Biology subjects

Lessner, D. J.

Publications and source records attributed to Lessner, D. J..

5 recordsLinked to original sources

bioRxiv2025

Cryo-EM structure of a methanogen nitrogenase-PII protein supercomplex

Nitrogenases are metalloenzymes that catalyze the reduction of atmospheric dinitrogen to ammonia, sustaining the global nitrogen cycle. While bacterial nitrogenase has been extensively characterized, the architecture and regulation of archaeal nitrogenases remain unknown despite longstanding evidence of nitrogen fixation in methanogens. Here we report a 3.2 [A] cryo-electron microscopy structure of a native nitrogenase-PII protein supercomplex from Methanosarcina acetivorans. The structure reveals an unprecedented assembly of three NifDK heterotetramers bridged by six NifI1,2 heterotrimeric PII complexes, which sterically block NifH association and lock the enzyme in an inactive state. The PII complexes display asymmetric binding of ADP and 2-oxoglutarate, coupling nitrogenase inhibition directly to cellular energy and nitrogen status. Addition of 2-oxoglutarate and ATP releases the NifI complexes, stimulating a threefold increase in NifDK activity in vitro. This higher-order architecture uncovers a previously unrecognized regulatory strategy in methanogens, in which PII proteins drive nitrogenase oligomerization to control activity. The discovery that nitrogenase activity may be modulated through direct assembly into higher-order structures opens new avenues for exploring nitrogenase evolution, regulation, and biotechnological applications. One sentence summaryDiscovery of a nitrogenase-PII protein supercomplex in methanogens, uncovering a metabolite-gated assembly mechanism for nitrogenase inhibition.

biochemistry↗

bioRxiv2023

The nitrogenase cofactor biogenesis enzyme NifB is essential for the viability of methanogens

Dinitrogen (N2) is only bioavailable to select bacteria and archaea that possess the metalloenzyme nitrogenase, which reduces N2 to NH3 in a process called nitrogen fixation or diazotrophy. A long-term goal is to engineer diazotrophy into plants to decrease the use of nitrogen fertilizers, saving billions of dollars annually and greatly reducing nutrient pollution. This goal has not been realized, in part due to the inability to produce the nitrogenase metallocofactor within plants. Biogenesis of the cofactor requires NifB, a radical S-adenosy-L-methionine (SAM) enzyme that generates a precursor [8Fe-9S-C] cluster that matures into the final metallocofactor. Although maturation of nitrogenase is the only known function of NifB in bacteria, bioinformatic analyses reveal that NifB is conserved across methanogens, including those lacking nitrogenase, which suggests NifB functions outside of nitrogenase maturation. Indeed, several lines of evidence show that NifB is essential for viability of the model diazotroph, Methanosarcina acetivorans. First, CRISPRi repression was unable to abolish NifB production, whereas CRISPRi repression abolishes non-essential nitrogenase production. Second, unlike nitrogenase production, NifB production is not controlled by fixed nitrogen availability. Finally, nifB could not be deleted from M. acetivorans unless complemented in trans with nifB from other methanogens, including Methanothrix thermoacetophila, a species that lacks nitrogenase. Notably, M. thermoacetophila NifB supported diazotrophy in M. acetivorans, demonstrating that NifB from a non-diazotrophic methanogen produces the [8Fe-9S-C] cluster. Overall, these results link the metallocofactor biogenesis function of NifB to nitrogen fixation and methanogenesis, two processes of global importance. SIGNIFICANCEMethanogens directly impact life on Earth since they produce methane, a potent greenhouse gas, and are the principal archaea capable of nitrogen fixation, a process that requires nitrogenase. In this study, we demonstrate that NifB, an enzyme required to produce the metallocofactor in non-essential nitrogenase, is essential to the viability of methanogens. This identifies NifB as a new potential target in the goal of inhibiting methanogens to reduce methane emissions. The discovery that NifB functions outside of nitrogenase maturation will also aid efforts to engineer nitrogen fixation in plants, since NifB is a key factor to achieve this goal. Realization of these goals would have immense economic, environmental, and societal benefits.

microbiology↗

bioRxiv2021

Vht hydrogenase is required for hydrogen cycling during nitrogen fixation by the non-hydrogenotrophic methanogen Methanosarcina acetivorans.

Methanosarcina acetivorans is the primary model to understand the physiology of methanogens that do not use hydrogenase to consume or produce hydrogen (H2) during methanogenesis. The genome of M. acetivorans encodes putative methanophenazine-reducing hydrogenases (Vht and Vhx), F420-reducing hydrogenase (Frh), and hydrogenase maturation machinery (Hyp), yet cells lack significant hydrogenase activity under all growth conditions tested to date. Thus, the importance of hydrogenase to the physiology of M. acetivorans has remained a mystery. M. acetivorans can fix dinitrogen (N2) using nitrogenase that is documented in bacteria to produce H2 during the reduction of N2 to ammonia. Therefore, we hypothesized that M. acetivorans uses hydrogenase to recycle H2 produced by nitrogenase during N2 fixation. Results demonstrate that hydrogenase expression and activity is higher in N2-grown cells compared to cells grown with fixed nitrogen (NH4Cl). To test the importance of each hydrogenase and the maturation machinery, the CRISPRi-dCas9 system was used to generate separate M. acetivorans strains where transcription of the vht, frh, vhx, or hyp operons is repressed. Repression of vhx and frh does not alter growth with either NH4Cl or N2 and has no effect on H2 metabolism. However, repression of vht or hyp results in impaired growth with N2 but not NH4Cl. Importantly, H2 produced endogenously by nitrogenase is detected in the headspace of culture tubes containing the vht or hyp repression strains. Overall, the results reveal that Vht hydrogenase recycles H2 produced by nitrogenase that is required for optimal growth of M. acetivorans during N2 fixation. IMPORTANCEThe metabolism of M. acetivorans and closely related Methanosarcinales is thought to not involve H2. Here we show for the first time M. acetivorans is capable of H2 cycling like hydrogenotrophic Methanosarcinales (e.g., Methanosarcina barkeri). However, unlike M. barkeri hydrogenase activity and H2 cycling is tightly regulated in M. acetivorans and is only utilized during N2 fixation to consume H2 production endogenously by nitrogenase. The in vivo production of H2 by nitrogenase during N2 reduction is also demonstrated for the first time in a methanogen. Overall, the results provide new insight into the evolution and diversity of methanogen metabolism and new details about methanogen nitrogenase that could be leveraged for practical applications, such as nitrogenase-dependent production of H2 as a biofuel.

