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Methanocaldococcus sp. strain FS406-22, a hyperthermophilic methanogen, fixes nitrogen with a minimal set of known nif genes. Only four structural nif genes, nifH , nifD , nifK , and nifE, are present in a cluster, and a nifB homolog is present elsewhere in the genome. nifN , essential for the final synthesis of the iron-molybdenum cofactor of nitrogenase in well-characterized diazotrophs, is absent from FS406-22. In addition, FS406-22 encodes four novel hypothetical proteins, and a ferredoxin, in the nif cluster. Here, we develop a set of genetic tools for FS406-22 and test the functionality of genes in the nif cluster by making markerless in-frame deletion mutations. Deletion of the gene for one hypothetical protein, designated Hp4, delayed the initiation of diazotrophic growth and decreased the growth rate, an effect we confirmed by genetic complementation. NifE also appeared to play a role in diazotrophic growth, and the encoding of Hp4 and NifE in a single operon suggested they may work together in some way in the synthesis of the nitrogenase cofactor. No role could be discerned for any of the other hypothetical proteins, nor for the ferredoxin, despite the presence of these genes in a variety of related organisms. Possible pathways and evolutionary scenarios for the synthesis of the nitrogenase cofactor in an organism that lacks nifN are discussed. IMPORTANCE Methanocaldococcus has been considered a model genus, but genetic tools have not been forthcoming until recently. Here, we develop and illustrate the utility of positive selection with either of two selective agents (simvastatin and neomycin), negative selection, generation of markerless in-frame deletion mutations, and genetic complementation. These genetic tools should be useful for a variety of related species. We address the question of the minimal set of nif genes, which has implications for how nitrogen fixation evolved.

Phenazines are a class of bacterially produced redox-active metabolites that are found in natural, industrial, and clinical environments. In Pseudomonas spp., phenazine-1-carboxylic acid (PCA)-the precursor of all phenazine metabolites-facilitates nutrient acquisition, biofilm formation, and competition with other organisms. While the removal of phenazines negatively impacts these activities, little is known about the genes or enzymes responsible for phenazine degradation by other organisms. Here, we report that the first step of PCA degradation by Mycobacterium fortuitum is catalyzed by a ph enazine- d egrading decarboxylase (PhdA). PhdA is related to members of the UbiD protein family that rely on a prenylated flavin mononucleotide cofactor for activity. The gene for PhdB, the enzyme responsible for cofactor synthesis, is present in a putative operon with the gene encoding PhdA in a region of the M. fortuitum genome that is essential for PCA degradation. PhdA and PhdB are present in all known PCA-degrading organisms from the Actinobacteria M. fortuitum can also catabolize other Pseudomonas -derived phenazines such as phenazine-1-carboxamide, 1-hydroxyphenazine, and pyocyanin. On the basis of our previous work and the current characterization of PhdA, we propose that degradation converges on a common intermediate: dihydroxyphenazine. An understanding of the genes responsible for degradation will enable targeted studies of phenazine degraders in diverse environments. IMPORTANCE Bacteria from phylogenetically diverse groups secrete redox-active metabolites that provide a fitness advantage for their producers. For example, phenazines from Pseudomonas spp. benefit the producers by facilitating anoxic survival and biofilm formation and additionally inhibit competitors by serving as antimicrobials. Phenazine-producing pseudomonads act as biocontrol agents by leveraging these antibiotic properties to inhibit plant pests. Despite this importance, the fate of phenazines in the environment is poorly understood. Here, we characterize an enzyme from Mycobacterium fortuitum that catalyzes the first step of phenazine-1-carboxylic acid degradation. Knowledge of the genetic basis of phenazine degradation will facilitate the identification of environments where this activity influences the microbial community structure., (Copyright © 2018 American Society for Microbiology.)

The opportunistic pathogen Pseudomonas aeruginosa produces colorful redox-active metabolites called phenazines, which underpin biofilm development, virulence, and clinical outcomes. Although phenazines exist in many forms, the best studied is pyocyanin. Here, we describe pyocyanin demethylase (PodA), a hitherto uncharacterized protein that oxidizes the pyocyanin methyl group to formaldehyde and reduces the pyrazine ring via an unusual tautomerizing demethylation reaction. Treatment with PodA disrupts P. aeruginosa biofilm formation similarly to DNase, suggesting interference with the pyocyanin-dependent release of extracellular DNA into the matrix. PodA-dependent pyocyanin demethylation also restricts established biofilm aggregate populations experiencing anoxic conditions. Together, these results show that modulating extracellular redox-active metabolites can influence the fitness of a biofilm-forming microorganism., (Copyright © 2017, American Association for the Advancement of Science.)

The H2 -dependent methylene-tetrahydromethanopterin dehydrogenase (Hmd), also known as the [Fe]-hydrogenase, is found only in methanogens without cytochromes. In contrast to the binuclear metal centers of the [NiFe]- and [FeFe]-hydrogenases, the [Fe]-hydrogenase contains only a single Fe atom, which is coordinated by a novel guanylylpyridinol cofactor in the active site. The biosynthesis of the cofactor is not well understood and the responsible genes are unknown. However, seven genes (hmd co-occurring genes, hcg) encoding proteins of unknown function are always associated with the hmd gene. In the model methanogen Methanococcus maripaludis, we used a genetic background in which a deletion of hmd had a distinct growth phenotype, and made null-mutations in each hcg gene as well as in a gene encoding the Hmd paralog HmdII, which is hypothesized to function as a scaffold for cofactor synthesis. Deletions in all seven hcg genes resulted in the same growth phenotype as a deletion in hmd, suggesting they are required for Hmd function. In all cases, genetic complementation of the mutation restored the wild-type phenotype. A deletion in hmdII had no effect., (© 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.)

Despite decades of study, electron flow and energy conservation in methanogenic Archaea are still not thoroughly understood. For methanogens without cytochromes, flavin-based electron bifurcation has been proposed as an essential energy-conserving mechanism that couples exergonic and endergonic reactions of methanogenesis. However, an alternative hypothesis posits that the energy-converting hydrogenase Eha provides a chemiosmosis-driven electron input to the endergonic reaction. In vivo evidence for both hypotheses is incomplete. By genetically eliminating all nonessential pathways of H(2) metabolism in the model methanogen Methanococcus maripaludis and using formate as an additional electron donor, we isolate electron flow for methanogenesis from flux through Eha. We find that Eha does not function stoichiometrically for methanogenesis, implying that electron bifurcation must operate in vivo. We show that Eha is nevertheless essential, and a substoichiometric requirement for H(2) suggests that its role is anaplerotic. Indeed, H(2) via Eha stimulates methanogenesis from formate when intermediates are not otherwise replenished. These results fit the model for electron bifurcation, which renders the methanogenic pathway cyclic, and as such requires the replenishment of intermediates. Defining a role for Eha and verifying electron bifurcation provide a complete model of methanogenesis where all necessary electron inputs are accounted for.