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ABSTRACT Efforts toward microbial conversion of lignin to value-added products face many challenges because lignin’s methoxylated aromatic monomers release toxic C₁byproducts such as formaldehyde. The ability to grow on methoxylated aromatic acids (e.g., vanillic acid) has been identified in certain clades of methylotrophs, bacteria characterized by their unique ability to tolerate and metabolize high concentrations of formaldehyde. Here, we use a phyllosphere methylotroph isolate,Methylobacterium extorquensSLI 505, as a model to identify the fate of formaldehyde during methylotrophic growth on vanillic acid.M. extorquensSLI 505 displays concentration-dependent growth phenotypes on vanillic acid without concomitant formaldehyde accumulation. We conclude thatM. extorquensSLI 505 overcomes metabolic bottlenecks from simultaneous assimilation of multicarbon and C₁intermediates by allocating formaldehyde toward dissimilation and assimilating the ring carbons of vanillic acid heterotrophically. We correlate this strategy with maximization of bioenergetic yields and demonstrate that formaldehyde dissimilation for energy generation rather than formaldehyde detoxification is advantageous for growth on aromatic acids.M. extorquensSLI 505 also exhibits catabolite repression during growth on methanol and low concentrations of vanillic acid, but no diauxic patterns during growth on methanol and high concentrations of vanillic acid. Results from this study outline metabolic strategies employed byM. extorquensSLI 505 for growth on a complex single substrate that generates both C₁and multicarbon intermediates and emphasizes the robustness ofM. extorquensfor biotechnological applications for lignin valorization.IMPORTANCELignin, one of the most abundant and renewable carbon sources on Earth, is a promising alternative to non-renewable fossil fuels used to produce petrochemicals. Degradation of lignin releases toxic C₁byproducts such as formaldehyde, and thus most microorganisms are not suitable for biorefining lignin. By contrast,Methylobacterium extorquensSLI 505 is capable of growth on high concentrations of aromatic acids without concomitant formaldehyde accumulation. In addition, we show that the growth ofM. extorquensSLI 505 on aromatic acids is coupled to the production of the bioplastic, polyhydroxybutyrate. Aromatic acids serve as a model by which to understand howM. extorquensSLI 505 balances methylotrophic and heterotrophic pathways during growth to provide strategies for growth optimization when using complex substrates in both ecological and industrial fermentation applications. 

ABSTRACT 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. 

ABSTRACT The phototrophic purple nonsulfur bacterium Rhodopseudomonas palustris is known for its metabolic versatility and is of interest for various industrial and environmental applications. Despite decades of research on R. palustris growth under diverse conditions, patterns of R. palustris growth and carbon utilization with mixtures of carbon substrates remain largely unknown. R. palustris readily utilizes most short-chain organic acids but cannot readily use lactate as a sole carbon source. Here we investigated the influence of mixed-substrate utilization on phototrophic lactate consumption by R. palustris . We found that lactate was simultaneously utilized with a variety of other organic acids and glycerol in time frames that were insufficient for R. palustris growth on lactate alone. Thus, lactate utilization by R. palustris was expedited by its coutilization with additional substrates. Separately, experiments using carbon pairs that did not contain lactate revealed acetate-mediated inhibition of glycerol utilization in R. palustris . This inhibition was specific to the acetate-glycerol pair, as R. palustris simultaneously utilized acetate or glycerol when either was paired with succinate or lactate. Overall, our results demonstrate that (i) R. palustris commonly employs simultaneous mixed-substrate utilization, (ii) mixed-substrate utilization expands the spectrum of readily utilized organic acids in this species, and (iii) R. palustris has the capacity to exert carbon catabolite control in a substrate-specific manner. IMPORTANCE Bacterial carbon source utilization is frequently assessed using cultures provided single carbon sources. However, the utilization of carbon mixtures by bacteria (i.e., mixed-substrate utilization) is of both fundamental and practical importance; it is central to bacterial physiology and ecology, and it influences the utility of bacteria as biotechnology. Here we investigated mixed-substrate utilization by the model organism Rhodopseudomonas palustris . Using mixtures of organic acids and glycerol, we show that R. palustris exhibits an expanded range of usable carbon substrates when provided substrates in mixtures. Specifically, coutilization enabled the prompt consumption of lactate, a substrate that is otherwise not readily used by R. palustris . Additionally, we found that R. palustris utilizes acetate and glycerol sequentially, revealing that this species has the capacity to use some substrates in a preferential order. These results provide insights into R. palustris physiology that will aid the use of R. palustris for industrial and commercial applications.