Bacterial autotrophs often rely on CO2 concentrating mechanisms (CCMs) to assimilate carbon. Although many CCM proteins have been identified, a systematic screen of the components of CCMs is lacking. Here, we performed a genome-wide barcoded transposon screen to identify essential and CCM-related genes in the γ-proteobacterium Halothiobacillus neapolitanus. Screening revealed that the CCM comprises at least 17 and probably no more than 25 genes, most of which are encoded in 3 operons. Two of these operons (DAB1 and DAB2) contain a two-gene locus that encodes a domain of unknown function (Pfam: PF10070) and a putative cation transporter (Pfam: PF00361). Physiological and biochemical assays demonstrated that these proteins—which we name DabA and DabB, for DABs accumulate bicarbonate—assemble into a heterodimeric complex, which contains a putative β-carbonic anhydrase-like active site and functions as an energy-coupled inorganic carbon (Ci) pump. Interestingly, DAB operons are found in a diverse range of bacteria and archaea. We demonstrate that functional DABs are present in the human pathogens Bacillus anthracis and Vibrio cholerae. On the basis of these results, we propose that DABs constitute a class of energized Ci pumps and play a critical role in the metabolism of Ci throughout prokaryotic phyla.
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All of the Illumina sequencing data are accessible at the NCBI SRA (BioProject accession number: PRJNA546024). All other data are available on GitHub at https://github.com/jackdesmarais/DabTransporterPaper.
All custom code is available on GitHub at https://github.com/jackdesmarais/DabTransporterPaper.
Bathellier, C., Tcherkez, G., Lorimer, G. H. & Farquhar, G. D. Rubisco is not really so bad. Plant Cell Environ. 41, 705–716 (2018).
Flamholz, A. et al. Revisiting tradeoffs in Rubisco kinetic parameters. Biochemistry-US https://doi.org/10.1021/acs.biochem.9b00237 (2019).
Tcherkez, G. The mechanism of Rubisco-catalysed oxygenation. Plant Cell Environ. 39, 983–997 (2016).
Bauwe, H., Hagemann, M. & Fernie, A. R. Photorespiration: players, partners and origin. Trends Plant Sci. 15, 330–336 (2010).
Tcherkez, G. G. B., Farquhar, G. D. & Andrews, T. J. Despite slow catalysis and confused substrate specificity, all ribulose bisphosphate carboxylases may be nearly perfectly optimized. Proc. Natl Acad. Sci. USA 103, 7246–7251 (2006).
Savir, Y., Noor, E., Milo, R. & Tlusty, T. Cross-species analysis traces adaptation of Rubisco toward optimality in a low-dimensional landscape. Proc. Natl Acad. Sci. USA 107, 3475–3480 (2010).
Mangan, N. M., Flamholz, A., Hood, R. D., Milo, R. & Savage, D. F. pH determines the energetic efficiency of the cyanobacterial CO2 concentrating mechanism. Proc. Natl Acad. Sci. USA 113, E5354–E5362 (2016).
Raven, J. A., Beardall, J. & Sánchez-Baracaldo, P. The possible evolution and future of CO2-concentrating mechanisms. J. Exp. Bot. 68, 3701–3716 (2017).
Rae, B. D., Long, B. M., Badger, M. R. & Price, G. D. Functions, compositions, and evolution of the two types of carboxysomes: polyhedral microcompartments that facilitate CO2 fixation in cyanobacteria and some proteobacteria. Microbiol. Mol. Biol. Rev. 77, 357–379 (2013).
Long, B. M., Rae, B. D., Rolland, V., Förster, B. & Price, G. D. Cyanobacterial CO2-concentrating mechanism components: function and prospects for plant metabolic engineering. Curr. Opin. Plant Biol. 31, 1–8 (2016).
Price, G. D., Badger, M. R. & von Caemmerer, S. The prospect of using cyanobacterial bicarbonate transporters to improve leaf photosynthesis in C3 crop plants. Plant Physiol. 155, 20–26 (2011).
McGrath, J. M. & Long, S. P. Can the cyanobacterial carbon-concentrating mechanism increase photosynthesis in crop species? A theoretical analysis. Plant Physiol. 164, 2247–2261 (2014).
Long, B. M. et al. Carboxysome encapsulation of the CO2-fixing enzyme Rubisco in tobacco chloroplasts. Nat. Commun. 9, 3570 (2018).
Price, G. D. & Badger, M. R. Expression of human carbonic anhydrase in the cyanobacterium Synechococcus PCC7942 creates a high CO2-requiring phenotype evidence for a central role for carboxysomes in the CO2 concentrating mechanism. Plant Physiol. 91, 505–513 (1989).
