Biology subjects

Shalvarjian, K. E.

Publications and source records attributed to Shalvarjian, K. E..

6 recordsLinked to original sources

bioRxiv2025

Genus-specific remodeling of carbon and energy metabolism facilitates acetoclastic methanogenesis in Methanosarcina spp. and Methanothrix spp.

Methanogenic archaea (methanogens) are microorganisms that obligately produce methane as a byproduct of their energy metabolism. While most methanogens grow on CO2+H2, isolates of the Genus Methanosarcina and Methanothrix can use acetate as the sole substrate for methanogenesis. Methanogenic growth on acetate, i.e., acetoclastic methanogenesis, is hypothesized to require two distinct genetic modules: one for the activation of acetate to acetyl-CoA and the other for producing a chemiosmotic gradient using electrons derived from ferredoxin. In Methanosarcina spp., the activation of acetate to acetyl-CoA is mediated by acetate kinase (Ack) and phosphotransacetylase (Pta) whereas Methanothrix spp. encode AMP-forming acetyl-CoA synthetases (Acs). The Rhodobacter nitrogen fixation complex (Rnf) or Energy converting hydrogenase (Ech) are critical for energy conservation in Methanosarcina spp. during growth on acetate, and a F420:phenazine oxidoreductase-like complex (Fpo) likely plays an analogous role in Methanothrix spp. Here, we tested the proposed modularity of these pathways to facilitate acetoclastic methanogenesis. First, we surveyed over a hundred genomes within the Class Methanosarcinia to show that the genomic potential for acetoclastic methanogenesis using distinct combinations of modules is widespread. We then used the genetically tractable strain, Methanosarcina acetivorans, to build all modular combinations for acetoclastic methanogenesis. Our results indicate that Acs, while functional, cannot replace Ack+Pta to rescue acetate growth in M. acetivorans. Similarly, the Fpo bioenergetic complex cannot replace Rnf. As such, our work suggests that, in addition to horizontal gene transfer of core catabolic modules, acetoclastic metabolism in methanogens requires changes in energetic modules too. IMPORTANCEA large fraction of biogenic methane is derived from acetate, yet acetoclastic methanogens, i.e., methanogens that grow on acetate, remain poorly characterized due to their slow growth. Two groups of methanogens, Methanosarcina spp. and Methanothrix spp., perform acetoclastic methanogenesis using distinct sets of genes for acetate activation and energy conservation. It is widely hypothesized that these genetic modules from Methanosarcina spp. and Methanothrix spp. are functionally analogous and would thus be interchangeable. To test this hypothesis, we engineered different combinations of modules for acetoclastic growth in Methanosarcina acetivorans. Our results challenge this hypothesized paradigm of modularity, and we posit that other changes to the carbon and electron transfer pathways were crucial for the emergence of acetoclastic methanogenesis.

microbiology↗

bioRxiv2025

The Ambiguous Genetic Code of Methanogenic Archaea that Grow on Methylamines

Natural genetic code expansion is a phenomenon wherein an additional amino acid is encoded by a stop codon. These non-standard amino acids are beneficial as they facilitate novel biochemical reactions. However, code expansion leads to ambiguity at the recoded stop codon, which can either be read through or terminated. Pyrrolysine (Pyl) is encoded by the amber codon (TAG/UAG) and is widespread in archaea, where it is required for methylamine-mediated methanogenesis, an environmentally important metabolism. Mechanisms to conditionally suppress the amber stop codon for Pyl installation during protein synthesis have not been identified. Using the model methanogen, Methanosarcina acetivorans, we demonstrate that Pyl-encoding archaea maintain an ambiguous genetic code wherein UAG encodes dual meaning as stop and Pyl. Our data suggest that expression of Pyl biosynthesis and incorporation genes is tuned to the cellular demand for Pyl, which allows these archaea to navigate ambiguous stop decoding in response to environmental cues.

microbiology↗

bioRxiv2025

Methanogenesis marker 16 metalloprotein is the primary coenzyme M synthase in Methanosarcina acetivorans

