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.

Catabolic Network of the Fermentative Gut Bacterium Phocaeicola vulgatus (Phylum Bacteroidota) from a Physiologic-Proteomic Perspective.

INTRODUCTION: Phocaeicola vulgatus (formerly Bacteroides vulgatus) is a prevalent member of human and animal guts, where it influences by its dietary-fiber-fueled, fermentative metabolism the microbial community as well as the host health. Moreover, the fermentative metabolism of P. vulgatus bears potential for a sustainable production of bulk chemicals. The aim of the present study was to refine the current understanding of the P. vulgatus physiology. METHODS: P. vulgatus was adapted to anaerobic growth with 14 different carbohydrates, ranging from hexoses, pentoses, hemicellulose, via an uronic acid to deoxy sugars. These substrate-adapted cells formed the basis to define the growth stoichiometries by quantifying growth/fermentation parameters and to reconstruct the catabolic network by applying differential proteomics. RESULTS: The determination of growth performance revealed, e.g., doubling times (h) from 1.39 (arabinose) to 14.26 (glucuronate), biomass yields (gCDW/mmolS) from 0.01 (fucose) to 0.27 (α-cyclodextrin), and ATP yields (mMATP/mMC) from 0.21 (rhamnose) to 0.60 (glucuronate/xylan). Furthermore, fermentation product spectra were determined, ranging from broad and balanced (with xylan: acetate, succinate, formate, and propanoate) to rather one sided (with rhamnose or fucose: mainly propane-1,2-diol). The fermentation network serving all tested compounds is composed of 56 proteins (all identified), with several peripheral reaction sequences formed with high substrate specificity (e.g., conversion of arabinose to d-xylulose-3-phosphate) implicating a fine-tuned regulation. By contrast, central modules (e.g., glycolysis or the reaction sequence from PEP to succinate) were constitutively formed. Extensive formation of propane-1,2-diol from rhamnose and fucose involves rhamnulokinase (RhaB), rhamnulose-1-phosphate kinase (RhaD), and lactaldehyde reductase (FucO). Furthermore, Sus-like systems are apparently the most relevant uptake systems and a complex array of transmembrane electron-transfer systems (e.g., Na+-pumping Rnf and Nqr complexes, fumarate reductase) as well as F- and V-type ATP-synthases were detected. CONCLUSIONS: The present study provides insights into the potential contribution of P. vulgatus to the gut metabolome and into the strain's biotechnological potential for sustainable production of short-chain fatty acids and alcohols.