Marine Proteobacteria metabolize glycolate via the β-hydroxyaspartate cycle

Abstract

One of the most abundant sources of organic carbon in the ocean is glycolate, the secretion of which by marine phytoplankton results in an estimated annual flux of one petagram of glycolate in marine environments1. Although it is generally accepted that glycolate is oxidized to glyoxylate by marine bacteria2,3,4, the further fate of this C2 metabolite is not well understood. Here we show that ubiquitous marine Proteobacteria are able to assimilate glyoxylate via the β-hydroxyaspartate cycle (BHAC) that was originally proposed 56 years ago5. We elucidate the biochemistry of the BHAC and describe the structure of its key enzymes, including a previously unknown primary imine reductase. Overall, the BHAC enables the direct production of oxaloacetate from glyoxylate through only four enzymatic steps, representing—to our knowledge—the most efficient glyoxylate assimilation route described to date. Analysis of marine metagenomes shows that the BHAC is globally distributed and on average 20-fold more abundant than the glycerate pathway, the only other known pathway for net glyoxylate assimilation. In a field study of a phytoplankton bloom, we show that glycolate is present in high nanomolar concentrations and taken up by prokaryotes at rates that allow a full turnover of the glycolate pool within one week. During the bloom, genes that encode BHAC key enzymes are present in up to 1.5% of the bacterial community and actively transcribed, supporting the role of the BHAC in glycolate assimilation and suggesting a previously undescribed trophic interaction between autotrophic phytoplankton and heterotrophic bacterioplankton.

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Fig. 1: The BHAC.
Fig. 2: Reaction sequence catalysed by β-hydroxyaspartate dehydratase (BhcB) and iminosuccinate reductase (BhcD).
Fig. 3: The BHAC during the spring phytoplankton bloom 2018 at Helgoland.

Data availability

The coordinates and structure factors of the crystal structures generated from this research are available at the PDB under accession numbers 6QKB and 6RQA. Mass spectrometry proteomics data are available via ProteomeXchange with the identifier PXD013274. MAGs are available under accession PRJEB28156 at the European Nucleotide Archive (ENA). All other relevant data are available in the Article and the Supplementary Information. Source Data for Figs. 2, 3 and Extended Data Fig. 1, 4, 5, 79 are provided with the paper.

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Acknowledgements

We thank L. Franzmeyer and K.-P. Rücknagel as well as the crew of the Aade and the staff at the Biological Station at Helgoland (BAH) for sample collection and processing; K. H. Wiltshire for providing Chl a data collected at BAH; S. Vidal-Melgosa, A. Bolte and J.-H. Hehemann for sharing samples; T. Ferdelman for help with radioactive tracer work; B. Vögeli, T. Schwander, G. Stoffel and S. Burgener for helpful discussions. We acknowledge the support from the staff scientists at the European Synchrotron Radiation Facility Grenoble, France (ESRF, beamlines ID29 & ID30B) as well as at the Deutsches Elektronen-Synchrotron Hamburg, Germany (DESY, beamline P13). Metagenome sequences were obtained within the COGITO (Coastal Microbe Genomic and Taxonomic Observatory) project granted to H. Teeling (MPI Bremen) as a community sequencing project by the Department of Energy’s Joint Genome Institute in Walnut Creek, CA, USA (Proposal ID 998; https://doi.org/10.25585/1488076). This study was funded by the Max-Planck-Society (R.I.A. and T.J.E.), FET-Open Grant 686330 (Future Agriculture) and the German Research Foundation (SFB987 ‘Microbial diversity in environmental signal response’ and FOR 2406 ‘Proteogenomics of marine polysaccharide utilization’).

