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 module's learning goals, describe course resources (available for public use at http://methylothon.com), and relate lessons learned from adapting Methylothon for remote learning during the pandemic in spring of 2021. This curriculum description is intended not only for instructors but also for microbial ecology researchers with an interest in conducting K-12 outreach. 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.

An Archaea-specific c-type cytochrome maturation machinery is crucial for methanogenesis in Methanosarcina acetivorans.

c-Type cytochromes (cyt c) are proteins that undergo post-translational modification to covalently bind heme, which allows them to facilitate redox reactions in electron transport chains across all domains of life. Genomic evidence suggests that cyt c are involved in electron transfer processes among the Archaea, especially in members that produce or consume the potent greenhouse gas methane. However, neither the maturation machinery for cyt c in Archaea nor their role in methane metabolism has ever been functionally characterized. Here, we have used CRISPR-Cas9 genome editing tools to map a distinct pathway for cyt c biogenesis in the model methanogenic archaeon Methanosarcina acetivorans, and have also identified substrate-specific functional roles for cyt c during methanogenesis. Although the cyt c maturation machinery from M. acetivorans is universally conserved in the Archaea, our evolutionary analyses indicate that different clades of Archaea acquired this machinery through multiple independent horizontal gene transfer events from different groups of Bacteria. Overall, we demonstrate the convergent evolution of a novel Archaea-specific cyt c maturation machinery and its physiological role during methanogenesis, a process which contributes substantially to global methane emissions.

Archaea are single-celled organisms that were discovered over half a century ago. Recently, there has been a renewed interest in these microbes because theyplay a key role in climate change by controlling greenhouse gas emissions, like methane. Indeed, methane-producing Archaea generate nearly 70% of the methane gas released into the atmosphere. A group of proteins called c-type cytochromes are essential to energy generation in several methane-producing archaea. However, it is a mystery how Archaea assemble their c-type cytochromes. In fact, genomic studies suggest that Archaea are missing some of the c-type cytochrome assembly machinery that bacteria use. This has led scientists to suspect that Archaea have an alternate mechanism for building these essential components. To solve this mystery, Gupta, Shalvarjian, and Nayak used CRISPR-Cas9 gene-editing tools to characterize which proteins are essential for c-type cytochrome production in Methanosarcina acetivorans, a species of Archaea that produces methane. These experiments showed that M. acetivorans discarded a few parts of the process used by bacteria to generate c-type cytochromes, streamlining the assembly of these proteins. By comparing the genes of different Archaeal species, Gupta, Shalvarjian and Nayak were able to determine that Archaea acquired the genes for producing c-type cytochromes from bacteria via horizontal gene transfer, a process in which genes move directly from one organism into another. The streamlining of the process took place later, as different Archaeal species evolved independently, but losing the same parts of the process. Gupta Shalvajiran and Nayak's experiments also showed that c-type cytochromes are essential for the growth and fitness of methane-producing Archaea like M. acetivorans. The role of c-type cytochromes in methane production varies in different species of Archaea depending on their growth substrate or where they live. These results provide vital information about how Archaea produce methane, and the tools and techniques developed will aid further investigation of the role of Archaea in climate change.

Genome-Wide Identification of Tomato Xylem Sap Fitness Factors for Three Plant-Pathogenic Ralstonia Species.

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: Ralstonia solanacearum IBSBF1503, Ralstonia pseudosolanacearum GMI1000, and Ralstonia 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 lipopolysaccharide (LPS) biosynthesis. Mutant strains with mutations 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. IMPORTANCE Traditional 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 the 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.

Transcriptional regulation of methanogenic metabolism in archaea.

Methanogenesis is a widespread metabolism of evolutionary and environmental importance that is likely to have originated on early Earth. Microorganisms that perform methanogenesis, termed methanogens, belong exclusively to the domain Archaea. Despite maintaining eukaryotic transcription machinery and homologs of bacterial regulators, archaeal transcription and gene regulation appear to be distinct from either domain. While genes involved in methanogenic metabolism have been identified and characterized, their regulation in response to both extracellular and intracellular signals is less understood. Here, we review recent reports on transcriptional regulation of methanogenesis using two model methanogens, Methanococcus maripaludis and Methanosarcina acetivorans, and highlight directions for future research in this nascent field.