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Exploring the molecular machinery of denitrification in Haloferax mediterranei through proteomics

ABSTRACT: 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.

INSTRUMENT(S): Agilent instrument model

ORGANISM(S): Haloferax Mediterranei Atcc 33500

TISSUE(S): Prokaryotic Cell

SUBMITTER: Javier Torregrosa-Crespo

LAB HEAD: Rosa María Martínez-Espinosa

PROVIDER: PXD022473 | Pride | 2020-12-07

REPOSITORIES: Pride

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Project description:Anaerobic benzene oxidation coupled to the reduction of Fe(III) was studied in Ferroglobus placidus in order to learn more about how such a stable molecule could be metabolized under strict anaerobic conditions. F. placidus conserved energy to support growth at 85°C in a medium with benzene provided as the sole electron donor and Fe(III) as the sole electron acceptor. The stoichiometry of benzene loss and Fe(III) reduction, as well as the conversion of [14C]-benzene to [14C]-carbon dioxide, was consistent with complete oxidation of benzene to carbon dioxide with electron transfer to Fe(III). Benzoate, but not phenol or toluene, accumulated at low levels during benzene metabolism and [14C]-benzoate was produced from [14C]-benzene. Analysis of gene transcript levels revealed increased expression of genes encoding enzymes for anaerobic benzoate degradation during growth on benzene versus growth on acetate, but genes involved in phenol degradation were not up-regulated during growth on benzene. A gene for a putative carboxylase that was more highly expressed in benzene- versus benzoate-grown cells was identified. These results suggest that benzene is carboxylated to benzoate and that phenol is not an important intermediate in the benzene metabolism of F. placidus. This is the first demonstration of a microorganism in pure culture that can grow on benzene under strict anaerobic conditions and for which there is strong evidence for degradation of benzene via clearly defined anaerobic metabolic pathways. Thus, F. placidus provides a much needed pure culture model for further studies on the anaerobic activation of benzene in microorganisms. A five-chip study using total RNA recovered from three separate cultures of Ferroglobus placidus DSM 10642 grown with 0.5 mM phenol (experimental condition) and two separate cultures of Ferroglobus placidus DSM 10642 grown on 1 mM benzoate (control condition). Each chip measures the expression level of 2,613 genes from Ferroglobus placidus DSM 10642 with nine 45-60-mer probe pairs (PM/MM) per gene, with three-fold technical redundancy.

Project description:Anaerobic benzene oxidation coupled to the reduction of Fe(III) was studied in Ferroglobus placidus in order to learn more about how such a stable molecule could be metabolized under strict anaerobic conditions. F. placidus conserved energy to support growth at 85°C in a medium with benzene provided as the sole electron donor and Fe(III) as the sole electron acceptor. The stoichiometry of benzene loss and Fe(III) reduction, as well as the conversion of [14C]-benzene to [14C]-carbon dioxide, was consistent with complete oxidation of benzene to carbon dioxide with electron transfer to Fe(III). Benzoate, but not phenol or toluene, accumulated at low levels during benzene metabolism and [14C]-benzoate was produced from [14C]-benzene. Analysis of gene transcript levels revealed increased expression of genes encoding enzymes for anaerobic benzoate degradation during growth on benzene versus growth on acetate, but genes involved in phenol degradation were not up-regulated during growth on benzene. A gene for a putative carboxylase that was more highly expressed in benzene- versus benzoate-grown cells was identified. These results suggest that benzene is carboxylated to benzoate and that phenol is not an important intermediate in the benzene metabolism of F. placidus. This is the first demonstration of a microorganism in pure culture that can grow on benzene under strict anaerobic conditions and for which there is strong evidence for degradation of benzene via clearly defined anaerobic metabolic pathways. Thus, F. placidus provides a much needed pure culture model for further studies on the anaerobic activation of benzene in microorganisms. A six-chip study using total RNA recovered from three separate cultures of Ferroglobus placidus DSM 10642 grown with 1 mM benzene (experimental condition) and three separate cultures of Ferroglobus placidus DSM 10642 grown on 0.5 mM phenol (control condition). Each chip measures the expression level of 2,613 genes from Ferroglobus placidus DSM 10642 with nine 45-60-mer probe pairs (PM/MM) per gene, with three-fold technical redundancy.

