Conspicuous chloroplast with light harvesting-photosystem I/II megacomplex in marine Prorocentrum cordatum.

Marine photosynthetic (micro)organisms drive multiple biogeochemical cycles and display a large diversity. Among them, the bloom-forming, free-living dinoflagellate Prorocentrum cordatum CCMP 1329 (formerly P. minimum) stands out with its distinct cell biological features. Here, we obtained insights into the structural properties of the chloroplast and the photosynthetic machinery of P. cordatum using microscopic and proteogenomic approaches. High-resolution FIB/SEM analysis revealed a single large chloroplast (∼40% of total cell volume) with a continuous barrel-like structure, completely lining the inner face of the cell envelope and enclosing a single reticular mitochondrium, the Golgi apparatus, as well as diverse storage inclusions. Enriched thylakoid membrane fractions of P. cordatum were comparatively analyzed with those of the well-studied model-species Arabidopsis (Arabidopsis thaliana) using 2D BN DIGE. Strikingly, P. cordatum possessed a large photosystem-light harvesting megacomplex (>1.5 MDa), which is dominated by photosystems I and II (PSI, PSII), chloroplast complex I, and chlorophyll a-b binding light harvesting complex proteins. This finding parallels the absence of grana in its chloroplast and distinguishes from the predominant separation of PSI and PSII complexes in A. thaliana, indicating a different mode of flux balancing. Except for the core elements of the ATP synthase and the cytb6f-complex, the composition of the other complexes (PSI, PSII, and pigment-binding proteins, PBPs) of P. cordatum differed markedly from those of A. thaliana. Furthermore, a high number of PBPs was detected, accounting for a large share of the total proteomic data (∼65%) and potentially providing P. cordatum with flexible adaptation to changing light regimes.

Multifaceted dinoflagellates and the marine model Prorocentrum cordatum.

BACKGROUND: Dinoflagellates are a monophyletic group within the taxon Alveolata, which comprises unicellular eukaryotes. Dinoflagellates have long been studied for their organismic and morphologic diversity as well as striking cellular features. They have a main size range of 10‒100 µm, a complex "cell covering", exceptionally large genomes (~1‒250 Gbp with a mean of 50,000 protein-encoding genes) spread over a variable number of highly condensed chromosomes, and perform a closed mitosis with extranuclear spindles (dinomitosis). Photosynthetic, marine, and free-living Prorocentrum cordatum is a ubiquitously occurring, bloom-forming dinoflagellate and an emerging model system, particularly with respect to systems biology. SUMMARY: Focused ion beam/scanning electron microscopy (FIB/SEM) analysis of P. cordatum recently revealed (i) a flattened nucleus with unusual structural features and a total of 62 tightly packed chromosomes, (ii) a single, barrel-shaped chloroplast devoid of grana and harboring multiple starch granules, (iii) a single, highly reticular mitochondrion, and (iv) multiple phosphate and lipid storage bodies. Comprehensive proteomics of subcellular fractions suggested (i) major basic nuclear proteins to participate in chromosome condensation, (ii) composition of nuclear pores to differ from standard knowledge, (iii) photosystems I and II, chloroplast complex I, and chlorophyll a-b binding light harvesting complex to form a large megacomplex (>1.5 MDa), and (iv) an extraordinary richness in pigment-binding proteins. Systems biology-level investigation of heat stress response demonstrated a concerted down-regulation of CO2-concentrating mechanisms, CO2-fixation, central metabolism, and monomer biosynthesis, which agrees with reduced growth yields. KEY MESSAGES: FIB/SEM analysis revealed new insights into the remarkable subcellular architecture of P. cordatum, complemented by proteogenomic unravelling of novel nuclear structures and a photosynthetic megacomplex. These recent findings are put in the wider context of current understanding of dinoflagellates.

The blue light-dependent LOV-protein LdaP of Dinoroseobacter shibae acts as antirepressor of the PpsR repressor, regulating photosynthetic gene cluster expression.

In the marine α-proteobacterium Dinoroseobacter shibae more than 40 genes of the aerobic anoxygenic photosynthesis are regulated in a light-dependent manner. A genome-wide screen of 5,605 clones from a D. shibae transposon library for loss of pigmentation and changes in bacteriochlorophyll absorbance identified 179 mutant clones. The gene encoding the LOV-domain containing protein Dshi_1135 was identified by its colorless phenotype. The mutant phenotype was complemented by the expression of a Dshi_1135-strep fusion protein in trans. The recombinantly produced and chromatographically purified Dshi_1135 protein was able to undergo a blue light-induced photocycle mediated by bound FMN. Transcriptome analyses revealed an essential role for Dshi_1135 in the light-dependent expression of the photosynthetic gene cluster. Interactomic studies identified the repressor protein PpsR as an interaction partner of Dshi_1135. The physical contact between PpsR and the Dshi_1135 protein was verified in vivo using the bacterial adenylate cyclase-based two-hybrid system. In addition, the antirepressor function of the Dshi_1135 protein was demonstrated in vivo testing of a bchF-lacZ reporter gene fusion in a heterologous Escherichia coli-based host system. We therefore propose to rename the Dshi_1135 protein to LdaP (light-dependent antirepressor of PpsR). Using the bacterial two-hybrid system, it was also shown that cobalamin (B12) is essential for the interaction of the antirepressor PpaA with PpsR. A regulatory model for the photosynthetic gene cluster in D. shibae was derived, including the repressor PpsR, the light-dependent antirepressor LdaP and the B12-dependent antirepressor PpaA.

