Methanogens use an unusual energy-conserving electron transport chain that involves reduction of a limited number of electron acceptors to methane gas. Previous biochemical studies suggested that the proton-pumping F 420H2 dehydrogenase (Fpo) plays a crucial role in this process during growth on methanol. However, Methanosarcina barkeri ⌬fpo mutants constructed in this study display no measurable phenotype on this substrate, indicating that Fpo plays a minor role, if any. In contrast, ⌬frh mutants lacking the cytoplasmic F 420-reducing hydrogenase (Frh) are severely affected in their ability to grow and make methane from methanol, and double ⌬fpo/⌬frh mutants are completely unable to use this substrate. These data suggest that the preferred electron transport chain involves production of hydrogen gas in the cytoplasm, which then diffuses out of the cell, where it is reoxidized with transfer of electrons into the energy-conserving electron transport chain. This hydrogen-cycling metabolism leads directly to production of a proton motive force that can be used by the cell for ATP synthesis. Nevertheless, M. barkeri does have the flexibility to use the Fpo-dependent electron transport chain when needed, as shown by the poor growth of the ⌬frh mutant. Our data suggest that the rapid enzymatic turnover of hydrogenases may allow a competitive advantage via faster growth rates in this freshwater organism. The mutant analysis also confirms the proposed role of Frh in growth on hydrogen/carbon dioxide and suggests that either Frh or Fpo is needed for aceticlastic growth of M. barkeri.hydrogen electron transport ͉ F420 ͉ H2 cycling ͉ methanogenesis

The production of biofuels from lignocellulose yields a substantial lignin by-product stream that currently has few applications. Biological conversion of lignin-derived compounds into chemicals and fuels has the potential to improve the economics of lignocellulose-derived biofuels, but few microbes are able both to catabolize lignin-derived aromatic compounds and to generate valuable products. While Escherichia coli has been engineered to produce a variety of fuels and chemicals, it is incapable of catabolizing most aromatic compounds. Therefore, we engineered E. coli to catabolize protocatechuate, a common intermediate in lignin degradation, as the sole source of carbon and energy via heterologous expression of a nine-gene pathway from Pseudomonas putida KT2440. We next used experimental evolution to select for mutations that increased growth with protocatechuate more than 2-fold. Increasing the strength of a single ribosome binding site in the heterologous pathway was sufficient to recapitulate the increased growth. After optimization of the core pathway, we extended the pathway to enable catabolism of a second model compound, 4-hydroxybenzoate. These engineered strains will be useful platforms to discover, characterize, and optimize pathways for conversions of ligninderived aromatics.IMPORTANCE Lignin is a challenging substrate for microbial catabolism due to its polymeric and heterogeneous chemical structure. Therefore, engineering microbes for improved catabolism of lignin-derived aromatic compounds will require the assembly of an entire network of catabolic reactions, including pathways from genetically intractable strains. Constructing defined pathways for aromatic compound degradation in a model host would allow rapid identification, characterization, and optimization of novel pathways. We constructed and optimized one such pathway in E. coli to enable catabolism of a model aromatic compound, protocatechuate, and then extended the pathway to a related compound, 4-hydroxybenzoate. This optimized strain can now be used as the basis for the characterization of novel pathways.KEYWORDS lignin, protocatechuic acid, ortho-cleavage pathway, experimental evolution, synthetic biology, ligninolysis, metabolic engineering, ortho-cleavage B iofuels derived from lignocellulose will likely play an important role in the transition to a sustainable and carbon-neutral economy (1). In a typical biotransformation, carbohydrate-rich cellulose and hemicellulose are extracted and fermented to yield the desired biofuel, such as bioethanol. The lignin, comprising up to 25% of the dry

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Citizen science is becoming even more accessible to the general public through technological advances in the development of mobile applications, facilitating information dissemination and data collection. With the advent of “big data,” many citizen-science projects designed to help researchers sift through piles of research data now exist entirely online, either in the form of playing a game or via other digital avenues. Recent trends in citizen science have also focused on “crowdsourcing” solutions from the general public to help solve societal issues, often requiring nothing more than brainstorming and a computer to submit ideas. Online citizen science thus provides an excellent platform to expand the accessibility of experiential learning opportunities for a broad range of nonmajor science students at institutions with limited resources (e.g., community colleges). I created an activity for a general microbiology lecture to engage students in hands-on experiences via participation in online citizen-science projects. The objectives of the assignment were for students to: 1) understand that everyone can be a scientist; 2) learn to be creative and innovative in designing solutions to health and science challenges; and 3) further practice science communication skills with a written report. This activity is designed for introductory science courses with nonmajor science students who have limited opportunities to participate in undergraduate research experiences.

Aims:  To develop a defined medium for Clostridium scatologenes ATCC 25775, which produces the malodorants 3‐methylindole (skatole) and 4‐methylphenol (p‐cresol). Methods and Results:  Clostridium scatologenes was cultured in anaerobic broth medium (pH 6·3) at 37°C containing ammonia, minerals and a commercial vitamin solution. Data indicate α‐ketoglutarate, l‐glutamate or l‐glutamine is a required nutrient that can also serve as a primary carbon and energy source. When cultured in defined medium containing glutamate; glucose, fructose and betaine served as primary carbon and energy sources. l‐Tryptophan, l‐tyrosine, sorbitol and indole acetic acid did not enhance growth. In the absence of tryptophan, cells produced indole when grown using glucose or fructose. 4‐Methylphenol was produced when growing cells were supplied with tyrosine. When supplied with tryptophan, 3‐methylindole was produced by glucose‐ or fructose‐growing cells but not from glutamate‐growing cells. Cells grown in the presence of pyruvate produced indole, 3‐methylindole and 4‐methylphenol. Conclusions:  Clostridium scatologenes requires α‐ketoglutarate, l‐glutamate, or l‐glutamine for growth in defined medium. Cells produce indole when glucose or fructose is included in defined medium. Significance and Impact of the Study:  The development of a defined medium will assist in physiology studies and genetic analysis of this strain.