Ligand cross-feeding resolves bacterial vitamin B12 auxotrophies.

Cobalamin (vitamin B12, herein referred to as B12) is an essential cofactor for most marine prokaryotes and eukaryotes1,2. Synthesized by a limited number of prokaryotes, its scarcity affects microbial interactions and community dynamics2-4. Here we show that two bacterial B12 auxotrophs can salvage different B12 building blocks and cooperate to synthesize B12. A Colwellia sp. synthesizes and releases the activated lower ligand α-ribazole, which is used by another B12 auxotroph, a Roseovarius sp., to produce the corrin ring and synthesize B12. Release of B12 by Roseovarius sp. happens only in co-culture with Colwellia sp. and only coincidently with the induction of a prophage encoded in Roseovarius sp. Subsequent growth of Colwellia sp. in these conditions may be due to the provision of B12 by lysed cells of Roseovarius sp. Further evidence is required to support a causative role for prophage induction in the release of B12. These complex microbial interactions of ligand cross-feeding and joint B12 biosynthesis seem to be widespread in marine pelagic ecosystems. In the western and northern tropical Atlantic Ocean, bacteria predicted to be capable of salvaging cobinamide and synthesizing only the activated lower ligand outnumber B12 producers. These findings add new players to our understanding of B12 supply to auxotrophic microorganisms in the ocean and possibly in other ecosystems.

Vitamin B12 is not shared by all marine prototrophic bacteria with their environment.

Vitamin B12 (cobalamin, herein B12) is an essential cofactor involved in amino acid synthesis and carbon resupply to the TCA cycle for most prokaryotes, eukaryotic microorganisms, and animals. Despite being required by most, B12 is produced by only a minor fraction of prokaryotes and therefore leads to complex interaction between prototrophs and auxotrophs. However, it is unknown how B12 is provided by prototrophs to auxotrophs. In this study, 33 B12 prototrophic alphaproteobacterial strains were grown in co-culture with Thalassiosira pseudonana, a B12 auxotrophic diatom, to determine the bacterial ability to support the growth of the diatom by sharing B12. Among these strains, 18 were identified to share B12 with the diatom, while nine were identified to retain B12 and not support growth of the diatom. The other bacteria either shared B12 with the diatom only with the addition of substrate or inhibited the growth of the diatom. Extracellular B12 measurements of B12-provider and B12-retainer strains confirmed that the cofactor could only be detected in the environment of the tested B12-provider strains. Intracellular B12 was measured by LC-MS and showed that the concentrations of the different B12-provider as well as B12-retainer strains differed substantially. Although B12 is essential for the vast majority of microorganisms, mechanisms that export this essential cofactor are still unknown. Our results suggest that a large proportion of bacteria that can synthesise B12 de novo cannot share the cofactor with their environment.

Simultaneous quantification of all B vitamins and selected biosynthetic precursors in seawater and bacteria by means of different mass spectrometric approaches.

B vitamins have high microbiological relevance in the marine environment, but their very low concentrations and the chemical heterogeneity of the individual vitamins make their analysis challenging. Mass spectrometric analysis of B vitamins in environmental samples at trace levels has mainly been performed using triple quadrupole mass spectrometers operated in targeted analysis mode. The development of such a method can be laborious and error prone. Additionally, high-resolution mass spectrometers can be used to measure a sample in full scan mode and subsequently search the total ion current chromatogram for extracted ion chromatograms of targeted vitamins. Three different analytical approaches for trace analysis of all B vitamins and some of their biosynthetic precursors were optimized and compared on two different mass spectrometers. A triple quadrupole mass spectrometer in selected reaction monitoring mode, and a high-resolution orbitrap mass spectrometer in parallel reaction monitoring, as well as in full scan mode were employed. Detection limits down to 10 ng/L were achieved with all three techniques. The methods were applied to a marine water sample from the North Sea and to the cell extract of a bacterial culture of Phaeobacter inhibens. Most vitamins and precursors were found in the bacterial cell extract and the seawater sample with all three measuring methods. The results of this study emphasize that, in addition to tandem mass spectrometry, high-resolution full scan mass spectrometry is a promising technique for the simultaneous detection of structurally diverse B vitamins in complex natural samples. This enables highly sensitive measurements without loss of detailed mass spectrometric information, which is inevitable when using a triple quadrupole system in MS/MS mode.

