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Metagenomics-Guided Analysis of Microbial Chemolithoautotrophic Phosphite Oxidation Yields Evidence of a Seventh Natural CO2 Fixation Pathway Israel A

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Metagenomics-Guided Analysis of Microbial Chemolithoautotrophic Phosphite Oxidation Yields Evidence of a Seventh Natural CO2 Fixation Pathway Israel A Metagenomics-guided analysis of microbial chemolithoautotrophic phosphite oxidation yields evidence of a seventh natural CO2 fixation pathway Israel A. Figueroaa, Tyler P. Barnuma, Pranav Y. Somasekhara, Charlotte I. Carlströma,1, Anna L. Engelbrektsona, and John D. Coatesa,2 aDepartment of Plant and Microbial Biology, University of California, Berkeley, CA 94720 Edited by David M. Karl, University of Hawaii, Honolulu, HI, and approved November 2, 2017 (received for review September 5, 2017) Dissimilatory phosphite oxidation (DPO), a microbial metabolism PtxD is a phosphite dehydrogenase that catalyzes the NAD- 2− 3− by which phosphite (HPO3 ) is oxidized to phosphate (PO4 ), is dependent oxidation of phosphite to phosphate, and PtxE is a the most energetically favorable chemotrophic electron-donating transcriptional regulator. The remaining five genes (ptdFCGHI) process known. Only one DPO organism has been described to have so far been found only in FiPS-3 (13, 22, 23). PtdC is an inner date, and little is known about the environmental relevance of this membrane transporter that facilitates phosphite uptake, possibly metabolism. In this study, we used 16S rRNA gene community anal- by functioning as a phosphite/phosphate antiporter (22, 23). ysis and genome-resolved metagenomics to characterize anaerobic PtdFGHI are likely involved in energy conservation during DPO, wastewater treatment sludge enrichments performing DPO coupled but their functions have yet to be experimentally confirmed (5). to CO2 reduction. We identified an uncultivated DPO bacterium, Whether this gene cluster is conserved in all DPOM is uncertain. Candidatus Phosphitivorax (Ca. P.) anaerolimi strain Phox-21, that belongs to candidate order GW-28 within the Deltaproteobacteria, Significance which has no known cultured isolates. Genes for phosphite oxida- tion and for CO reduction to formate were found in the genome of 2 2− Ca. P. anaerolimi, but it appears to lack any of the known natural Phosphite (HPO3 ) is the most energetically favorable bi- carbon fixation pathways. These observations led us to propose a ological electron donor known, but only one organism capable metabolic model for autotrophic growth by Ca. P. anaerolimi of growing by phosphite oxidation has been previously iden- tified. Here, we describe a phosphite-oxidizing bacterium that whereby DPO drives CO2 reduction to formate, which is then assim- ilated into biomass via the reductive glycine pathway. can grow with CO2 as its sole electron acceptor, and we pro- pose a metabolic model in which inorganic carbon is assimi- phosphite oxidation | carbon fixation | metagenomics | formatotrophic | lated via the reductive glycine pathway. Although the reductive glycine pathway reductive glycine pathway has previously been identified as a “synthetic” carbon fixation pathway, this study provides evi- dence that it may actually function as a natural autotrophic lthough phosphorus has long been recognized as an essen- pathway. Our results suggest that phosphite may serve as a Atial nutrient for life, the role of phosphorus redox chemistry driver of microbial growth and carbon fixation in energy- in biology remains poorly understood. Most of the phosphorus 3− limited environments, particularly in aphotic environments on extant Earth exists as phosphate (PO4 ), but recent work has lacking alternative terminal electron acceptors. detected the reduced inorganic phosphorus compound phosphite 2− (HPO3 ) in many environments, including rivers, lakes, swamps, Author contributions: I.A.F. and J.D.C. designed research; I.A.F. and P.Y.S. performed re- geothermal pools, and wastewater treatment plants (1–4). search; T.P.B., C.I.C., and A.L.E. contributed new reagents/analytic tools; I.A.F. and J.D.C. Phosphite appears to be a key intermediate in the phosphorus analyzed data; and I.A.F., T.P.B., A.L.E., and J.D.C. wrote the paper. redox cycle, accounting for up to 30% of the total dissolved The authors declare no conflict of interest. phosphorus in some ecosystems (1). It can be produced naturally This article is a PNAS Direct Submission. through geothermal reactions, volcanic eruptions, lightning dis- Published under the PNAS license. charges, and metal corrosion and can also be present in some Data deposition: The full 16S rRNA gene sequence of Ca. Phosphitivorax anaerolimi Phox- industrial products, such as fertilizers and fungicides (3–7). A 21 has been deposited in the GenBank (GB) database (accession no. KU898264). MiSeq reads from community 16S rRNA gene amplicon sequencing have been deposited in the variety of microorganisms have evolved the ability to utilize National Center for Biotechnology Information Sequence Read Archive (accession no. phosphite as a phosphorus source by oxidizing phosphite to SRP071909). The combined metagenomic assembly of all four enrichment community phosphate, a process known as assimilatory