microbiology↗

bioRxiv2021

Methanosarcina acetivorans simultaneously produces molybdenum, vanadium, and iron-only nitrogenases in response to fixed nitrogen and molybdenum depletion

All nitrogen-fixing bacteria and archaea (diazotrophs) use molybdenum (Mo) nitrogenase to reduce dinitrogen (N2) to ammonia. Some diazotrophs also contain alternative nitrogenases that lack Mo: vanadium (V) and iron-only (Fe) nitrogenases. Among diazotrophs, the regulation and usage of the alternative nitrogenases in methanogens is largely unknown. Methanosarcina acetivorans contains nif, vnf, and anf gene clusters encoding putative Mo-, V-, and Fe-nitrogenases, respectively. This study investigated the effect of fixed nitrogen and Mo/V availability on nitrogenase expression and growth by M. acetivorans. The availability of Mo and V did not affect growth of M. acetivorans with fixed nitrogen but significantly affected growth with N2. M. acetivorans exhibited the fastest growth rate and highest cell yield during growth with N2 in medium containing Mo. Depletion of Mo (Fe-only condition) resulted in a significant decrease in growth rate and cell yield. The addition of V to Mo-depleted medium stimulated diazotrophic growth but was still less than growth in Mo-replete medium. qPCR analysis revealed transcription of the nif operon is only moderately affected by depletion of fixed nitrogen and Mo. However, vnf and anf transcription increased significantly when fixed nitrogen and Mo were depleted, with removal of Mo being the key factor. Immunoblot analysis revealed Mo-nitrogenase is produced when fixed nitrogen is depleted regardless of Mo availability, while V- and Fe-nitrogenases are produced only in the absence of fixed nitrogen and Mo. These results reveal that alternative nitrogenase production in M. acetivorans is tightly controlled and that all three nitrogenases can be simultaneously produced. IMPORTANCEMethanogens and closely related methanotrophs are the only archaea known or predicted to possess nitrogenase. As such, methanogens play critical roles in both the global biological nitrogen and carbon cycles. Moreover, methanogens are an ancient microbial lineage and nitrogenase likely originated in methanogens. An understanding of the usage and properties of nitrogenases in methanogens can provide new insight into the evolution of nitrogen fixation and aid in the development nitrogenase-based biotechnology. This study provides the first evidence that a methanogen can produce all three forms of nitrogenases, even simultaneously. Surprisingly, Mo-nitrogenase was produced in cells grown in the absence of Mo, indicating components of Mo-nitrogenase regulate or are needed to produce V- and Fe-nitrogenases in methanogens. The results provide a foundation to understanding the assembly, regulation, and activity of the alternative nitrogenases in methanogens.

microbiology↗

bioRxiv2020

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 Methanosarcina acetivorans CRISPR-Cas9 system was repurposed by replacing Cas9 with the catalytically dead Cas9 (dCas9) to generate a CRISPRi-dCas9 system for targeted gene repression. To test the utility of the system, genes involved in nitrogen (N2) fixation were targeted for dCas9-mediated repression. First, the nif operon (nifHI1I2DKEN) that encodes molybdenum nitrogenase was targeted by separate guide RNAs (gRNA), one targeting the promoter and the other nifD. Remarkably, growth of M. acetivorans with N2 was abolished by dCas9-mediated repression of the nif operon with each gRNA. The abundance of nif transcripts was >90% reduced in both strains expressing the gRNAs, and NifD was not detected in cell lysate. Next, we targeted NifB, which is required for nitrogenase cofactor biogenesis. Expression of a gRNA targeting the coding sequence of NifB decreased nifB transcript abundance >85% and impaired but did not abolish growth of M. acetivorans with N2. Finally, to ascertain the ability to study gene regulation using CRISPRi-dCas9, nrpR1 encoding a subunit of the repressor of the nif operon was targeted. The nrpR1 repression strain grew normally with N2 but had increased nif operon transcript abundance consistent with a NrpR1 as repressor. These results highlight the utility of the system, whereby a single gRNA when expressed with dCas9 can block transcription of targeted genes and operons in M. acetivorans. IMPORTANCEGenetic tools are needed to understand and manipulate the biology of archaea, which serve critical roles in the biosphere. Methanogenic archaea (methanogens) are essential for the biological production of methane, an intermediate in the global carbon cycle, an important greenhouse gas and a biofuel. The CRISPRi-dCas9 system in the model methanogen M. acetivorans is the first Cas9-based CRISPR interference system in archaea. Results demonstrate that the system is remarkably efficient in targeted gene repression and provide new insight into nitrogen fixation by methanogens, the only archaea with nitrogenase. Overall, the CRISPRi-dCas9 system provides a simple, yet powerful, genetic tool to control the expression of target genes and operons in methanogens.

microbiology↗