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Affiliations

Authors

Contributions

L.S.v.B. identified the bhc gene cluster, purified proteins, performed enzyme kinetic analysis, qPCR, phylogenetic analysis and analysis of Tara Oceans metagenomes, generated and characterized mutant P. denitrificans strains and measured glycolate uptake rates. F. Severi performed enzyme kinetic analysis, crystallization of BhcD and enzyme assays in P. denitrificans cell-free extracts. K.K. performed phylogenetic analysis and analysis of Helgoland metagenomes. L.H. performed gel shift assays with BhcR. A.G. performed crystallization of BhcC. F. Sippel performed enzyme kinetic analysis. B.P. generated mutant P. denitrificans strains. P.C. and N.S.C. performed small-molecule mass spectrometry. T.G. performed mass spectrometry for proteomics. J.Z. collected X-ray datasets, solved, refined and analysed crystal structures. B.M.F. and R.I.A. planned and supervised fieldwork at Helgoland and provided reagents. L.S.v.B., E.B., S.Z., U.G.M., R.I.A. and T.J.E. planned experiments, analysed data and supervised the project. L.S.v.B. and T.J.E. wrote the manuscript, with contributions from all other authors.

Corresponding authors

Correspondence to Lennart Schada von Borzyskowski or Tobias J. Erb.

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Competing interests

The Max-Planck-Gesellschaft zur Förderung der Wissenschaften is the patent applicant for the following three patents. All patent applications are pending. L.S.v.B. and T.J.E. have filed European patent no. EP 19190404.4 for the production of plants with altered photorespiration due to implementation of the BHAC. L.S.v.B., J.Z. and T.J.E. have filed European patent no. EP 18167406.0 for the production of photoautotrophic organisms with altered photorespiration due to implementation of the BHAC. L.S.v.B. and T.J.E. have filed European patent no. 18211454.6 for the enantioselective preparation of primary amine compounds using the enzyme BhcD or its homologues.

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Extended data figures and tables

Extended Data Fig. 1 Previously reported glycolate concentrations in environmental samples and cultures of photosynthetic organisms.

a, Bar diagram of glycolate concentrations as previously reported in environmental samples. For details on samples, replicates, and analytics see b and the literature cited therein. b, Table of glycolate concentrations as previously reported in environmental samples (E1, E2 and so on) and cultures of photosynthetic organisms (C1, C2 and so on). When reported in the reference1,2,4,9,10,11,30,31,32,33,34,35,36,37,38,39, the mean value ± error is given. Source data

Extended Data Fig. 2 Crystal structure and phylogenetic analysis of the β-hydroxyaspartate aldolase BhcC.

a, Cartoon representation of the β-hydroxyaspartate aldolase homodimer (PDB 6QKB) with superimposed protein surface (left, side view; right, top view). b, Active site of β-hydroxyaspartate aldolase with covalently bound PLP (light cyan). Active site residues highlighted in pink (A160, A195 and S313) are completely conserved only among β-hydroxyaspartate aldolases, but differ in d-threonine aldolases. c, Active site of d-threonine aldolase (PDB 4V15). The corresponding conserved residues among d-threonine aldolases (Q155, S190 and C303) are highlighted as in b. d, Maximum likelihood phylogenetic tree of the type III PLP-dependent protein superfamily. Sequences of the β-hydroxyaspartate aldolase BhcC and its homologues form a distinct clade (blue) within the d-threonine aldolase branch of this superfamily. Bootstrap values of at least 50 are given on the respective nodes.

Extended Data Fig. 3 Crystal structure and phylogenetic analysis of the iminosuccinate reductase BhcD.

a, Cartoon representation of the iminosuccinate reductase homodimer (PDB 6RQA) with superimposed protein surface (left, side view; right, top view). b, Active site of BhcD with bound NAD+ (light cyan). Residues highlighted in pink (V39, R41, G52, K54 and H83) may contribute to substrate binding and are conserved among iminosuccinate reductases, but differ in l-alanine dehydrogenases. c, Active site of l-alanine dehydrogenase (PDB 1OMO). The corresponding conserved residues among l-alanine dehydrogenases (K41, Y43, R52, M54 and V81) are highlighted as in b. d, Maximum likelihood phylogenetic tree of the ornithine cyclodeaminase/µ-crystalline protein superfamily. Sequences of the iminosuccinate reductase BhcD and its homologues form a distinct clade (red) within this superfamily. Bootstrap values of at least 50 are given on the respective nodes. e, Sequence similarity network of 1,614 sequences from the ornithine cyclodeaminase/µ-crystalline protein superfamily. Connected sequences with more than 80% identity are clustered into nodes. The number in each node gives the number of sequences contained within. Nodes with more than 50% identity are connected by edges. Similar to the phylogenetic analysis shown in d, sequences of the iminosuccinate reductase BhcD and its homologues form a distinct clade (red) within this superfamily.