phosphite oxidation samples has been deposited in the Integrated Microbial Genomes (IMG) database (acces- (APO) (8–17). sion no. Ga0100964). Individual genomes recovered from the combined assembly are available in the IMG and GB databases under the following accession nos.: Ca. Phos- Phosphite is also the most energetically favorable chemo- phitivorax anaerolimi Phox-21, Ga0115057 (IMG), MPOS00000000 (GB); Tepidanaero- trophic electron donor known due to the extremely low redox bacter sp. EBM-38, Ga0115060 (IMG), MPOT00000000 (GB); unclassified bacterium EBM- ′ potential of the phosphate/phosphite couple (Eo = −650 mV) 40, Ga0115061 (IMG), MPOU00000000 (GB); Proteiniphilum sp. EBM-39, Ga0115062 (IMG), MPOV00000000 (GB); Thermotogales bacterium EBM-19, Ga0115064 (IMG), (18). This property, together with its high solubility and chemical MPOW00000000 (GB); Proteiniphilum sp. EBM-41, Ga0115065 (IMG), MPOX00000000 stability, allows it to drive cellular growth in a process known as (GB); Methanoculleus sp. EBM-46, Ga0115067 (IMG), MPOY00000000 (GB); Coprothermo- dissimilatory phosphite oxidation (DPO) since the phosphate bacter sp. EBM-25, Ga0115069 (IMG), MPOZ00000000 (GB); Methanococcoides sp. EBM- produced is excreted from the cells as an end product of energy 47, Ga0115071 (IMG), MPPA00000000 (GB); Spirochaeta sp. EBM-43, Ga0115073 (IMG), MPPB00000000 (GB); Aminobacterium sp. EBM-42, Ga0115070 (IMG), MPPC00000000 metabolism rather than being incorporated into biomass (13, 19). (GB); Thermotogales bacterium EBM-38, Ga0115076 (IMG), MPPD00000000 (GB); Tepid- However, the only dissimilatory phosphite-oxidizing microor- anaerobacter sp. EBM-49, Ga0115075 (IMG), MPPE00000000 (GB). ganism (DPOM) described to date is Desulfotignum phosphitox- See Commentary on page 7. idans FiPS-3, which grows by coupling phosphite oxidation to 1Present address: Institute for Microbiology, ETH Zürich, Zürich, Switzerland. either sulfate or CO2 reduction (19, 20). Its ability to oxidize 2To whom correspondence should be addressed. Email: [email protected]. phosphite is conferred by the ptx-ptd gene cluster. Two of these This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. genes (ptxED) can also be found in APO bacteria (13, 14, 17, 21). 1073/pnas.1715549114/-/DCSupplemental. E92–E101 | PNAS | Published online November 28, 2017 www.pnas.org/cgi/doi/10.1073/pnas.1715549114 Downloaded by guest on September 28, 2021 PNAS PLUS Here, we expand our understanding of DPOM through 16S A 10 ribosomal RNA (rRNA) gene community analysis and genome- resolved metagenomics of phosphite-oxidizing enrichments from 8 anaerobic wastewater treatment sludge grown with CO2 as the sole electron acceptor. We reveal the presence in our enrich- ments of an uncultivated DPOM possessing a ptx-ptd gene cluster (mM) 6 3- and propose a metabolic model for phosphite oxidation coupled 4 to CO reduction and carbon fixation in this organism based on , PO 2 2- genomic evidence. Our results suggest that inorganic carbon is 3 4 assimilated via the reductive glycine pathway. Although this path- HPO way has been previously proposed as a viable “synthetic” route for SEE COMMENTARY 2 carbon fixation (24), this study provides evidence that it may ac- tually function in a natural system as an autotrophic pathway. 0 Results 02468101214161820 Enrichment for Dissimilatory Phosphite-Oxidizing Microorganisms Time (days) from Wastewater Treatment Sludge. To enrich for DPOM, phos- phite and CO2 were added as the sole electron donor and electron Phosphite + Rumen Fluid Rumen Fluid acceptor, respectively, to defined anaerobic media inoculated with B 100 C 100 wastewater treatment sludge. Killed controls (autoclaved in- 90 ty 90 oculum with phosphite added) and no-phosphite controls (live 80 80 70 70 mmuni inoculum with no phosphite added) were also prepared. The live 60 o 60 50 50 phosphite-fed enrichment cultures completely oxidized 10 mM of c 40 40 phosphite after 29 wk of incubation (Fig. S1). The rate of phos- tage ± μ 30 30 phite oxidation was 50 1 M/d and was accompanied by a 20 20 Percentage of community of Percentage 10 10 stoichiometric increase in the phosphate concentration in the Percen 0 0 media. No phosphite oxidation or phosphate accumulation was 0 5 11 16 19 0 5 11 16 19 seen in the killed controls over the same time period (Fig. S1). Time (days) Time (days) DNA samples were collected at 0, 20, and 29 wk after inoculation from both the live phosphite-fed cultures and the no-phosphite Phosphite Phosphite + Rumen Fluid + Molybdate D 100 E 100 controls for microbial community composition analysis. Three y 90 t 90 operational taxonomic units (OTUs) were prevalent (≥1% of the 80 80 70 70 community) only in the phosphite-fed cultures: OTU 21 (Pro- mmunity o 60 60 communi ± ± teobacteria, 4.4 2.4%), OTU 28 (Thermovirga, 1.3 0.7%), and f 50 o 50 ± c e of OTU 33 (Desulfomonile, 1.0 1.0%) (Fig. S2). 40 ge 40 MICROBIOLOGY Given the slow overall rate of phosphite oxidation in our en- 30 nta 30 20 20 richments and the presence of an OTU belonging to the Desul- erce Percentag 10 10 P fomonile (OTU 33) among phosphite-enriched taxa, we decided 0 0 to test variables known to enhance or inhibit growth of this or- 05111619 0 5 11 16 19 ganism in pure culture.
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