2-mercaptoethanesulfonate (Coenzyme M, CoM) is an organic sulfur-containing cofactor used for hydrocarbon metabolism in Archaea and Bacteria. In Archaea, CoM serves as an alkyl group carrier for enzymes belonging to the alkyl-CoM reductase family, including methyl-CoM reductase, which catalyzes methane formation in methanogens. Two pathways for the biosynthesis of CoM are present in methanogenic archaea. The initial steps of these pathways are distinct but the last two reactions, leading up to CoM formation, are universally conserved. The final step is proposed to be mediated by methanogenesis marker metalloprotein 16 (MMP16), a putative sulfurtransferase, that replaces the aldehyde group of sulfoacetaldehyde with a thiol to generate CoM. The assignment of MMP16 as CoM synthase (ComF) is not widely accepted as deletion mutants have been shown to grow without any CoM dependence. Here, we investigate the role of MMP16 in the model methanogen, Methanosarcina acetivorans. We show that a mutant lacking MMP16 has a CoM-dependent growth phenotype and a global transcriptomic profile reflective of CoM-starvation. Additionally, the {Delta}MMP16 mutant is a CoM auxotroph in sulfide-free medium. These data reinforce prior claims that MMP16 is a bona fide ComF but point to backup pathway(s) that can conditionally compensate for its absence. We found that L- aspartate semialdehyde sulfurtransferase (L-ASST), catalyzing a sulfurtransferase reaction during homocysteine biosynthesis in methanogens, is potentially involved in genetic compensation of the MMP16 deletion. Even though, both, L-ASST and MMP16 are members of the COG1900 family, site-directed mutagenesis of conserved cysteine residues implicated in catalysis reveal that the underlying reaction mechanisms may be distinct. Altogether, we have provided concrete evidence that MMP16 is the primary ComF in methanogenic archaea. Author SummaryMethane is a high energy renewable fuel that is the primary constituent of natural gas and a potent greenhouse gas. A significant fraction of global methane emissions is generated by the activity of methanogenic archaea. These microorganisms use a cofactor called Coenzyme M (CoM) as a methyl carrier for methane production mediated by the enzyme Methyl-Coenzyme M reductase. Since methane production is essential for energy conservation in methanogens, they need to synthesize or import CoM. Accordingly, most methanogens encode either one of two CoM biosynthesis pathways. Methanogenesis marker 16 metalloprotein (MMP16) is proposed to catalyze the last step of CoM biosynthesis in both pathways however experimental evidence to this effect is lacking. Here we demonstrate that MMP16 is, indeed, the primary CoM synthase (ComF) in the model methanogen, Methanosarcina acetivorans. Since MMP16 is uniquely and widely distributed in methanogens, it can serve as an ideal candidate for the design of anti- methanogenic chemical inhibitors.

microbiology↗

bioRxiv2022

An Archaea-Specific c-type Cytochrome Maturation Machinery is Crucial for Methanogenesis in Methanosarcina acetivorans

C-type cytochromes (cyt c) are proteins that covalently bind heme and are integral to electron transport chains. A growing body of evidence suggests that cyt c play a vital role in both intra- and extra-cellular electron transfer processes in Archaea, especially in members that metabolize methane and other short chain alkanes. Elaborate mechanisms for the biogenesis of cyt c are known in Bacteria and Eukarya but this process remains largely uncharacterized in Archaea. Here, we have used the model methanogenic archaeon Methanosarcina acetivorans to characterize a distinct form of the system I cyt c maturation machinery (referred to as the Ccm machinery henceforth) that is broadly distributed in members of the Archaea. Phenotypic analyses of M. acetivorans mutants deficient in essential components of the Ccm machinery reveal that cyt c are broadly important for growth and methanogenesis, but the magnitude of their impact can vary substantially depending on the growth substrate. Heterologous expression of a synthetic operon with the Ccm machinery (CcmABCEF) from M. acetivorans is both necessary and sufficient for cyt c biogenesis in a non-native host (M. barkeri Fusaro) that is incapable of cyt c biogenesis. Even though components of the Ccm machinery are universally conserved across the Archaea, our evolutionary analyses indicate that different clades of Archaea acquired this pathway through multiple independent horizontal gene transfer events from different groups of Bacteria. Overall, we have demonstrated the convergent evolution of a novel Archaea-specific Ccm machinery for cyt c biogenesis and its role in methane metabolism. Significance StatementMicroorganisms belonging to the domain Archaea play an especially important role in regulating atmospheric methane levels. Specifically, methanogens are the primary source of biogenic methane and anaerobic methanotrophic archaea (ANME) consume a substantial proportion of methane released in marine sediments. Genomic studies have implicated a class of electron-transfer proteins called c-type cytochromes as being crucial in mediating archaeal methane metabolism in the environment. However, neither the biogenesis nor the role of c-type cytochromes in methane metabolism has ever been investigated. Here, we have used a model methanogen, Methanosarcina acetivorans, to characterize a distinct pathway for maturation of c-type cytochromes that seems to be uniformly conserved across the Archaea and have also identified substrate-specific functional roles for c-type cytochromes during methanogenesis.

