Abstract
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 dynamics2,3,4. 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.
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Data availability
Complete genome sequences of Colwellia sp. M166 and Roseovarius sp. M141 were uploaded into the NCBI and annotated by JGI Gold. Genomes can be inquired by their IMG genome identifiers 2828890045 for Colwellia and 2828508468 for Roseovarius. Transcriptomic sequences generated in this study have been deposited into the ENA at EMBL-EBI with the accession number PRJEB43320, using the data brokerage service of the GFBio in compliance with the Minimal Information about any (X) Sequence (MIxS) standard. The data from the sampling stations (ANT-XXVIII/4 and ANT-XXVIII/5) are available from PANGEA under the accession number PANGAEA.906247, and respective sequence data can be retrieved from the ENA under the INSDC accession number PRJEB34453. The SILVA and rfam database was used for transcriptome analysis. For bacterial gene annotation, the ProGenome database was used, and for phage gene and protein annotation, the NR database (from the NCBI), the Virus Orthologous Group database, the InterPro database and the HMM database were used. Source data are provided with this paper.
Code availability
No specialized in-house code was used for this study. All software used for the data analyses in this study are publicly available and cited in the Methods. Custom scripts and the pipeline used have been deposited into GitHub (https://github.com/LeonDlugosch/MetaSeq-Toolkit).
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Acknowledgements
We are grateful to C. Alejandre-Colomo, R. Mosseló-Móra, R. Amann and V. Bischoff for providing the North Sea collection of bacterial isolates; B. Kuerzel and M. Wolterink for technical assistance in the HPLC analysis of amino acids and enumerating VLPs; the captains, their crews and the scientific groups of the cruises ANT XXVIII/4 and XXVIII/5 of RV Polarstern and RV Senckenberg for their support; S. Sañudo-Wilhelmy for stimulating discussions regarding B12 analyses; and D. Kirchman for carefully revising a previous version of this publication This work was supported by Deutsche Forschungsgemeinschaft within the Transregional Collaborative Research Center Roseobacter (TRR51) and the Gordon and Betty Moore Foundation (to F.A.).
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G.W. designed the study, carried out the experiments, the genome analyses for B12 biosynthetic pathways, the transcriptomic analyses and wrote the manuscript. C.M. and G.W. carried out the direct-geneFISH analyses for prophage induction and the prophage-related genomic analyses. D.Q.T. contributed to the CARD-FISH analyses. S.B. and H.W. analysed B12. S.S. assisted in the tripartite consortium experiment. L.D. carried out the transcriptome mapping and mapping of taxa with different B12 genetic traits to the Atlantic Ocean microbiome. F.A. partially supervised the co-culture experiments. M.S. assisted in designing the study, partially supervised the experiments and data analyses and wrote the manuscript, together with G.W. All authors revised the manuscript to finalize it.
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Extended data figures and tables
Extended Data Fig. 1 Growth characteristics of Colwellia and Roseovarius when supplemented with and without B12 and respective building blocks and methionine and Chaetoceros muelleri in tri-culture with Colwellia and Roseovarius.
a, Maximum growth yield as means of triplicates ± SD as total cell numbers per ml assessed by flow cytometry of Roseovarius; b, of Colwellia when supplemented with B12 (at 1, 10 or 100 pM) or DMB / Cbi (at 1, 10 or 100 pM); Maximum growth yield as means of triplicates ± SD as determined by optical density of c, Roseovarius and d, Colwellia. Each isolate was supplemented with B12 (100 pM), no addition (plain bar) and methionine (1 µM); e, Chlorophyll a fluorescence and total cell numbers enumerated by hemocytometer of axenic Chaetoceros muelleri over time when cultivated without any addition, f, supplemented with B12 at 10 pM and g, 100 pM final concentration and h, when grown in tri-culture with Colwellia or Roseovarius. Experiments a-d were conducted in n = 3 and experiments e-h in n = 4 biological independent samples each in 1 independent experiment.
Extended Data Fig. 2 Metabolic pathways related to cobalamin biosynthesis and transcript expression patterns of Colwellia and Roseovarius grown in co-culture.
Differential gene expression between the control, represented by the mono-cultures (Roseovarius & Colwellia) with B12 added, and the co-culture, without B12 addition, was quantified with the DESeq2 package as implemented in R v4.0.5 (R-Core-Team, 2022). In each treatment n = 3 biological independent samples were used. Coloring of genes refers to their LOG-2FC regulation, defined as downregulated (light red to red), upregulated (light green to dark green) and similarly regulated (orange). Genes that are significantly regulated (LOG2-FC larger than 2 and smaller than −2 with an BH adj. p-value < 0.05) are marked with an asterisk. When an illustration represents more than one gene (all genes > (-) 2 LOG-2FC and adj. p-value < 0.05), the asterisk is placed in brackets. Genes that were not identified in the genome (grey). Since the bluB gene in Roseovarius is missing a large section of the sequence, we have indicated this by outlining the gene in gray. Corresponding gene expression significances (adjusted p-value) and further details on genes are presented in Supplementary Data 2. Our documented microbial interactions including the coincidence of prophage induction, host cell lysis and release of B12, whose causative relationships still need to be proven, are illustrated and explained by the dashed arrows and the explanations 1–6 in the legend.
