithoautotrophic thiosulfate oxidation by Thiobacillus denitrificans under aerobic vs. denitrifying conditions. Journal of Bacteriology 188:7005-7015. Thiobacillus denitrificans is one of the few known obligate chemolithoautotrophic bacteria capable of energetically coupling thiosulfate oxidation to denitrification as well as aerobic respiration. As very little is known about the differential expression of genes associated with key chemolithoautotrophic functions (such as sulfur-compound oxidation and CO2 fixation) under aerobic versus denitrifying conditions, we conducted whole-genome, cDNA microarray studies to explore this topic systematically. The microarrays identified 277 genes (approximately ten percent of the genome) as differentially expressed using Robust Multi-array Average statistical analysis and a 2-fold cutoff. Genes upregulated (ca. 6- to 150-fold) under aerobic conditions included a cluster of genes associated with iron acquisition (e.g., siderophore-related genes), a cluster of cytochrome cbb3 oxidase genes, cbbL and cbbS (encoding the large and small subunits of form I ribulose 1,5-bisphosphate carboxylase/oxygenase, or RubisCO), and multiple molecular chaperone genes. Genes upregulated (ca. 4- to 95-fold) under denitrifying conditions included nar, nir, and nor genes (associated respectively with nitrate reductase, nitrite reductase, and nitric oxide reductase, which catalyze successive steps of denitrification), cbbM (encoding form II RubisCO), and genes involved with sulfur-compound oxidation (including two physically separated but highly similar copies of sulfide:quinone oxidoreductase and of dsrC, associated with dissimilatory sulfite reductase). Among genes associated with denitrification, relative expression levels (i.e., degree of upregulation with nitrate) tended to decrease in the order nar > nir > nor > nos. Reverse transcription, quantitative PCR analysis was used to validate these trends. Keywords: bacterial metabolism
2006-10-06 | GSE5256 | GEO
Project description:Dimitry Y. Sorokin et al., (2021, Russian Academy of Sciences, Russia and Delft University of Technology, The Netherlands) describe the isolation and physiological and genomic properties of a fifth functional group of sulfur-respiring haloarchaea enriched from hypersaline lake sediments with CO as the electron donor. Additional shotgun proteomic profiling of the described strains has been performed.
2022-12-25 | PXD028241 | Pride
Project description:Genome-wide transcription profiling of B. diazoefficiens (formerly B. japonicum) wild type and nnrR mutant strain which were grown in free-living anoxic denitrifying conditions (using nitrate as final electron aceptor). This study includes also the expression data of the wild type strain grown oxically (samples GSM210269 to GSM210283 in GEO record number GSE8478). Keywords: CRP/FNR proteins, in vitro transcription, microoxia, nitrogen oxides, Rhizobia, transcriptomics
2019-08-22 | GSE130684 | GEO
Project description:Halohasta litchfieldiae and Halorubrum lacusprofundi are important members of Deep Lake in Antarctica, representing ~44% (the most abundance) and ~10% (the 3rd most abundance) of the lake population. Deep Lake is in the Vestfold Hills, East Antarctica (68°33’36.8S, 78°11’48.7E) and is 36 m deep, monomictic, and perennially cold (down to -20C) (DeMaere et al., 2013; Williams et al., 2014; Tschitschko et al., 2015; Tschitschko et al., 2016). In this project, using quantitative proteomics (8-plex iTRAQ labeling), proteins, pathways and cellular processes important for cold adaptation were determined for both Hht. litchfieldiae and Hrr. lacusprofundi. The data provided a new level of understanding about molecular mechanisms and regulatory networks involved in adaptation of the Antarctic haloarchaea to cold environment. DeMaere, M. Z., Williams, T. J., Allen, M. A., Brown, M. V., Gibson, J. A., Rich, J., et al. (2013) High level of intergenera gene exchange shapes the evolution of haloarchaea in an isolated Antarctic lake. Proc Natl Acad Sci USA 110: 16939-16944. Tschitschko, B., Williams, T. J., Allen, M. A., Páez-Espino, D., Kyrpides, N., Zhong, L., et al. (2015) Antarctic archaea–virus interactions: metaproteome-led analysis of invasion, evasion and adaptation. ISME J 9: 2094-2107. Tschitschko, B., Williams, T. J., Allen, M. A., Zhong, L., Raftery, M. J., and Cavicchioli, R. (2016) Ecophysiological distinctions of haloarchaea from a hypersaline Antarctic lake determined using metaproteomics. Appl Environ Microbiol 82: 3165 – 3173. Williams, T. J., Allen, M. A., DeMaere, M. Z., Kyrpides, N. C., Tringe, S. G., Woyke, T., and Cavicchioli, R. (2014) Microbial ecology of an Antarctic hypersaline lake: genomic assessment of ecophysiology among dominant haloarchaea. ISME J 8: 1645-1658.