Extended Data Fig. 4 Michaelis–Menten kinetics of all enzyme reactions characterized in this study.

a, Michaelis–Menten kinetics for aspartate–glyoxylate aminotransferase (BhcA). b, Michaelis–Menten kinetics for β-hydroxyaspartate dehydratase (BhcB). c, Michaelis–Menten kinetics for β-hydroxyaspartate aldolase (BhcC). d, Michaelis–Menten kinetics for iminosuccinate reductase (BhcD). ad, Data are shown from n = 3 independent experiments at different substrate concentrations. The data are summarized in Table 1. Source data

Extended Data Fig. 5 Physiological role of the BHAC in P. denitrificans DSM 413.

a, Growth rate of wild-type P. denitrificans DSM 413 on the BHAC substrates glycolate and glyoxylate. The middle line and box are the median and interquartile range of n = 6 independent experiments and the whiskers indicate the maximum range of the dataset. b, c, Representative growth curves of wild-type P. denitrificans DSM 413 (grey) and bhc deletion strains (coloured) grown in the presence of 60 mM glycolate (b) or 60 mM glyoxylate (c). Deletion of any single gene in the bhc gene cluster is sufficient to completely abolish growth in the presence of glycolate and glyoxylate. These experiments were repeated three times independently with similar results. df, Growth rates (μ) of wild-type P. denitrificans DSM 413 (grey) and BHAC deletion strains (coloured) grown in the presence of 60 mM acetate (d), 30 mM succinate (e) or 20 mM glucose (f). Deletion of any single gene in the bhc gene cluster, or of the whole bhc gene cluster, still permits growth on acetate, succinate or glucose with comparable growth rates as for the wild type. Data are the mean ± s.d. of n = 3 independently grown cultures. g, Analysis of the proteome of glycolate-grown compared to succinate-grown P. denitrificans DSM 413. All proteins that were quantified by at least three unique peptides are shown. The 15 proteins that showed the strongest increase in abundance are marked in the volcano plot. The four enzymes of the BHAC are marked in red, the three subunits of glycolate oxidase in orange, the proteins of a putative operon for lactate utilization in white and the proteins directly downstream of the bhc gene cluster in light red. h, The abundance of these proteins, given as the percentage of the intensity-based absolute quantification (iBAQ) value. Data are the mean ± s.d. of n = 4 independently grown cultures. i, Specific activities of BHAC enzymes in cell-free extracts of glycolate-grown P. denitrificans DSM 413, as measured spectrophotometrically. Note that the activity of BhcD is plotted on the right y axis and consists of the actual iminosuccinate reductase activity (iminosuccinate to l-aspartate) as well as endogenous malate dehydrogenase activity (oxaloacetate to l-malate). j, Ratio of malate to aspartate determined by LC–MS during the enzyme assay for BhcD activity. The ratio remains approximately constant at 12:1, indicating that only approximately 8% of the activity (around 1.3 U mg−1) shown in i can be ascribed to iminosuccinate reductase. i, j, Data are the mean ± s.d. of n = 3 independently grown cultures; each data point represents the mean of n = 3 technical replicates. k, DNA-binding properties of BhcR. Left, a fluorescently labelled DNA fragment carrying the putative promoter region of the bhc gene cluster (Pbhc) was incubated with increasing amounts of purified BhcR protein and subsequently separated by electrophoresis to visualize DNA bound to BhcR and free DNA; a DNA fragment derived from the coding region of bhcA was used as a negative control. BhcR specifically forms a complex with the DNA fragment containing the putative promoter region of the bhc gene cluster. Right, the Pbhc–BhcR complex was incubated with increasing concentrations of glyoxylate and subsequently separated by electrophoresis to assess the effect of glyoxylate on complex formation; the bhcA DNA fragment together with BhcR was used as a negative control. Increasing concentrations of glyoxylate decrease the binding of BhcR to the Pbhc DNA fragment. For gel source data, see Supplementary Fig. 1. Source data

