ModE Regulates Alternative Nitrogenase Expression in the Methanogen Methanosarcina acetivorans.

All methanogens that can fix nitrogen use molybdenum (Mo) nitrogenase. Some methanogens, including Methanosarcina acetivorans , also contain alternative vanadium- and iron-nitrogenases, encoded by the vnf and anf operons, respectively. These nitrogenases are produced when there is insufficient Mo to support Mo-nitrogenase activity.

The minimal SUF system is not required for Fe-S cluster biogenesis in the methanogenic archaeon Methanosarcina acetivorans.

Iron-sulfur (Fe-S) proteins are essential for the ability of methanogens to carry out methanogenesis and biological nitrogen fixation (diazotrophy). Nonetheless, the factors involved in Fe-S cluster biogenesis in methanogens remain largely unknown. The minimal SUF Fe-S cluster biogenesis system (i.

Expression of V-nitrogenase and Fe-nitrogenase in is controlled by molybdenum, fixed nitrogen, and the expression of Mo-nitrogenase.

All nitrogen-fixing bacteria and archaea (diazotrophs) use molybdenum (Mo) nitrogenase to reduce dinitrogen (N) to ammonia, with some also containing vanadium (V) and iron-only (Fe) nitrogenases that lack Mo. Among diazotrophs, the regulation and usage of the alternative V-nitrogenase and Fe-nitrogenase in methanogens are largely unknown. contains , , and gene clusters encoding putative Mo-nitrogenase, V-nitrogenase, and Fe-nitrogenase, respectively.

Methanosarcina acetivorans contains a functional ISC system for iron-sulfur cluster biogenesis.

Background: The production of methane by methanogens is dependent on numerous iron-sulfur (Fe-S) cluster proteins; yet, the machinery involved in Fe-S cluster biogenesis in methanogens remains largely unknown. Methanogen genomes encode uncharacterized homologs of the core components of the ISC (IscS and IscU) and SUF (SufBC) Fe-S cluster biogenesis systems found in bacteria and eukaryotes. Methanosarcina acetivorans contains three iscSU and two sufCB gene clusters.

A CRISPRi-dCas9 System for Archaea and Its Use To Examine Gene Function during Nitrogen Fixation by Methanosarcina acetivorans.

CRISPR-based systems are emerging as the premier method to manipulate many cellular processes. In this study, a simple and efficient CRISPR interference (CRISPRi) system for targeted gene repression in archaea was developed. The CRISPR-Cas9 system was repurposed by replacing Cas9 with the catalytically dead Cas9 (dCas9) to generate a CRISPRi-dCas9 system for targeted gene repression.

Toward a mechanistic and physiological understanding of a ferredoxin:disulfide reductase from the domains Archaea and Bacteria.

Disulfide reductases reduce other proteins and are critically important for cellular redox signaling and homeostasis. is a methane-producing microbe from the domain Archaea that produces a ferredoxin:disulfide reductase (FDR) for which the crystal structure has been reported, yet its biochemical mechanism and physiological substrates are unknown. FDR and the extensively characterized plant-type ferredoxin:thioredoxin reductase (FTR) belong to a distinct class of disulfide reductases that contain a unique active-site [4Fe-4S] cluster.

Methanosarcina acetivorans utilizes a single NADPH-dependent thioredoxin system and contains additional thioredoxin homologues with distinct functions.

The thioredoxin system plays a central role in the intracellular redox maintenance in the majority of cells. The canonical system consists of an NADPH-dependent thioredoxin reductase (TrxR) and thioredoxin (Trx), a disulfide reductase. Although Trx is encoded in almost all sequenced genomes of methanogens, its incorporation into their unique physiology is not well understood.

The [4Fe-4S] clusters of Rpo3 are key determinants in the post Rpo3/Rpo11 heterodimer formation of RNA polymerase in Methanosarcina acetivorans.

Subunits Rpo3 and Rpb3/AC40 of RNA polymerase (RNAP) from many archaea and some eukaryotes, respectively, contain a ferredoxin-like domain (FLD) predicted to bind one or two [4Fe-4S] clusters postulated to play a role in regulating the assembly of RNAP. To test this hypothesis, the two [4Fe-4S] cluster Rpo3 from Methanosarcina acetivorans was modified to generate variants that lack the FLD or each [4Fe-4S] cluster. Viability of gene replacement mutants revealed that neither the FLD nor the ability of the FLD to bind either [4Fe-4S] cluster is essential.

The Methanosarcina acetivorans thioredoxin system activates DNA binding of the redox-sensitive transcriptional regulator MsvR.

The production of biogas (methane) by an anaerobic digestion is an important facet to renewable energy, but is subject to instability due to the sensitivity of strictly anaerobic methanogenic archaea (methanogens) to environmental perturbations, such as oxygen. An understanding of the oxidant-sensing mechanisms used by methanogens may lead to the development of more oxidant tolerant (i.e.

Molecular characterization of the thioredoxin system from Methanosarcina acetivorans.

The thioredoxin system, composed of thioredoxin reductase (TrxR) and thioredoxin (Trx), is widely distributed in nature, where it serves key roles in electron transfer and in the defense against oxidative stress. Although recent evidence reveals Trx homologues are almost universally present among the methane-producing archaea (methanogens), a complete thioredoxin system has not been characterized from any methanogen. We examined the phylogeny of Trx homologues among methanogens and characterized the thioredoxin system from Methanosarcina acetivorans.

Expression of a bacterial catalase in a strictly anaerobic methanogen significantly increases tolerance to hydrogen peroxide but not oxygen.

