Supplementary Material for: Global growth phase response of the gut bacterium Phocaeicola vulgatus (phylum Bacteroidota)

Phocaeicola vulgatus belongs to the intestinal microbiome, where it fermentatively breaks down of food-derived biopolymers , thereby, contributing to the gut metabolome. Moreover, due to its product range, P. vulgatus is a potential nonstandard platform organism for sustainable production of basic organic chemicals. Complementing a recent physiologic-proteomic report deciphering the strain's versatile fermentation network [1], the present study focuses on the global growth phase-dependent response.

P. vulgatus was anaerobically cultivated with glucose in process-controlled bioreactors. Close sampling was conducted to measure growth parameters (OD, CDW, ATP content, substrate/product profiles) to determine growth stoichiometry. A coarser sampling (½ODmax, ODmax, and ODstat) served the molecular analysis of the global growth phase-dependent response, applying proteomics (soluble and membrane fractions, nanoLC-ESI-MS/MS) and targeted/untargeted metabolome analyses.

The determined growth performance of features 1.74 h doubling time, 0.06 gCDW/mmolGlc biomass yield, 0.36 (succinate) and 0.61 (acetate) mmolP/mmolGlc as predominant fermentation product yields, and 0.43 mmolATP/mmolC as theoretically calculated ATP yield. The fermentation pathway displayed growth phase-dependent dynamics: the levels of proteins and their accompanying metabolites constituting the upper part of glycolysis peaked at ½ODmax, whereas those of the lower part of glycolysis and of the fermentation routes in particular towards predominant acetate and succinate were highest at ODmax and ODstat. While identified proteins of monomer biosynthesis displayed rather unspecific profiles, most of the intracellular amino acids peaked at ODmax. By contrast, proteins and metabolites related to stress response and quorum sensing showed increased abundances at ODmax and ODstat. The composition of the exometabolome expanded from 2,317 molecular formulas at ½ODmax via 4,258 at ODmax to 4,501 at ODstat, with growth phase-specific subsets increasing in parallel.

The present study provides insights into the distinct growth phase-dependent behavior of P. vulgatus during cultivation in bioreactors. This could serve as a valuable knowledge base for further developing P. vulgatus as a non-conventional platform organism for biotechnological applications. In addition, the findings shed new light on the potential growth phase-dependent imprints of P. vulgatus on the gut microbiome environment, e.g. by indicating interactions via quorum sensing and by unraveling the complex exometabolic background against which fermentation products and secondary metabolites are formed.