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.

Rich microbial and depolymerising diversity in Antarctic krill gut.

With almost a quadrillion individuals, the Antarctic krill processes five million tons of organic carbon every day during austral summer. This high carbon flux requires a broad range of hydrolytic enzymes to decompose the diverse food-derived biopolymers. While krill itself possesses numerous such enzymes, it is unclear, to what extent the endogenous microbiota contribute to the hydrolytic potential of the gut environment. Here we applied amplicon sequencing, shotgun metagenomics, cultivation, and physiological assays to characterize the krill gut microbiota. The broad bacterial diversity (273 families, 919 genera, and 2,309 species) also included a complex potentially anaerobic sub-community. Plate-based assays with 198 isolated pure cultures revealed widespread capacities to utilize lipids (e.g., tributyrin), followed by proteins (casein) and to a lesser extent by polysaccharides (e.g., alginate and chitin). While most isolates affiliated with the genera Pseudoalteromonas and Psychrobacter, also Rubritalea spp. (Verrucomicrobia) were observed. The krill gut microbiota growing on marine broth agar plates possess 13,012 predicted hydrolyses; 15-fold more than previously predicted from a transcriptome-proteome compendium of krill. Cultivation-independent and -dependent approaches indicated members of the families Flavobacteriaceae and Pseudoalteromonadaceae to dominate the capacities for lipid/protein hydrolysis and to provide a plethora of carbohydrate-active enzymes, sulfatases, and laminarin- or porphyrin-depolymerizing hydrolases. Notably, also the potential to hydrolyze plastics such as polyethylene terephthalate and polylactatide was observed, affiliating mostly with Moraxellaceae. Overall, this study shows extensive microbial diversity in the krill gut, and suggests that the microbiota likely play a significant role in the nutrient acquisition of the krill by enriching its hydrolytic enzyme repertoire.IMPORTANCEThe Antarctic krill (Euphausia superba) is a keystone species of the Antarctic marine food web, connecting the productivity of phyto- and zooplankton with the nutrition of the higher trophic levels. Accordingly, krill significantly contributes to biomass turnover, requiring the decomposition of seasonally varying plankton-derived biopolymers. This study highlights the likely role of the krill gut microbiota in this ecosystem function by revealing the great number of diverse hydrolases that microbes contribute to the krill gut environment. The here resolved repertoire of hydrolytic enzymes could contribute to the overall nutritional resilience of krill and to the general organic matter cycling under changing environmental conditions in the Antarctic sea water. Furthermore, the krill gut microbiome could serve as a valuable resource of cold-adapted hydrolytic enzymes for diverse biotechnological applications.

Global Growth Phase Response of the Gut Bacterium Phocaeicola vulgatus (Phylum Bacteroidota).

INTRODUCTION: Phocaeicola vulgatus (basonym Bacteroides vulgatus) belongs to the intestinal microbiome of healthy humans and animals, where it participates in the fermentative breakdown of biopolymers ingested with food. In doing so, P. vulgatus contributes to the shaping of the gut metabolome, which benefits the host health. Moreover, considering the fermentation product range (short chain fatty acids), P. vulgatus suggests itself as a potential nonstandard platform organism for a sustainable production of basic organic chemicals. Complementing a recent physiologic-proteomic report deciphering the strain's versatile fermentation network, the present study focusses on the global growth phase-dependent response of P. vulgatus. METHODS: P. vulgatus was anaerobically cultivated with glucose as sole source of carbon and energy in process-controlled bioreactors operated in parallel. Close sampling was conducted to measure growth parameters (OD, CDW, ATP-content, substrate/product profiles) as basis for determining growth stoichiometry in detail. A coarser sampling (½ODmax, ODmax, and ODstat) served the molecular analysis of the global growth phase-dependent response, studied by means of differential proteomics (soluble and membrane fractions, nanoLC-ESI-MS/MS) as well as targeted metabolite (GC-MS and LC-MS/MS) and untargeted exometabolome (FT-ICR-MS) analyses. RESULTS: The determined growth performance of P. vulgatus 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 distinct 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 toward the predominant products 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. Finally, 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. CONCLUSIONS: The present study provides insights into the distinct growth phase-dependent behavior of P. vulgatus during cultivation in bioreactors on the physiological and molecular levels. This could serve as a valuable knowledge-base for further developing P. vulgatus as a nonconventional 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.