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.


Preparation and purity verification of α-ribazole
Alpha-ribazole was prepared by alkaline hydrolysis of B12, purified [23] and validated applying nuclear magnetic resonance (NMR) spectra, recorded with a Avance DRX 500 MHz and a Avance III 500 MHz spectrometer (Bruker, Bremen, Germany) at room temperature ( Supplementary Fig. S1). 1 H-and 13 Csignals were assigned using DEPT, H,H-COSY and HMQC experiments. HPLC-UV-ESI-MS was carried out on a Alliance 2695 system (Waters, Eschborn, Germany) equipped with a NUCLEODUR C18 Pyramid column (Macherey-Nagel, Dueren, Germany; particle size 3 µm, length 125 mm, inner diameter 3 mm), a 996 PDA detector and a Micromass Q-ToF-MS (Waters). Water (solvent A) and methanol (solvent B), each acidified with 0.5% formic acid, served as eluents. Run conditions were initially 5% B at a flow rate of 0.8 ml min -1 . Concentration of B was raised linearly to 100% within 10 min and kept for further 6 min. UV-VIS detection was set in a wavelength range from 210 to 650 nm at a scan rate of 1 spectrum per second. The mass spectrometer was run in ESI positive mode covering a m/z range from 150 to 1400 at a scan rate of 1.7 spectra per second. High resolution (about 5 ppm) was achieved using lock spray (sodium formiate solution) for mass calibration.

Microbial community analysis
The composition of the prokaryotic and eukaryotic microbial communities was analysed in all experiments after three hours of the onset and at days 3 and 6. Five hundred ml of water were withdrawn from the mesocosms and concentrated by vacuum filtration on a 0.2 µm polycarbonate filter (Millipore, Burlington, MA, USA), immediately deep-frozen in liquid Nitrogen and stored at − 80 °C. DNA and RNA were extracted simultaneously as described elsewhere [Schneider et al. 2017].
Prokaryotic and eukaryotic microbial communities were analysed targeting the variable regions V3-V4 of the 16S and V9 region of the 18S gene by specific primer sets as indicated in the Materials and Methods section. Extracted DNA (5 ng) was used as template for PCR amplification with each reaction (25 µl) also containing dNTPs (100 µM of each), MgSO4 (1.5 mM), Platinum Taq DNA polymerase HF (0.5 U/reaction), Platinum High Fidelity buffer (1X) (Thermo Fisher Scientific, Waltham, MA, USA) and tailed primer mix (400 nM of each forward and reverse primer). Thermocycling was 95 °C for 2 min, 30 cycles of amplification (95 °C for 15 s, 55 °C for 15 s, 72 °C for 50 s) and finally 72 °C for 5 min. Additionally for the prokaryote communities cDNA was prepared from RNA extracts and used as PCR template with the SuperScript™ IV One-Step RT-PCR system and tailed primer mix (400 nM of each forward and reverse primer). Tails of the forward and reverse primers were designed to be compatible with sequencing according to the standard Illumina protocols. The resulting amplicon libraries were purified using Agencourt Ampure XP Beads (Beckman Coulter, Brea, CA, USA).
Sequencing libraries were prepared from the amplicon libraries using a second PCR reaction each (25 µL) containing PCRBIO HiFi buffer (1x), PCRBIO HiFi Polymerase (1 U/reaction) (PCR Biosystems, London, UK), sequencing adaptor mix (400 nM of each forward and reverse) and up to 10 ng template.
Thermocycling was 95 °C for 2 min, 8 cycles of amplification (95 °C for 20 s, 55 °C for 30 s, 72 °C for 60 s) and finally 72 °C for 5 min. The resulting sequencing libraries were purified using Agencourt Ampure XP Beads. Purified sequencing libraries were pooled in equimolar concentrations, diluted to 2 nM, and paired-end sequenced (2x300 bp) on a MiSeq (Illumina) using a MiSeq Reagent kit v3 (Illumina).
Throughout library preparation the DNA concentrations were measured using Qubit dsDNA HS Assay kit (Thermo Fisher Scientific), and DNA size distributions and purity using Tapestation 2200 with D1000/High sensitivity screentapes (Agilent Technologies, Santa Clara, CA, USA).
All sequencing and bioinformatic analyses were carried out by DNASense (Aalborg, Denmark).

Metagenome and metatranscriptome library preparation and sequencing
For metagenome sequencing, DNA extract concentrations were measured using Qubit (Thermo Fisher Scientific, Waltham, MA, USA ) and DNA fragmented using a Covaris M220 with microTUBE AFA Fiber screw tubes and the settings: Duty Factor 10 %, peak/displayed power 75 W, cycles/burst 200, duration 40 s and temperature 20 °C. The fragmented DNA was used for metagenome preparation using the NEB Next Ultra II DNA library preparation kit, and the resulting libraries were paired-end sequenced (2 x 150 bp) on a HiSeq system (Illumina, San Diego, USA). For metatranscriptome sequencing, RNA extract concentrations were measured using the Qubit HS RNA assay. The RNA quality and integrity were evaluated using TapeStation with RNA ScreenTape (Agilent Technologies, Santa Clara, CA, USA).
Sequencing libraries were prepared using the NEB Next Ultra II RNA library preparation kit (New England Biolabs, Ipswich, MA, USA). Library concentrations were measured using Qubit HS DNA assay and library size distributions using TapeStation D1000 ScreenTapes (Agilent Technologies). The 36 sample libraries were pooled in equimolar concentrations and paired-end sequenced (2 x 150 bp) on a NovaSeq system (Illumina). The library preparation and DNA/RNA sequencing were done by DNASense (Aalborg, Denmark).
Supplementary Table S1 | Vitamin and micronutrient supplementation in the mesocosm experiments M1, M2 and M3. B12 and α-ribazole were added at 100 pM final concentration, Listed are micronutrient additions (), in order to circumvent co-limitations, as well as vitamin B12 and α-ribazole supplementation. Furthermore, temperature and mesocosm duration times of each mesocosm experiment are listed.