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Mechanisms and Novel Proteins for Iron Oxide Mineral Reduction in the Hyperthermophilic Crenarchaeon Pyrodictium delaneyi

ABSTRACT: Dissimilatory iron reduction by hyperthermophilic archaea occurs in many geothermal environments and generally relies on microbe-mineral interactions that transform various iron oxide minerals. In this study, the physiology of dissimilatory iron and nitrate reduction was examined in the hyperthermophilic crenarchaeon Pyrodictium delaneyi Su06T. Protein electrophoresis showed that the c-type cytochrome and general protein compositions of P. delaneyi changed when grown on ferrihydrite relative to nitrate. Differential proteomic analyses using tandem mass tagged protein fragments and mass spectrometry detected 660 proteins and differential production of 127 proteins. Among these, two putative membrane-bound molybdopterin-dependent oxidoreductase complexes increased in relative abundance 60- to 3,000-fold and 50-100-fold in cells grown on iron oxide. A putative 8-heme c-type cytochrome was 60-fold more abundant in iron grown cells and was unique to the Pyrodictiaceae. There was also a >14,700-fold increase in a membrane transport protein in iron grown cells. There were no changes in the abundances of flagellin proteins nor a putative nitrate reductase, but a membrane nitric oxide reductase was more abundant on nitrate. These data help to elucidate the mechanisms by which hyperthermophilic crenarchaea generate energy and transfer electrons across the membrane to iron oxide minerals.

INSTRUMENT(S): Orbitrap Fusion

ORGANISM(S): Pyrodictium Delaneyi

SUBMITTER: Srishti Kashyap

LAB HEAD: James F Holden

PROVIDER: PXD020757 | Pride | 2020-12-23

REPOSITORIES: Pride

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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.

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

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Mechanisms and Novel Proteins for Iron Oxide Mineral Reduction in the Hyperthermophilic Crenarchaeon Pyrodictium delaneyi

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