Unknown,Transcriptomics,Genomics,Proteomics

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Transcription profiling of Methanosarcina mazei Goe1 in response to bismuth nitrate.

ABSTRACT: Pattern of gene expression of M. mazei in response to 10 uM bismuth nitrate was analyzed relative to gene expression pattern of M. mazei grown in the presence of 30 uM potassium nitrate (control cultures). Therefore, five pre-cultures of M. mazei was grown to mid-exponential growth-phase. Then, the pre-cultures were divided into aliquotes, one half spiked with bismuth nitrate and the other half with potassium nitrate (control) to match nitrate concentration of the bismuth spike. Samples were propagated for two days. Then, total RNA was isolated and transformed into Cy3 or Cy5 labeled cDNA. Labeled cDNA derived from one culture treated with bismuth and from one control culture, both obtained from the same pre-culture, was hybridized with microarray chips. Hybridized chips were scanned, derived data processed and relative expression levels of orfs from M. mazei in response to bismuth calculated.

ORGANISM(S): Methanosarcina mazei

SUBMITTER: Frank Thomas

PROVIDER: E-MTAB-787 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Here, we examined the ramifications of between-species diversity by documenting the transcriptional response of three marine diatoms - Thalassiosira pseudonana, Fragilariopsis cylindrus, and Pseudo-nitzschia multiseries - to the onset of nitrate limitation of growth, a common limiting nutrient in the ocean. Less than 5% of orthologous genes, shared across the three diatoms, displayed the same transcriptional responses across species when growth was limited by nitrate availability. Orthologs, such as those involved in nitrogen uptake and assimilation, as well as carbon metabolism, were differently expressed across the three species. The two pennate diatoms, F. cylindrus and P. multiseries, shared 3,839 clusters without orthologs in the genome of the centric diatom T. pseudonana. A majority of these pennate-clustered genes, as well as the non-orthologous genes in each species, had minimal annotation information, but were often significantly differentially expressed under nitrate limitation, indicating their potential importance in the response to nitrogen availability. Despite these variations in the specific transcriptional response of each diatom, overall transcriptional patterns suggested that all three diatoms displayed a common physiological response to nitrate limitation that consisted of a general reduction in carbon fixation and carbohydrate and fatty acid metabolism and an increase in nitrogen recycling. Transcriptomes were collected for diatom cultures harvested at the onset of stationary phase in low nitrate media (55 M-NM-<M NaNO3, 212 M-NM-<M Na2SiO3, 72.4 M-NM-<M NaH2PO4) or during mid-exponential growth in nutrient-replete media (882 M-NM-<M NaNO3, 106 M-NM-<M Na2SiO3, 36.2 M-NM-<M NaH2PO4) in artificial seawater, maintaining three biological replicates per condition and per diatom (N=18). The SOLiD sequencer (version 4) was used to generate the transcriptomes and the SEAStAR software package was used to process the SOLiD reads and to calculate gene counts. Pooled counts for the nitrate-limited treatment were normalized to pooled counts for the nutrient-replete M-bM-^@M-^\controlM-bM-^@M-^] treatment to generate log fold changes in gene transcription using the R software package edgeR from Bioconductor.

Project description:Cellular respiration is a fundamental process undergone within energy-transducing membranes through the redox activity of multimeric protein assemblies, so-called respiratory complexes. In most cases, electron transport is ensured by lipophilic molecules, quinones, serving as electron shuttles. This redox activity is coupled to the net translocation of protons across the membrane thereby establishing an electrochemical gradient, the protonmotive force (pmf). Such a gradient powers the transport of molecules (such as proteins, ions or antibiotics) and ATP synthesis but was also shown to participate in protein localization in prokaryotes. Importantly, energy-transducing membranes show a high level of organization with heterogeneous distribution of respiratory complexes as observed across several bacterial lineages. Although electron transport in energy-transducing membranes is considered to be a kinetic process coupled to the diffusion of all reactants and in particular quinones, it is not clear to which extent subcellular distribution of respiratory complexes can have an influence on the overall kinetics. We previously evidenced polar clustering of nitrate reductase, NarGHI, an anaerobic respiratory complex in the gut bacterium Escherichia coli. Such spatial organization was shown to directly impact the electron flux of the associated respiratory chain. While dynamic localization of such complex plays an important role in controlling respiration, the mechanism by which it impacts the electron flux is not understood. Yet, under such conditions where nitrate is the sole terminal electron acceptor, functioning of the electron transport chain in enteric bacteria such as E. coli and Salmonella typhimurium is associated with the production of nitric oxide (NO) mainly resulting from the reduction of nitrite by nitrate reductase. As a consequence, both E. coli and S. typhimurium produce a multitude of enzymes controlling NO homeostasis, the primary source of nitrosative stress. Protein S-nitrosylation is a ubiquitous NO-dependent post-translational modification of cysteine that regulates protein structure and function. Recently, Seth et al demonstrated that, in absence of oxygen, NO oxidation is mainly catalyzed by the hybrid cluster protein Hcp allowing its further reaction with thiols of a broad spectrum of proteins. Among Hcp-dependent S-nitrosylated targets are the nitrate reductase complex, multiple metabolic enzymes and the transcription factor OxyR whose S-nitrosylation entails a distinct nitrosative stress regulon. Optimizing electron transport through clustering of nitrate reductase will lead to nitrite accumulation which may in turn be responsible for NO production. Here, the electron-donating respiratory complex, the formate dehydrogenase, FdnGHI was shown to cluster at the poles under nitrate respiring conditions. Its proximity with the nitrate reductase complex was confirmed by evaluating the interactome of the later under distinct metabolic conditions reported to impact its subcellular organization. All the identified partners were exclusively found when cells were grown under nitrate respiration. The proximity of two respiratory complexes provides mechanistic explanation on the importance of subcellular organization onto quinone pool turnover. Noteworthy is the identification of several components playing key roles in the protection against endogenous nitrosative stress.

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Transcription profiling of Methanosarcina mazei Goe1 in response to bismuth nitrate.

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