Extended Data Fig. 3 Growth and substrate use of Colwellia and Roseovarius when supplemented with B12 building blocks.
Presented are total bacterial cell numbers assessed by flow cytometry and glutamate (glutamic acid) concentrations of a-d, Colwellia and e-h, Roseovarius supplemented with a, e, B12, b, f, DMB and c, g, Cbi at 1 nM final concentrations each and d, h, without any supplementation and of as well as i, Colwellia + Roseovarius when cultivated in co-culture without any addition. Shown are means of triplicates ± SD. Each condition was carried out in n = 3 biological independent samples and growth characteristics were confirmed in independently conducted experiments.
Extended Data Fig. 4 Growth characteristics of Thalassiosira pseudonana and Colwellia and Roseovarius cultivated in consortium.
a, Growth of Colwellia and Roseovarius as monitored by flow cytometric cell counts and calculated by CARD-FISH analyses. Further, numbers of virus-like particles measured by flow cytometry are presented. b, cell numbers of T. pseudonana, assessed microscopically using a hemocytometer, and bacterial cells (black circle) assessed by flow cytometry. c, Total bacterial cell abundance and the ratio of virus-like particle counts to the number of Colwellia. d, Total bacterial cell abundance and the ratio of virus-like particle counts to the number of Roseovarius. e, Relative proportions of Colwellia and Roseovarius cells (% of total abundance) during the culturing time. f. Descriptive illustration of the growth progression of the participating partners of the consortium, T. pseudonana, Roseovarius and Colwellia as well as the phage induction over a period of ten weeks. g, Maximum relative fluorescence of T. pseudonana when cultured with Colwellia and Roseovarius, with addition of 100 pM B12, without any addition, in co-culture with Roseovarius and in co-culture with Colwellia. a-f, Means of triplicates g, and ± SD. Measured values of the respective triplicates can be viewed in the corresponding source data file. Each treatment was carried out in n = 3 biological independent samples and growth characteristics were confirmed in independently conducted tests.
Extended Data Fig. 5 Lower ligand building block concentration in the exudate of Colwellia at B12 (20 pM) depleted growth conditions and in North Sea seawater samples.
a, means of triplicates + SD of growth of Colwellia supplemented with 1 nM B12, 20 pM B12 and no addition, monitored by flow cytometric cell counts over time. Extracellular concentrations of α-ribazole and 5,6-dimethylbenzimidazole (DMB) were measured for the growth-limited culture when supplemented with 20 pM B12. b, concentrations of B12, cobinamide, DMB and α -ribazole in North Sea seawater samples as means of triplicates + SD. c, Map of the German Bight of the North Sea. Points mark the sampling stations of the analyzed seawater samples. Experiment a was conducted in n = 3 biological independent samples. Experiment b was conducted on n = 3 independent sampling locations and each sample was detected in n = 3 technical replicates. Experiments a-b were conducted in 1 independent experiment. The map was created using Ocean Data View.
Extended Data Fig. 6 Predicted cobamide biosynthesis phenotypes in genomes of marine bacteria and their relative abundance in the Atlantic Ocean.
a, Map showing 22 stations (black dots) from 62°S in the Southern Ocean sector to 47°N in the north Atlantic Ocean sampled at 20 m depth during cruises ANTXXVIII/4 and /5 with RV Polarstern. The percentage given along each pie chart represents the proportion of genome-sequenced prokaryotes of the entire prokaryotic community (identified by 16S rRNA gene). The pie chart of every station sampled presents relative proportions of prokaryotes encoding i) the complete B12 biosynthesis pathway (B12 producers; red), ii) corrin ring biosynthesiser (dark red), iii) cobinamide salvagers (lower ligand producers) (pink) iv) prokaryotes identified as B12 non-producers (grey). Stations are overlayed on a map with annual mean concentrations of chlorophyll a at the surface (https://oceandata.sci.gsfc.nasa.gov). b-e, B12 biosynthesis genes of 1,904 genomes of marine bacteria were analyzed and classified into cobamide biosynthesis phenotypes: as described above for a. Presented are relative abundance of b, all analyzed bacteria, c, phyla and classes, d, orders of Alphaproteobacteria and e, orders of Gammaproteobacteria.