Extended Data Fig. 6 Phylogenetic analysis of the bhc gene cluster.

a, Genome-based maximum likelihood phylogenetic tree of bacterial strains with the bhc gene cluster. The bhc gene cluster is found in Gammaproteobacteria (green), and in the alphaproteobacterial orders Rhizobiales (blue) and Rhodobacterales (red), as well as in one member each of Sphingomonadales and Kiloniellales. The phylogenetic tree is based on an alignment of 120 bacterial marker genes from 264 publicly available bacterial genomes and 5 MAGs and was calculated using GTDB-Tk64 (https://github.com/Ecogenomics/GtdbTk). If several strains from the same genus cluster together, nodes are collapsed at the genus level, and the size of the resulting circle corresponds to the respective number of strains. Loktanella*: collapsed node contains the MAGs 20110516_Bin_8_1 and 20110523_Bin_9_1; Planktotalea**: collapsed node contains the MAG 20110523_Bin_97_1; Litoricola***: collapsed node contains the MAG 20110526_Bin_19_1. b, Maximum likelihood phylogenetic tree of concatenated BHAC enzyme sequences. Colour code is the same as in a. Phylogenetic groups that were mostly isolated from terrestrial or freshwater habitats are marked with a black dot. Comparison with a reveals that the sequences of the BHAC enzymes are not phylogenetically representative, as, for example, alpha- and gammaproteobacterial sequences form a common branch and sequences from terrestrial or freshwater Rhizobiales and Rhodobacterales form another common branch. This suggests that the bhc gene cluster might have been subject to horizontal gene transfer between distantly related strains in shared habitats. The environmental bhc gene cluster sequence that could not be binned successfully is marked in bold and clusters together with isolated representatives of Pseudoruegeria, Litoreibacter and Pseudooceanicola. The phylogenetic tree is based on the concatenated alignments of the 4 enzymes (BhcA–BhcD) from 264 publicly available bacterial genomes and from 6 metagenome contigs. It was calculated using raxmlGUI67. Bootstrap values of at least 50 are given on the respective nodes; calculated branch lengths of the tree are ignored for the sake of better visualization. If several strains from the same genus cluster together, nodes are collapsed at the genus level, and the size of the resulting circle corresponds to the respective number of strains. If strains from the same genus cluster in more than one node, the respective branches are labelled as Genus_1, Genus_2, and so on, in a clockwise manner. Loktanella_2*: collapsed node contains the MAGs 20110516_Bin_8_1 and 20110523_Bin_9_1; Planktotalea**: collapsed node contains the MAG 20110523_Bin_97_1; Litoricola***: collapsed node contains the MAG 20110526_Bin_19_1. a, b, Taxonomy is based on GTDB (release 03-RS86; http://gtdb.ecogenomic.org/). All strains contained in the phylogenetic trees are listed in Supplementary Data 1.

Extended Data Fig. 7 Glyoxylate assimilation pathways in marine metagenomes.