Project description:Anaerobic benzene oxidation coupled to the reduction of Fe(III) was studied in Ferroglobus placidus in order to learn more about how such a stable molecule could be metabolized under strict anaerobic conditions. F. placidus conserved energy to support growth at 85°C in a medium with benzene provided as the sole electron donor and Fe(III) as the sole electron acceptor. The stoichiometry of benzene loss and Fe(III) reduction, as well as the conversion of [14C]-benzene to [14C]-carbon dioxide, was consistent with complete oxidation of benzene to carbon dioxide with electron transfer to Fe(III). Benzoate, but not phenol or toluene, accumulated at low levels during benzene metabolism and [14C]-benzoate was produced from [14C]-benzene. Analysis of gene transcript levels revealed increased expression of genes encoding enzymes for anaerobic benzoate degradation during growth on benzene versus growth on acetate, but genes involved in phenol degradation were not up-regulated during growth on benzene. A gene for a putative carboxylase that was more highly expressed in benzene- versus benzoate-grown cells was identified. These results suggest that benzene is carboxylated to benzoate and that phenol is not an important intermediate in the benzene metabolism of F. placidus. This is the first demonstration of a microorganism in pure culture that can grow on benzene under strict anaerobic conditions and for which there is strong evidence for degradation of benzene via clearly defined anaerobic metabolic pathways. Thus, F. placidus provides a much needed pure culture model for further studies on the anaerobic activation of benzene in microorganisms. A six-chip study using total RNA recovered from three separate cultures of Ferroglobus placidus DSM 10642 grown with 1 mM benzene (experimental condition) and three separate cultures of Ferroglobus placidus DSM 10642 grown on 10 mM acetate (control condition). Each chip measures the expression level of 2,613 genes from Ferroglobus placidus DSM 10642 with nine 45-60-mer probe pairs (PM/MM) per gene, with three-fold technical redundancy.

Project description:Anaerobic benzene oxidation coupled to the reduction of Fe(III) was studied in Ferroglobus placidus in order to learn more about how such a stable molecule could be metabolized under strict anaerobic conditions. F. placidus conserved energy to support growth at 85°C in a medium with benzene provided as the sole electron donor and Fe(III) as the sole electron acceptor. The stoichiometry of benzene loss and Fe(III) reduction, as well as the conversion of [14C]-benzene to [14C]-carbon dioxide, was consistent with complete oxidation of benzene to carbon dioxide with electron transfer to Fe(III). Benzoate, but not phenol or toluene, accumulated at low levels during benzene metabolism and [14C]-benzoate was produced from [14C]-benzene. Analysis of gene transcript levels revealed increased expression of genes encoding enzymes for anaerobic benzoate degradation during growth on benzene versus growth on acetate, but genes involved in phenol degradation were not up-regulated during growth on benzene. A gene for a putative carboxylase that was more highly expressed in benzene- versus benzoate-grown cells was identified. These results suggest that benzene is carboxylated to benzoate and that phenol is not an important intermediate in the benzene metabolism of F. placidus. This is the first demonstration of a microorganism in pure culture that can grow on benzene under strict anaerobic conditions and for which there is strong evidence for degradation of benzene via clearly defined anaerobic metabolic pathways. Thus, F. placidus provides a much needed pure culture model for further studies on the anaerobic activation of benzene in microorganisms. A five-chip study using total RNA recovered from three separate cultures of Ferroglobus placidus DSM 10642 grown with 1 mM benzene (experimental condition) and two separate cultures of Ferroglobus placidus DSM 10642 grown on 1 mM benzoate (control condition). Each chip measures the expression level of 2,613 genes from Ferroglobus placidus DSM 10642 with nine 45-60-mer probe pairs (PM/MM) per gene, with three-fold technical redundancy.

Project description:Insight into the mechanisms for the anaerobic metabolism of aromatic compounds by the hyperthermophilic archaeon Ferroglobus placidus is expected to improve understanding of the degradation of aromatics in hot (> 80 °C) environments and to identify enzymes that might have biotechnological applications. Analysis of the F. placidus genome revealed genes predicted to encode enzymes homologous to those previously identified as playing a role in benzoate and phenol metabolism in mesophilic bacteria. Surprisingly, F. placidus lacks genes for an ATP-independent class II benzoyl-CoA reductase found in all strictly anaerobic bacteria, but instead has two sets of genes for ATP-consuming class I benzoyl-CoA reductases, similar to those found in facultative bacteria. The lower portion of the benzoate degradation pathway appears to be more similar to that found in the phototroph Rhodopseudomonas palustris, than the pathway reported for all heterotrophic anaerobic benzoate degraders. Many of the genes predicted to be involved in benzoate metabolism were found in one of two gene clusters. Genes for a phenol carboxylation proceeding through a phenylphosphate intermediate and for conversion of p-hydroxybenzoate to benzoyl-CoA were identified in a single gene cluster. Analysis of transcript abundance with a whole-genome microarray and quantitative PCR demonstrated that most of the genes predicted to be involved in benzoate or phenol metabolism had higher transcript abundance during growth on those substrates versus growth on acetate. These results suggest that the general strategies for benzoate and phenol metabolism may be highly conserved between microorganisms living in moderate and hot environments, and that anaerobic metabolism of aromatic compounds might be analyzed in a wide range of environments with similar molecular targets. A four chip study using total RNA recovered from two separate cultures of Ferroglobus placidus DSM 10642 grown with 1 mM sodium benzoate (experimental condition) and two separate cultures of Ferroglobus placidus DSM 10642 grown on 10 mM acetate (control condition). Each chip measures the expression level of 2613 genes from Ferroglobus placidus DSM 10642 with nine 45-60-mer probe pairs (PM/MM) per gene, with three-fold technical redundancy.

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Exploring the molecular machinery of denitrification in Haloferax mediterranei through proteomics

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