2017-02-27 | PXD005076 | Pride
Project description:Small RNAs have been studied in detail in Bacteria and Eukarya domain, but in the case of Archaea domain the knowledge is scarce and the physiological function of the majority is still uncertain. To extend the knowledge of sRNAs in Archaea domain and its possible role in the regulation of the nitrogen assimilation metabolism in haloarchaea, Haloferax mediterranei has been used as a model microorganism. Bioinformatic approach has allowed to predict 295 putative sRNAs genes in the genome of H. mediterranei, 88 of which have been verified by means of RNA-seq. The secondary structure of putative sRNAs and its possible targets have been identified. Curiously, some of them present as possible targets genes related to the nitrogen assimilation, as glutamate dehydrogenase or regulatory nitrogen protein PII. Analysis of RNA-seq data has also revealed differences in the expression pattern of 16 sRNAs according to the nitrogen source. Consequently, RNomic and the bioinformatic approaches used in this work have allowed the identification of new sRNAs in Hfx. mediterranei, some of which show different expression pattern depending on the nitrogen source. It suggests that these sRNAs could be involved in the regulation of nitrogen assimilation, being able to constitute important gene regulatory network.
2018-02-20 | GSE108616 | GEO
Project description:Halorubrum lacusprofundi is an important member of Deep Lake in Antarctica, representing ~10% of the lake population (DeMaere et al., 2013). Deep Lake is in the Vestfold Hills, East Antarctica (68°33’36.8S, 78°11’48.7E) and is 36 m deep, monomictic, and perennially cold (down to -20°C) (DeMaere et al., 2013; Williams et al., 2014; Tschitschko et al., 2015; Tschitschko et al., 2016). In this project, using quantitative proteomics (8-plex iTRAQ labeling), proteins, pathways and cellular processes important for biofilm formation were determined for Hrr. lacusprofundi. The data provided a new level of understanding about molecular mechanisms and regulatory networks involved in adaptation of Hrr. lacusprofundi to biofilm formation. DeMaere, M. Z., Williams, T. J., Allen, M. A., Brown, M. V., Gibson, J. A., Rich, J., et al. (2013) High level of intergenera gene exchange shapes the evolution of haloarchaea in an isolated Antarctic lake. Proc Natl Acad Sci USA 110: 16939-16944. Tschitschko, B., Williams, T. J., Allen, M. A., Páez-Espino, D., Kyrpides, N., Zhong, L., et al. (2015) Antarctic archaea–virus interactions: metaproteome-led analysis of invasion, evasion and adaptation. ISME J 9: 2094-2107. Tschitschko, B., Williams, T. J., Allen, M. A., Zhong, L., Raftery, M. J., and Cavicchioli, R. (2016) Ecophysiological distinctions of haloarchaea from a hypersaline Antarctic lake determined using metaproteomics. Appl Environ Microbiol 82: 3165 – 3173. Williams, T. J., Allen, M. A., DeMaere, M. Z., Kyrpides, N. C., Tringe, S. G., Woyke, T., and Cavicchioli, R. (2014) Microbial ecology of an Antarctic hypersaline lake: genomic assessment of ecophysiology among dominant haloarchaea. ISME J 8: 1645-1658.
2016-12-13 | PXD004202 | Pride