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The enzyme methyl-coenzyme M reductase (MCR) plays an important role in mediating global levels of methane by catalyzing a reversible reaction that leads to the production or consumption of this potent greenhouse gas in methanogenic and methanotrophic archaea. In methanogenic archaea, the alpha subunit of MCR (McrA) typically contains four to six posttranslationally modified amino acids near the active site. Recent studies have identified enzymes performing two of these modifications (thioglycine and 5-[S]-methylarginine), yet little is known about the formation and function of the remaining posttranslationally modified residues. Here, we provide in vivo evidence that a dedicated S-adenosylmethionine-dependent methyltransferase encoded by a gene we designated methylcysteine modification (mcmA) is responsible for formation of S-methylcysteine in Methanosarcina acetivorans McrA. Phenotypic analysis of mutants incapable of cysteine methylation suggests that the S-methylcysteine residue might play a role in adaption to mesophilic conditions. To examine the interactions between the S-methylcysteine residue and the previously characterized thioglycine, 5-(S)-methylarginine modifications, we generated M. acetivorans mutants lacking the three known modification genes in all possible combinations. Phenotypic analyses revealed complex, physiologically relevant interactions between the modified residues, which alter the thermal stability of MCR in a combinatorial fashion that is not readily predictable from the phenotypes of single mutants. High-resolution crystal structures of inactive MCR lacking the modified amino acids were indistinguishable from the fully modified enzyme, suggesting that interactions between the posttranslationally modified residues do not exert a major influence on the static structure of the enzyme but rather serve to fine-tune the activity and efficiency of MCR.

In 2015, Duke Energy and Dominion Energy joined forces to begin work on a massive natural gas pipeline extending through Appalachia. The proposed path of the Atlantic Coast Pipeline, as the project was named, ran through several low-income, minority communities and also traversed a section of the Appalachian Trail located in George Washington National Forest. The U.S. Forest Service granted the energy companies a permit to build the pipeline across this national forest and the Appalachian Trail, but before work could begin, a group of environmental stakeholders filed an action challenging the Forest Service's authority to issue the permit. The Supreme Court adjudicated that challenge in U.S. Forest Service v. Cowpasture River Preservation Ass'n. The dispositive issue in the case was whether the Appalachian Trail is actually 'land.' If so, then under established federal law, the Forest Service would lack the authority to grant the permit; if not, then the grant of the permit would be valid. To make its decision on the subject, the Court turned to private easement law principles and, in doing so, concluded that the permit grant must be upheld because the Appalachian Trail is not land. As the Court explained, '[a] trail is a trail, and land is land'--the two are distinct from one another. Or are they? This Recent Development answers that question in the negative, positing that the Cowpasture Court reached an erroneous conclusion by incorrectly invoking the private law of easements to decide the case. And although Duke and Dominion eventually abandoned the Atlantic Coast Pipeline project, the legal precedent set by the Court's decision remains. This precedent will result in increased future harms to the environment and low-income, minority communities and, in that way, ultimately constitutes lost ground for environmental advocates in the fight against environmental injustice., INTRODUCTION With its narrow progression along mountain ridgelines, slow descents into forested valleys, and meandering turns through sprawling fields, the Appalachian Trail ('the Trail') provides a glimpse into some of [...]

Pantaphos is small molecule virulence factor made by the plant pathogen Pantoea ananatis. An 11 gene operon, designated hvr for high virulence, is required for production of this phosphonic acid natural product, but the metabolic steps used in its production have yet to be established. Herein, we determine the complete biosynthetic pathway using a combination of bioinformatics, in vitro biochemistry and in vivo heterologous expression. Only 6 of the 11 hvr genes are needed to produce pantaphos, while a seventh is likely to be required for export. Surprisingly, the pathway involves a series of O‐methylated intermediates, which are then hydrolyzed to produce the final product. The methylated intermediates are produced by an irreversible S‐adenosylmethione (SAM)‐dependent methyltransferase that is required to drive a thermodynamically unfavorable dehydration in the preceding step, a function not previously attributed to members of this enzyme class. Methylation of pantaphos by the same enzyme is also likely to limit its toxicity in the producing organism. The pathway also involves a novel flavin‐dependent monooxygenase that differs from homologous proteins due to its endogenous flavin‐reductase activity. Heterologous production of pantaphos by Escherichia coli strains expressing the minimal gene set strongly supports the in vitro biochemical data. [ABSTRACT FROM AUTHOR]