Haem-dependent catalase is an antioxidant enzyme that degrades H2O2, producing H2O and O2, and is common in aerobes. Catalase is present in some strictly anaerobic methane-producing archaea (methanogens), but the importance of catalase to the antioxidant system of methanogens is poorly understood. We report here that a survey of the sequenced genomes of methanogens revealed that the majority of species lack genes encoding catalase.

Redox-sensitive DNA binding by homodimeric Methanosarcina acetivorans MsvR is modulated by cysteine residues.

Background: Methanoarchaea are among the strictest known anaerobes, yet they can survive exposure to oxygen. The mechanisms by which they sense and respond to oxidizing conditions are unknown. MsvR is a transcription regulatory protein unique to the methanoarchaea.

Assessment of the oxidant tolerance of Methanosarcina acetivorans.

All methane-producing Archaea (methanogens) are strict anaerobes, but the majority of species are tolerant to oxidants. Methanosarcina species are important environmental and industrial methanogens as they are one of only two genera capable of producing methane with acetate. Importantly, Methanosarcina species appear to be the most oxidant-tolerant; however, the mechanisms underlying this tolerance are poorly understood.

Subunit D of RNA polymerase from Methanosarcina acetivorans contains two oxygen-labile [4Fe-4S] clusters: implications for oxidant-dependent regulation of transcription.

Subunit D of multisubunit RNA polymerase from many species of archaea is predicted to bind one to two iron-sulfur (Fe-S) clusters, the function of which is unknown. A survey of encoded subunit D in the genomes of sequenced archaea revealed six distinct groups based on the number of complete or partial [4Fe-4S] cluster motifs within domain 3. Only subunit D from strictly anaerobic archaea, including all members of the Methanosarcinales, are predicted to bind two [4Fe-4S] clusters.

An engineered methanogenic pathway derived from the domains Bacteria and Archaea.

A plasmid-based expression system wherein mekB was fused to a constitutive Methanosarcina acetivorans promoter was used to express MekB, a broad-specificity esterase from Pseudomonas veronii, in M. acetivorans. The engineered strain had 80-fold greater esterase activity than wild-type M.

Methanogenesis in marine sediments.

The anaerobic conversion of complex organic matter to CH(4) is an essential link in the global carbon cycle. In freshwater anaerobic environments, the organic matter is decomposed to CH(4) and CO(2) by a microbial food chain that terminates with methanogens that produce methane primarily by reduction of the methyl group of acetate and also reduction of CO(2). The process also occurs in marine environments, particularly those receiving large loads of organic matter, such as coastal sediments.

The archaeon Methanosarcina acetivorans contains a protein disulfide reductase with an iron-sulfur cluster.

Methanosarcina acetivorans, a strictly anaerobic methane-producing species belonging to the domain Archaea, contains a gene cluster annotated with homologs encoding oxidative stress proteins. One of the genes (MA3736) is annotated as a gene encoding an uncharacterized carboxymuconolactone decarboxylase, an enzyme required for aerobic growth with aromatic compounds by species in the domain Bacteria. Methane-producing species are not known to utilize aromatic compounds, suggesting that MA3736 is incorrectly annotated.

An unconventional pathway for reduction of CO2 to methane in CO-grown Methanosarcina acetivorans revealed by proteomics.

Methanosarcina acetivorans produces acetate, formate, and methane when cultured with CO as the growth substrate [Rother M, Metcalf WW (2004) Proc Natl Acad Sci USA 101:], which suggests novel features of CO metabolism. Here we present a genome-wide proteomic approach to identify and quantify proteins differentially abundant in response to growth on CO versus methanol or acetate. The results indicate that oxidation of CO to CO2 supplies electrons for reduction of CO2 to a methyl group by steps and enzymes of the pathway for CO2 reduction determined for other methane-producing species.

Electron transport in the pathway of acetate conversion to methane in the marine archaeon Methanosarcina acetivorans.

A liquid chromatography-hybrid linear ion trap-Fourier transform ion cyclotron resonance mass spectrometry approach was used to determine the differential abundance of proteins in acetate-grown cells compared to that of proteins in methanol-grown cells of the marine isolate Methanosarcina acetivorans metabolically labeled with 14N versus 15N. The 246 differentially abundant proteins in M. acetivorans were compared with the previously reported 240 differentially expressed genes of the freshwater isolate Methanosarcina mazei determined by transcriptional profiling of acetate-grown cells compared to methanol-grown cells.

Structural insight into the dioxygenation of nitroarene compounds: the crystal structure of nitrobenzene dioxygenase.

Nitroaromatic compounds are used extensively in many industrial processes and have been released into the environment where they are considered environmental pollutants. Nitroaromatic compounds, in general, are resistant to oxidative attack due to the electron-withdrawing nature of the nitro groups and the stability of the benzene ring. However, the bacterium Comamonas sp.

Expression of the nitroarene dioxygenase genes in Comamonas sp. strain JS765 and Acidovorax sp. strain JS42 is induced by multiple aromatic compounds.

This work reports a genetic analysis of the expression of nitrobenzene dioxygenase (NBDO) in Comamonas sp. strain JS765 and 2-nitrotoluene dioxygenase (2NTDO) in Acidovorax sp. strain JS42.

Molecular characterization and substrate specificity of nitrobenzene dioxygenase from Comamonas sp. strain JS765.

Comamonas sp. strain JS765 can grow with nitrobenzene as the sole source of carbon, nitrogen, and energy. We report here the sequence of the genes encoding nitrobenzene dioxygenase (NBDO), which catalyzes the first step in the degradation of nitrobenzene by strain JS765.