Selection, drift and community interactions shape microbial biogeographic patterns in the Pacific Ocean.

Despite accumulating data on microbial biogeographic patterns in terrestrial and aquatic environments, we still lack a comprehensive understanding of how these patterns establish, in particular in ocean basins. Here we show the relative significance of the ecological mechanisms selection, dispersal and drift for shaping the composition of microbial communities in the Pacific Ocean over a transect of 12,400 km between subantarctic and subarctic regions. In the epipelagic, homogeneous selection contributes 50-60% and drift least to the three mechanism for the assembly of prokaryotic communities whereas in the upper mesopelagic, drift is relatively most important for the particle-associated subcommunities. Temperature is important for the relative significance of homogeneous selection and dispersal limitation for community assembly. The relative significance of both mechanisms was inverted with increasing temperature difference along the transect. For eukaryotes >8 µm, homogeneous selection is also the most important mechanisms at two epipelagic depths whereas at all other depths drift is predominant. As species interactions are essential for structuring microbial communities we further analyzed co-occurrence-based community metrics to assess biogeographic patterns over the transect. These interaction-adjusted indices explained much better variations in microbial community composition as a function of abiotic and biotic variables than compositional or phylogenetic distance measures like Bray-Curtis or UniFrac. Our analyses are important to better understand assembly processes of microbial communities in the upper layers of the largest ocean and how they adapt to effectively perform in global biogeochemical processes. Similar principles presumably act upon microbial community assembly in other ocean basins.

The overlooked role of a biotin precursor for marine bacteria - desthiobiotin as an escape route for biotin auxotrophy.

Biotin (vitamin B7) is involved in a wide range of essential biochemical reactions and a crucial micronutrient that is vital for many pro- and eukaryotic organisms. The few biotin measurements in the world's oceans show that availability is subject to strong fluctuations. Numerous marine microorganisms exhibit biotin auxotrophy and therefore rely on supply by other organisms. Desthiobiotin is the primary precursor of biotin and has recently been detected at concentrations similar to biotin in seawater. The last enzymatic reaction in the biotin biosynthetic pathway converts desthiobiotin to biotin via the biotin synthase (BioB). The role of desthiobiotin as a precursor of biotin synthesis in microbial systems, however, is largely unknown. Here we demonstrate experimentally that bacteria can overcome biotin auxotrophy if they retain the bioB gene and desthiobiotin is available. A genomic search of 1068 bacteria predicts that the biotin biosynthetic potential varies greatly among different phylogenetic groups and that 20% encode solely bioB and thus can potentially overcome biotin auxotrophy. Many Actino- and Alphaproteobacteria cannot synthesize biotin de novo, but some possess solely bioB, whereas the vast majority of Gammaproteobacteria and Flavobacteriia exhibit the last four crucial biotin synthesis genes. We detected high intra- and extracellular concentrations of the precursor relative to biotin in the prototrophic bacterium, Vibrio campbellii, with extracellular desthiobiotin reaching up to 1.09 ± 0.15*106 molecules per cell during exponential growth. Our results provide evidence for the ecological role of desthiobiotin as an escape route to overcome biotin auxotrophy for bacteria in the ocean and presumably in other ecosystems.

Availability of vitamin B12 and its lower ligand intermediate α-ribazole impact prokaryotic and protist communities in oceanic systems.