a, Metagenomes collected during the Tara Oceans expedition were searched for the presence of BhcC as representative enzyme of the BHAC. Dots on the map mark sampling locations of metagenomes containing BhcC sequences; the colour of the dot corresponds to BhcC abundance in samples from surface water (0.22–3-µm size fraction), as shown in the legend. The map was made with Ocean Data View 5.1.5 (Schlitzer, R., Ocean Data View, odv.awi.de, 2018). b, Phylogenetic distribution of 104 unique BhcC sequences found in Tara Oceans metagenomes. c, Phylogenetic distribution of 32 unique Gcl (as representative enzyme of the glycerate pathway) sequences found in Tara Oceans metagenomes. Whereas BhcC is mainly found in Alphaproteobacteria, Gcl is largely restricted to Gammaproteobacteria. b, c, Taxonomy is based on GTDB64 (release 03-RS86; http://gtdb.ecogenomic.org/). d, Ratio of the abundances (in percentage of total reads) of BhcC to Gcl in Tara Oceans metagenomes. BhcC:Gcl ratios from n = 210 samples are plotted together (left) and clustered by sampling depth (SRF, upper layer zone (n = 101); DCM, deep chlorophyll maximum layer (n = 68); MES, mesopelagic zone (n = 41)). Samples from the 0.22–3-µm size fraction are denoted by a black dot; samples from the <0.22-µm size fraction are denoted by a blue dot. The median is shown in orange as centre value and error bars represent interquartile ranges. The median BhcC:Gcl ratio of all samples is 18.7. The highest BhcC:Gcl ratio is found in surface water samples (median = 41.8), with the ratio generally being higher in the 0.22–3-µm size fraction than in the <0.22-µm size fraction. Sequence IDs, abundances and BhcC:Gcl ratios are given in Supplementary Data 2. Source data

Extended Data Fig. 8 Abundance of the bhc gene cluster in Helgoland metagenomes.

a, The location of Helgoland Island approximately 40 km offshore the northern German coastline in the North Sea is marked with a red dot. The map was made with Ocean Data View 5.1.5 (R. Schlitzer, Ocean Data View, odv.awi.de, 2018). b, The long-term ecological research site ‘Kabeltonne’ (red dot: 54° 11.3′ N, 7° 54.0′ E) is located between Helgoland Island (left) and the small island Düne (right). Satellite image from WorldWind Explorer (B. Schubert, worldwind.earth/explorer, 2016–2018); the image was adapted to indicate the sampling site. ce, Abundance of the bhc gene cluster (in RPKM) was calculated in 38 metagenomes from samples collected during the algal spring blooms of 2010 to 2012 in the North Sea close to Helgoland28. Six different sequences were investigated, five of which could be assigned to metagenome bins (Extended Data Fig. 6 and Supplementary Data 3), whereas the remaining, most abundant sequence (black) could not be binned successfully. Source data

Extended Data Fig. 9 Validation of degenerate bhcC primers.

Degenerate primers for bhcC were used for qPCR with different amounts of genomic DNA from P. denitrificans DSM 413, Rhodobacter sphaeroides 2.4.1, and E. coli K-12 MG1655 as template. While the bhcC gene from P. denitrificans DSM 413 is amplified, genomic DNA from organisms that lack the bhc gene cluster does not result in reliable amplification. Data are mean ± s.d.; n = 3 independent experiments. Source data

Extended Data Table 1 X-ray diffraction data collection and model refinement statistics

Supplementary information

Supplementary Figure 1

This file contains a figure showing an SDS-PAGE gel of all purified proteins of the bhc gene cluster, as well as uncropped gel data scans of this figure and Extended Data Figure 5k.

Reporting Summary

Supplementary Tables 1-3

This file contains Supplementary Table 1 (an overview of known natural net glyoxylate assimilation pathways), Supplementary Table 2 (a list of the strains and plasmids used in this study) and Supplementary Table 3 (a list of the primers used in this study).

Supplementary Data 1

This dataset contains a list of bacterial isolates with the bhc gene cluster, as well as their taxonomic classification and the Gene IDs for their bhcC and glcD genes.

Supplementary Data 2

This dataset contains the Gene IDs, taxonomic classifications, abundances, and ratios of BhcC and Gcl sequences found in Tara Oceans metagenomes.

Supplementary Data 3

This dataset contains information on the quality of Helgoland metagenome bins containing the bhc gene cluster.

Supplementary Data 4

This dataset contains information on the yields of DNA and RNA extractions from North Sea water samples.

Source data

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Schada von Borzyskowski, L., Severi, F., Krüger, K. et al. Marine Proteobacteria metabolize glycolate via the β-hydroxyaspartate cycle. Nature 575, 500–504 (2019). https://doi.org/10.1038/s41586-019-1748-4

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