microbiology↗

bioRxiv2021

Methylothon: a versatile course-based high school research experience in microbiology and bioinformatics-- with pink bacteria

Methylothon is an inquiry-based high school learning module in microbial ecology, molecular biology, and bioinformatics that centers around pink-pigmented plant-associated methylotrophic bacteria. Here we present an overview of the modules learning goals, describe course resources (available for public use on http://methylothon.com), and relate lessons learned from adapting Methylothon for remote learning during the pandemic in spring of 2021. The original in-person version of the module allows students to isolate their own strains of methylotrophic bacteria from plants they sample from the environment, to identify these using PCR, sequencing, and phylogenetic analysis, and to contribute their strains to original research in a university lab. The adapted version strengthens the focus on bioinformatics and increases its flexibility and accessibility by making the lab portion optional and adopting free web-based tools. Student feedback and graded assignments from Spring 2021 revealed that the lesson was especially effective at introducing the concepts of BLAST and phylogenetic trees, and that students valued and felt inspired by the opportunity to conduct hands-on work and to participate in community science.

scientific communication and education↗

bioRxiv2020

Genome-wide identification of tomato xylem sap fitness factors for Ralstonia pseudosolanacearum and Ralstonia syzygii

Plant pathogenic Ralstonia spp. colonize plant xylem and cause wilt diseases on a broad range of host plants. To identify genes that promote growth of diverse Ralstonia strains in xylem sap from tomato plants, we performed genome-scale genetic screens (random barcoded transposon mutant sequencing screens; RB-TnSeq) in three strains spanning the genetic, geographical, and physiological range of plant pathogenic Ralstonia: R. solanacearum IBSBF1503, R. pseudosolanacearum GMI1000, and R. syzygii PSI07. Contrasting mutant fitness phenotypes in culture media versus in xylem sap suggest that Ralstonia strains are adapted to ex vivo xylem sap and that culture media impose foreign selective pressures. Although wild-type Ralstonia grew in sap and in rich medium with similar doubling times and to a similar carrying capacity, more genes were essential for growth in sap than in rich medium. Each strain required many genes associated with envelope remodeling and repair processes for full fitness in xylem sap. These genes were associated with peptidoglycan peptide formation (murI), secretion of periplasmic proteins (tatC), periplasmic protein folding (dsbA), synthesis of osmoregulated periplasmic glucans (mdoGH), and LPS biosynthesis. Mutants in four genes had strong, sap-specific fitness defects in all strain backgrounds: murI, thiC, purU, and a lipoprotein (RSc2007). Many amino acid biosynthesis genes were required for fitness in both minimal medium and xylem sap. Multiple mutants with insertions in virulence regulators had gains-of-fitness in culture media and neutral fitness in sap. Our genome-scale genetic screen identified Ralstonia fitness factors that promote growth in xylem sap, an ecologically relevant condition. ImportanceTraditional transposon mutagenesis genetic screens pioneered molecular plant pathology and identified core virulence traits like the type III secretion system. TnSeq approaches that leverage next-generation sequencing to rapidly quantify transposon mutant phenotypes are ushering in a new wave of biological discovery. Here we have adapted a genome-scale approach, random barcoded transposon mutant sequencing (RB-TnSeq), to discover fitness factors that promote growth of three related bacterial strains in a common niche, tomato xylem sap. Fitness of wild-type and mutants show that Ralstonia spp. are adapted to grow well in xylem sap from their natural host plant, tomato. Our screen identified multiple sap-specific fitness factors with roles in maintaining the bacterial envelope. These factors include putative adaptations to resist plant defenses that may include antimicrobial proteins and specialized metabolites that damage bacterial membranes.

microbiology↗