Extended Data Fig. 7 Proportions of genes encoding reactions of B12 biosynthesis in genomes of Rhodobacterales, Alteromonadales, Vibrionales and Betaproteobacteria.
Presence (as percentage) of individual B12 biosynthesis genes in Rhodobacterales (220), Alteromonadales (96), Vibrionales (52) and Betaproteobacteria (126) genomes. Percent of gene abundance is given in color-coded 10% increments from red (0%) to dark green (100%). Genes depicted in grey were not identified in examined genomes.
Extended Data Fig. 8 (Pro)-phage targeting direct-geneFISH signal intensities.
Shown are the mean relative intensities of the (pro)-phage targeting direct-geneFISH signals of Roseovarius in triplicate co-culture samples withdrawn after 96 h and 120 h and the mono-culture Roseovarius samples withdrawn after 96 h as boxplots. Individual, small dots represent the (pro)-phage targeting direct-geneFISH signal intensities of the individually measured regions of interest (ROI; approximate one cell). At least 465 ROIs were defined and validated for each sample. The red dashed line defines the threshold for the definition of induced cells. The percentage below the samples indicates the proportion of induced cells in the total cells analyzed. In each treatment n = 3 biological independent samples were run. The lower and upper hinges of the box correspond to the first and third quartiles (the 25th and 75th percentiles). The central line in the box is the median. The upper whisker extends from the hinge to the largest value no further than 1.5 * IQR from the hinge (where IQR is the inter-quartile range, or distance between the first and third quartiles). The lower whisker extends from the hinge to the smallest value at most 1.5 * IQR of the hinge. Data beyond the end of the whiskers are called “outlying” points and are plotted individually. Prophage induction events, indicated by a significant increase of the phage signal intensity in regions of interest (ROI), were observed in 2 independent experiments.
Extended Data Fig. 9 Enumeration of bacterial cells and virus-like particles of Colwellia and Roseovarius grown in co- and mono-culture.
a, Bacterial cell numbers (white triangle) and ratio of virus-like particles to bacterial cell number (black circle) of Colwellia and Roseovarius growing in co-culture, c, Roseovarius and e, Colwellia in mono-culture with B12 supplementation. b, Bacterial cell numbers (white triangle) and virus-like particles (black circle) of Colwellia and Roseovarius growing in co-culture, d, Roseovarius and f, Colwellia in mono-culture with B12 supplementation. Shown are means of triplicates + SD. Each treatment was carried out in n = 3 biological independent samples and growth characteristics were confirmed in independently conducted tests.
Supplementary information
Supplementary Figures
Supplementary Figs. 1–5.
Supplementary Table 1
Growth rate, growth yield and required molecules per cell of Colwellia and Roseovarius growing in mono-culture when supplemented with B12 or respective B12 building blocks.
Supplementary Table 2
Intracellular vitamin B12 recovery (Hydroxycobalamin, Cyanocobalamin, Adenosylcobalamin, Methylcobalamin) by LC–MS of Colwellia and Roseovarius growing in mono-culture when supplemented with B12 or respective B12 building blocks.
Supplementary Table 3
Growth rate and growth yield of C. muelleri growing in mono-culture when supplemented with B12 or in consortium with Colwellia and Roseovarius.
Supplementary Table 4
Composition of synASW medium and synASW medium trace elements solution, used for combined diatom–bacteria cultivation of C. muellieri or T. pseudonana with Roseovarius and Colwellia.
Supplementary Table 5
Settings used for selected reaction monitoring.
Supplementary Data 1
Identification of B12 biosynthesis genes, B12-dependent reactions, B12-indipendent reactions, B12 transporter genes and B12 salvage genes in the genomes of Roseovarius and Colwellia.
Supplementary Data 2
Transcriptional gene regulation of both bacterial isolates in co-culture compared with gene regulation when supplemented with the addition of B12 (1 nM) cultivated in mono-culture. Genes transcribed at a log2(FC) below –2 and above 2 are outlined in separate sheets, highlighting respective cellular functions.
Supplementary Data 3
Classification of B12 pathway synthesizing groups of publicly available aquatic bacterial genomes.
Supplementary Data 4
Gene annotation of the prophage Roseophage ICBM167.
Supplementary Data 5
Polynucleotide probe mixture for the detection of Roseophage ICBM167 by targeted direct-geneFISH.
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Wienhausen, G., Moraru, C., Bruns, S. et al. Ligand cross-feeding resolves bacterial vitamin B12 auxotrophies. Nature (2024). https://doi.org/10.1038/s41586-024-07396-y
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DOI: https://doi.org/10.1038/s41586-024-07396-y
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