Desulfotignum phosphitoxidans is a strictly anaerobic, Gram-negative bacterium that utilizes phosphite as the sole electron source for homoacetogenic C[O.sub.2] reduction or sulfate reduction. A genomic library of D. phosphitoxidans, constructed using the fosmid vector pJK050, was screened for clones harboring the genes involved in phosphite oxidation via PCR using primers developed based on the amino acid sequences of phosphite-induced proteins. Sequence analysis of two positive clones revealed a putative operon of seven genes predicted to be involved in phosphite oxidation. Four of these genes (ptxD-ptdFCG) were cloned and heterologously expressed in Desulfotignum balticum, a related strain that cannot use phosphite as either an electron donor or as a phosphorus source. The ptxD.ptdFCG gene cluster was sufficient to confer phosphite uptake and oxidation ability to the D. balticum host strain but did not allow use of phosphite as an electron donor for chemolithotrophic growth. Phosphite oxidation activity was measured in cell extracts of D. balticum transconjugants, suggesting that all genes required for phosphite oxidation were cloned. Genes of the phosphite gene cluster were assigned putative functions on the basis of sequence analysis and enzyme assays. doi: 10.1128/JB.00541-10

Methanosarcina species possess three operons (mtaCB1, mtaCB2, and mtaCB3) encoding methanol-specific methyltransferase 1 (MT1) isozymes and two genes (mtaA1 and mtaA2) with the potential to encode a methanol-specific methyltransferase 2 (MT2). Previous genetic studies showed that these genes are differentially regulated and encode enzymes with distinct levels of methyltransferase activity. Here, the effects of promoter strength on growth and on the rate of methane production were examined by constructing strains in which the mtaCB promoters were exchanged. When expressed from the strong PmtaC1 or PmtaC2 promoter, each of the MtaC and MtaB proteins supported growth and methane production at wild-type levels. In contrast, all mtaCB operons exhibited poorer growth and lower rates of methane production when PmtaC3 controlled their expression. Thus, previously observed phenotypic differences can be attributed largely to differences in promoter activity. Strains carrying various combinations of mtaC, mtaB, and mtaA expressed from the strong, tetracycline-regulated PmcrB(tetO1) promoter exhibited similar growth characteristics on methanol, showing that all combinations of MtaC, MtaB, and MtaA can form functional MT1/MT2 complexes. However, an in vitro assay of coupled MT1/MT2 activity showed significant variation between the strains. Surprisingly, these variations in activity correlated with differences in protein abundance, despite the fact that all the encoding genes were expressed from the same promoter. Quantitative reverse transcriptase PCR and reporter gene fusion data suggest that the mtaCBA transcripts show different stabilities, which are strongly influenced by the growth substrate. doi: 10.1128/JB.00947-09

During phenotypic characterization of various Escherichia coli mutants, we observed that [DELTA]phoA strains are capable of using glycerol-2-phosphate (G2P) as a sole source of phosphorus. Mutations in the ugpBAECQ operon eliminated this phenotype, suggesting that G2P is a previously unrecognized substrate for the binding protein-dependent Ugp transporter.

Natural products containing phosphorus-carbon bonds have found widespread use in medicine and agriculture (1). One such compound, phosphinothricin tripeptide, contains the unusual amino acid phosphinothricin attached to two alanine residues. Synthetic phosphinothricin (glufosinate) is a component of two top-selling herbicides (Basta and Liberty), and is widely used with resistant transgenic crops including corn, cotton and canola. Recent genetic and biochemical studies showed that during phosphinothricin tripeptide biosynthesis 2-hydroxyethylphosphonate (HEP) is converted to hydroxymethylphosphonate (HMP) (2). Here we report the in vitro reconstitution of this unprecedented C([sp.sup.3])-C([sp.sup.3]) bond cleavage reaction and X-ray crystal structures of the enzyme. The protein is a mononuclear non-haem iron(n)-dependent dioxygenase that converts HEP to HMP and formate. In contrast to most other members of this family, the oxidative consumption of HEP does not require additional cofactors or the input of exogenous electrons. The current study expands the scope of reactions catalysed by the 2-His-1-carboxylate mononuclear non-haem iron family of enzymes., Hydroxyethylphosphonate dioxygenase (HEPD) was overexpressed in Escherichia coli with an amino-terminal hexa-histidine tag and purified by [Ni.sup.2+]-affinity chromatography. As isolated the protein was inactive, but in the presence of Fe(II) [...]

