Cross-feeding of Vitamin B1, purines, and their biosynthesis intermediates in Escherichia coli co-cultures

Abstract

A diverse array of microorganisms inhabits majority of the ecosystems on Earth. These microbes are often auxotrophic for essential nutrients and thus are engaged in a dynamic exchange of metabolites with other members in their community. Such a cross-talk gives rise to an intricate web of interactions, which shape the structure and function of microbial communities. The complexity of multiple interactions obscures our ability to understand how a microbial community is formed and sustained, and how its members proliferate to reach a stable composition. Thus, building a synthetic microbial community with a set of defined microbes and proposing hypotheses regarding the interactions, followed by testing which ones hold true using tools in microbiology and analytical biochemistry is a systematic approach to initiate a probe into more complex communities. In this thesis, we analyzed the exchange of Vitamin B1 (thiamin), purines, and their biosynthesis intermediates in microbial communities. In Escherichia coli, thiamin biosynthesis intermediates 4-amino-5-hydroxymethyl-2-methylpyrimidine (HMP) and 4-methyl-5-(2-hydroxyethyl)thiazole (THZ) are synthesized de novo in two distinct branches of the pathway. HMP is synthesized by ThiC in a single step rearrangement reaction from 5’-phosphoribosyl aminoimidazole (AIR), which is also an intermediate shared by the purine biosynthesis pathway. AIR is synthesized by PurM and is further committed to purine biosynthesis by the enzyme PurK. THZ is synthesized by ThiG, which is coupled with HMP by ThiE to form thiamin. To analyze the molecular cross-talk between thiamin and purine biosynthesis pathways, we designed a system based on synthetic co-cultures of E. coli. Towards this goal, we created single gene knockout mutants delta-thiC, delta-thiE, delta-thiG, delta-purK, and delta-purM of E. coli str. K-12 substr. MG1655. We observed that delta-thiC-delta-thiE and delta-thiC-delta-thiG co-cultures of E. coli survived in minimal media in the absence of exogenous thiamin, whereas the delta-thiE-delta-thiG co-culture did not. Further analysis confirmed that the survival of these co-cultures is based on the exchange of HMP and thiamin, whereas THZ exchange in them is limited. We also co-cultured delta-purK and delta-purM mutants with these three thiamin mutants and observed that only the delta-thiC-delta-purK co-culture survived in the minimal medium in the absence of external purine and thiamin supply. These results further support the exchange of HMP in co-cultures of E. coli mutants. We also quantified the ratio of mutants in two- and three-membered co-cultures of thiamin mutants, and observed that it changed in the absence of exogenous thiamin, suggestive of a cross-talk. The percentage of the delta-thiC strain under these conditions increased and stabilized to constitute about 80% of total cells, which could support a population of other cells 1/4th of their own population size. But in the presence of thiamin, their ratios did not deviate much from the starting ratio, indicating that the mutants did not interact. We also visualized the spatial patterns generated by these co-cultures in the presence and absence of thiamin supplementation, and observed that the mutants co-localized in the absence of thiamin, indicating their need for interactions based on metabolite exchange. The findings from this thesis highlight the importance of exchange of essential metabolites in shaping and stabilizing the interactions in microbial communities. Such studies can be used to determine the underlying principles of specific molecular-level interactions and to design synthetic communities for various biotechnological applications.