Exchange of Vitamin B1 and Its Biosynthesis Intermediates Shapes the Composition of Synthetic Microbial Cocultures and Reveals Complexities of Nutrient Sharing.

Microbial communities occupy diverse niches in nature, and community members routinely exchange a variety of nutrients among themselves. While large-scale metagenomic and metabolomic studies shed some light on these exchanges, the contribution of individual species and the molecular details of specific interactions are difficult to track. In this study, we follow the exchange of vitamin B1 (thiamin) and its intermediates between microbes within synthetic cocultures of Escherichia coli and Vibrio anguillarum. Thiamin contains two moieties, 4-amino-5-hydroxymethyl-2-methylpyrimidine (HMP) and 4-methyl-5-(2-hydroxyethyl)thiazole (THZ), which are synthesized by distinct pathways using enzymes ThiC and ThiG, respectively, and then coupled by ThiE to form thiamin. Even though E. coli ΔthiC, ΔthiE, and ΔthiG mutants are thiamin auxotrophs, we observed that cocultures of ΔthiC-ΔthiE and ΔthiC-ΔthiG mutants are able to grow in a thiamin-deficient medium, whereas the ΔthiE-ΔthiG coculture does not. Further, the exchange of thiamin and its intermediates in V. anguillarum cocultures and in mixed cocultures of V. anguillarum and E. coli revealed that there exist specific patterns for thiamin metabolism and exchange among these microbes. Our findings show that HMP is shared more frequently than THZ, concurrent with previous observations that free HMP and HMP auxotrophy is commonly found in various environments. Furthermore, we observe that the availability of exogenous thiamin in the media affects whether these strains interact with each other or grow independently. These findings collectively underscore the importance of the exchange of essential metabolites as a defining factor in building and modulating synthetic or natural microbial communities. IMPORTANCE Vitamin B1 (thiamin) is an essential nutrient for cellular metabolism. Microorganisms that are unable to synthesize thiamin either fully or in part exogenously obtain it from their environment or via exchanges with other microbial members in their community. In this study, we created synthetic microbial cocultures that rely on sharing thiamin and its biosynthesis intermediates and observed that some of them are preferentially exchanged. We also observed that the coculture composition is dictated by the production and/or availability of thiamin and its intermediates. Our studies with synthetic cocultures provide the molecular basis for understanding thiamin sharing among microorganisms and lay out broad guidelines for setting up synthetic microbial cocultures by using the exchange of an essential metabolite as their foundation.

Leveraging an enzyme/artificial substrate system to enhance cellular persulfides and mitigate neuroinflammation.

Persulfides and polysulfides, collectively known as the sulfane sulfur pool along with hydrogen sulfide (H2S), play a central role in cellular physiology and disease. Exogenously enhancing these species in cells is an emerging therapeutic paradigm for mitigating oxidative stress and inflammation that are associated with several diseases. In this study, we present a unique approach of using the cell's own enzyme machinery coupled with an array of artificial substrates to enhance the cellular sulfane sulfur pool. We report the synthesis and validation of artificial/unnatural substrates specific for 3-mercaptopyruvate sulfurtransferase (3-MST), an important enzyme that contributes to sulfur trafficking in cells. We demonstrate that these artificial substrates generate persulfides in vitro as well as mediate sulfur transfer to low molecular weight thiols and to cysteine-containing proteins. A nearly 100-fold difference in the rates of H2S production for the various substrates is observed supporting the tunability of persulfide generation by the 3-MST enzyme/artificial substrate system. Next, we show that the substrate 1a permeates cells and is selectively turned over by 3-MST to generate 3-MST-persulfide, which protects against reactive oxygen species-induced lethality. Lastly, in a mouse model, 1a is found to significantly mitigate neuroinflammation in the brain tissue. Together, the approach that we have developed allows for the on-demand generation of persulfides in vitro and in vivo using a range of shelf-stable, artificial substrates of 3-MST, while opening up possibilities of harnessing these molecules for therapeutic applications.