Methanosarcina acetivorans C2A encodes three putative hydrogenases, including one cofactor [F.sub.420]-linked (frh) and two methanophenazine-linked (vht) enzymes. Comparison of the amino acid sequences of these putative hydrogenases to those of Methanosarcina barkeri and Methanosarcina mazei shows that each predicted subunit contains all the known residues essential for hydrogenase function. The DNA sequences upstream of the genes in M. acetivorans were aligned with those in other Methanosarcina species to identify conserved transcription and translation signals. The M. acetivorans vht promoter region is well conserved among the sequenced Methanosarcina species, while the second vht-type homolog (here called vhx) and frh promoters have only limited similarity. To experimentally determine whether these promoters are functional in vivo, we constructed and characterized both M. acetivorans and M. barkeri strains carrying reporter gene fusions to each of the M. acetivorans and M. barkeri hydrogenase promoters. Generally, the M. acetivorans gene fusions are not expressed in either organism, suggesting that cis-acting mutations inactivated the M. acetivorans promoters. The M. barked hydrogenase gene fusions, on the other hand, are expressed in both organisms, indicating that M. acetivorans possesses the machinery to express hydrogenases, although it does not express its own hydrogenases. These data are consistent with specific inactivation of the M. acetivorans hydrogenase promoters and highlight the importance of testing hypotheses generated by using genomic data.

The entry of methanol into the methylotrophic pathway of methanogenesis is mediated by the concerted effort of two methyltransferases, namely, methyltransferase 1 (MTI) and methyltransferase 2 (MT2). The mtaA1, mtaA2, and mtbA genes of Methanosarcina acetivorans C2A encode putative methanol- or methylamine-specific MT2 enzymes. To address the in vivo roles of these genes in growth and methanogenesis from known substrates, we constructed and characterized mutants with deletions of each of these genes. The mtaA1 gene is required for growth on methanol, whereas mtaA2 was dispensable. However, the mtaA2 mutant had a reduced rate of methane production from methanol. Surprisingly, deletion of mtaA1 in combination with deletions of the genes encoding three methanol-specific MT1 isozymes led to lack of growth on acetate, suggesting that MT1 and MT2 enzymes might play an important role during growth on this substrate. The mtbA gene was required for dimethylamine and monomethylamine (MMA) utilization and was important, but not required, for trimethylamine utilization. Analysis of reporter gene fusions revealed that both mtaA1 and mtbA were expressed on all methanogenic substrates tested. However, mtaA1 expression was induced on methanol, while mtbA expression was down-regulated on MMA and acetate, mtaA2 was expressed at very low levels on all substrates. The mtaA1 transcript had a large 5' untranslated region (UTR) (275 bp), while the 5' UTR of the mtbA transcript was only 28 bp long.

A series of Methanosarcina barkeri mutants lacking the genes encoding the enzymes involved in the C1 oxidation/reduction pathway were constructed. Mutants lacking the methyl-tetrahydromethanopterin ([H.sub.4]MPT):coenzyme M (CoM) methyltransferase-encoding operon ([DELTA]mtr), the methylene-[H.sub.4]MPT reductase-encoding gene ([DELTA]mer), the methylene-[H.sub.4]MPT dehydrogenase-encoding gene ([DELTA]mtd), and the formyl-methanofuran:[H.sub.4]MPT formyl-transferase-encoding gene ([DELTA]flr) all failed to grow using either methanol or [H.sub.2]/C[O.sub.2] as a growth substrate, indicating that there is an absolute requirement for the C1 oxidation/reduction pathway for hydrogenotrophic and methylotrophic methanogenesis. The mutants also failed to grow on acetate, and we suggest that this was due to an inability to generate the reducing equivalents needed for biosynthetic reactions. Despite their lack of growth on methanol, the [DELTA]mtr and [DELTA]mer mutants were capable of producing methane from this substrate, whereas the [DELTA]mtd and [DELTA]ftr mutants were not. Thus, there is an Mtr/Mer bypass pathway that allows oxidation of methanol to the level of methylene-[H.sub.4]MPT in M. barkeri. The data further suggested that formaldehyde may be an intermediate in this bypass; however, no methanol dehydrogenase activity was found in [DELTA]mtr cell extracts, nor was there an obligate role for the formaldehyde-activating enzyme (Fae), which has been shown to catalyze the condensation of formaldehyde and [H.sub.4]MPT in vitro. Both the [DELTA]mer and [DELTA]mtr mutants were able to grow on a combination of methanol plus acetate, but they did so by metabolic pathways that are clearly distinct from each other and from previously characterized methanogenic pathways.

The article discusses biochemical pathways and molecular genetics of reduced bacterial phosphorus (P) metabolism. The presence of microbially mediated phosphorus redox cycle is observed.