Genome analyses predict that the cofactor cobalamin (vitamin B12, called B12 herein) is produced by only one-third of all prokaryotes but almost all encode at least one B12-dependent enzyme, in most cases methionine synthase. This implies that the majority of prokaryotes relies on exogenous B12 supply and interacts with producers. B12 consists of a corrin ring centred around a cobalt ion and the lower ligand 5'6-dimethylbenzimidazole (DMB). It has never been tested whether availability of this pivotal cofactor, DMB or its intermediate α-ribazole affect growth and composition of prokaryotic microbial communities. Here we show that in the subtropical, equatorial and polar frontal Pacific Ocean supply of B12 and α-ribazole enhances heterotrophic prokaryotic production and alters the composition of prokaryotic and heterotrophic protist communities. In the polar frontal Pacific, the SAR11 clade and Oceanospirillales increased their relative abundances upon B12 supply. In the subtropical Pacific, Oceanospirillales increased their relative abundance upon B12 supply as well but also downregulated the transcription of the btuB gene, encoding the outer membrane permease for B12. Surprisingly, Prochlorococcus, known to produce pseudo-B12 and not B12, exhibited significant upregulation of genes encoding key proteins of photosystem I + II, carbon fixation and nitrate reduction upon B12 supply in the subtropical Pacific. These findings show that availability of B12 and α-ribazole affect growth and composition of prokaryotic and protist communities in oceanic systems thus revealing far-reaching consequences of methionine biosynthesis and other B12-dependent enzymatic reactions on a community level.

Does the Chemodiversity of Bacterial Exometabolomes Sustain the Chemodiversity of Marine Dissolved Organic Matter?

Marine dissolved organic matter (DOM) is a complex mixture of chemical compounds. At 750 Pg C, it is one of the biggest pools of reduced carbon on Earth. It has been proposed that the diversity of DOM is responsible for its recalcitrance. We hypothesize that the chemodiversity of marine DOM is a reflection of the chemodiversity of bacterial exometabolomes. To test this, we incubated two model strains of the Roseobacter group; Phaeobacter inhibens and Dinoroseobacter shibae in pure culture using three different simple organic compounds as sole carbon sources (glutamate, glucose, and acetate and succinate for P. inhibens and D. shibae, respectively). The exometabolome of the model organisms was characterized using Fourier Transform Ion Cyclotron Resonance Mass Spectrometry (FT-ICR-MS) and ecological diversity measures. We detected thousands of molecular masses in the exometabolomes of P. inhibens and D. shibae (21,105 and 9,386, respectively), reflecting the capability of single bacterial strains to diversify simple organic compounds. The chemical composition of the exometabolomes changed with growth phase and also differed according to the strain incubated and the utilized substrate. We mimicked a higher diversity of substrates, bacterial species and heterogeneous growth (different growth phases) to approach the complexity of natural environments, by computationally creating combinations of detected exometabolomes. We compared the chemodiversity of these combinations, indicative for chemodiversity of freshly produced microbial DOM to that of refractory DOM from one of the oldest oceanic water masses (North Equatorial Pacific Intermediate Water). Some combinations of exometabolomes showed higher richness than the deep ocean refractory DOM, and all the combinations showed higher functional diversity. About 15% of the 13,509 molecular formulae detected in exometabolomes and refractory oceanic DOM were shared, i.e., occurred in Roseobacter exometabolomes and in deep water samples. This overlap provides further support for our hypothesis that marine bacteria from the Roseobacter group contribute to the sustainability of marine DOM chemodiversity and stability.

The Exometabolome of Two Model Strains of the Roseobacter Group: A Marketplace of Microbial Metabolites.

Recent studies applying Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS) showed that the exometabolome of marine bacteria is composed of a surprisingly high molecular diversity. To shed more light on how this diversity is generated we examined the exometabolome of two model strains of the Roseobacter group, Phaeobacter inhibens and Dinoroseobacter shibae, grown on glutamate, glucose, acetate or succinate by FT-ICR-MS. We detected 2,767 and 3,354 molecular formulas in the exometabolome of each strain and 67 and 84 matched genome-predicted metabolites of P. inhibens and D. shibae, respectively. The annotated compounds include late precursors of biosynthetic pathways of vitamins B1, B2, B5, B6, B7, B12, amino acids, quorum sensing-related compounds, indole acetic acid and methyl-(indole-3-yl) acetic acid. Several formulas were also found in phytoplankton blooms. To shed more light on the effects of some of the precursors we supplemented two B1 prototrophic diatoms with the detected precursor of vitamin B1 HET (4-methyl-5-(ß-hydroxyethyl)thiazole) and HMP (4-amino-5-hydroxymethyl-2-methylpyrimidine) and found that their growth was stimulated. Our findings indicate that both strains and other bacteria excreting a similar wealth of metabolites may function as important helpers to auxotrophic and prototrophic marine microbes by supplying growth factors and biosynthetic precursors.