Elucidating the role of electron transport to improve the understanding and potential of syntrophic Clostridium co-cultures
Date
2022
Authors
Journal Title
Journal ISSN
Volume Title
Publisher
University of Delaware
Abstract
A better understanding of interactions within microbial communities, both natural and synthetic, is of emerging interest. Elucidating the motivation for exchange of cellular material within microbial communities can help us to prepare for emerging microbial strains of medical interest, and can also help us to better engineer organisms of industrial interest. Clostridium spp. in particular are of interest industrially due to the large variety of substrates that different species can utilize. Most recently, synthetic co-cultures using Clostridium spp. have been developed to maximize the carbon feedstock utilization. One particular pair, Clostridium acetobutylicum and Clostridium ljungdahlii, have shown evidence of exchange of both metabolites and intracellular materials like protein and RNA. To better understand these co-culture interactions, reliable methods for tracking the two species’ populations within the coculture are necessary. Many previous co-culture studies have used fluorescent dyes and fluorescent proteins to track each individual population within the co-culture. However, due to the anaerobic nature of Clostridium spp. traditional fluorescent proteins that require oxygen for fluorophore maturation cannot be used. Here we describe several anaerobic fluorescent proteins that are used to further investigate microbial interactions within co-cultures. ☐ To design fluorescent systems compatible with anaerobic environments, we screened genetic parts like promoters, ribosome binding sites and reporters for engineering bright and stable expression of fluorescent proteins in both C.acetobutylicum and C. ljungdahlii. Successful candidates were then used to tag proteins of interest and observe localization within C. acetobutylicum and C.ljungdahlii cells for a better understanding of these proteins’ functions. With the fluorescent proteins FAST, SNAP-tag and HaloTag, we can further elucidate the mechanisms that may be involved in exchange of intracellular materials within cocultures. ☐ In addition to developing anaerobic environment-compatible fluorescent proteins, a novel anaerobic swim assay was developed to track microbial movement towards gaseous attractants. In co-culture, we hypothesize that C. ljungdahlii can sense waste gases produced by C. acetobutylicum and utilize its flagellar motility to move towards these gaseous attractants. In this study, we show evidence of targeted movement towards gaseous headspace of CO2 and H2, leading us to believe that C.ljungdahlii can utilize chemotaxis to move towards gaseous attractants. We examine several candidates for CO2 sensors and propose novel systems for deletion and further characterization of these proteins of interest.
Description
Keywords
Anaerobic, Clostridium, Fluorescence, Gas sensing, Reporters