Hopkinson, B. M., Young, J. N., Tansik, A. L. & Binder, B. J. The minimal CO2-concentrating mechanism of Prochlorococcus spp. MED4 is effective and efficient. Plant Physiol. 166, 2205–2217 (2014).
Whitehead, L., Long, B. M., Price, G. D. & Badger, M. R. Comparing the in vivo function of α-carboxysomes and β-carboxysomes in two model cyanobacteria. Plant Physiol. 165, 398–411 (2014).
Holthuijzen, Y. A., van Dissel-Emiliani, F. F. M., Kuenen, J. G. & Konings, W. N. Energetic aspects of CO2 uptake in Thiobacillus neapolitanus. Arch. Microbiol. 147, 285–290 (1987).
Price, G. D. & Badger, M. R. Isolation and characterization of high CO2-requiring-mutants of the cyanobacterium Synechococcus PCC7942: two phenotypes that accumulate inorganic carbon but are apparently unable to generate CO2 within the carboxysome. Plant Physiol. 91, 514–525 (1989).
Marcus, Y., Schwarz, R., Friedberg, D. & Kaplan, A. High CO2 requiring mutant of Anacystis nidulans R2. Plant Physiol. 82, 610–612 (1986).
Bonacci, W. et al. Modularity of a carbon-fixing protein organelle. Proc. Natl Acad. Sci. USA 109, 478–483 (2012).
Jorda, J., Lopez, D., Wheatley, N. M. & Yeates, T. O. Using comparative genomics to uncover new kinds of protein-based metabolic organelles in bacteria. Protein Sci. 22, 179–195 (2013).
Axen, S. D., Erbilgin, O. & Kerfeld, C. A. A taxonomy of bacterial microcompartment loci constructed by a novel scoring method. PLoS Comput. Biol. 10, e1003898 (2014).
Shibata, M., Ohkawa, H., Katoh, H., Shimoyama, M. & Ogawa, T. Two CO2 uptake systems in cyanobacteria: four systems for inorganic carbon acquisition in Synechocystis sp. strain PCC6803. Funct. Plant Biol. 29, 123–129 (2002).
Price, G. D. Inorganic carbon transporters of the cyanobacterial CO2 concentrating mechanism. Photosynth. Res. 109, 47–57 (2011).
Heinhorst, S., Cannon, G. C. & Shively, J. M. in Complex Intracellular Structures in Prokaryotes (ed. Shively, J. M.) 141–165 (Springer, 2006).
Shively, J. M., Ball, F., Brown, D. H. & Saunders, R. E. Functional organelles in prokaryotes: polyhedral inclusions (carboxysomes) of Thiobacillus neapolitanus. Science 182, 584–586 (1973).
Cannon, G. C. et al. Microcompartments in prokaryotes: carboxysomes and related polyhedra. Appl. Environ. Microbiol. 67, 5351–5361 (2001).
Mangiapia, M. et al. Proteomic and mutant analysis of the CO2 concentrating mechanism of hydrothermal vent chemolithoautotroph Thiomicrospira crunogena. J. Bacteriol. 199, e00871-16 (2017).
Scott, K. M. et al. Genomes of ubiquitous marine and hypersaline Hydrogenovibrio, Thiomicrorhabdus and Thiomicrospira spp. encode a diversity of mechanisms to sustain chemolithoautotrophy in heterogeneous environments. Environ. Microbiol. 20, 2686–2708 (2018).
Scott, K. M. et al. Diversity in CO2-concentrating mechanisms among chemolithoautotrophs from the genera Hydrogenovibrio, Thiomicrorhabdus, and Thiomicrospira, ubiquitous in sulfidic habitats worldwide. Appl. Environ. Microbiol. 85, e02096-18 (2019).
Wetmore, K. M. et al. Rapid quantification of mutant fitness in diverse bacteria by sequencing randomly bar-coded transposons. mBio 6, e00306–e00315 (2015).
Chaijarasphong, T. et al. Programmed ribosomal frameshifting mediates expression of the α-carboxysome. J. Mol. Biol. 428, 153–164 (2016).
Cai, F. et al. The pentameric vertex proteins are necessary for the icosahedral carboxysome shell to function as a CO2 leakage barrier. PLoS ONE 4, e7521 (2009).
Roberts, E. W., Cai, F., Kerfeld, C. A., Cannon, G. C. & Heinhorst, S. Isolation and characterization of the Prochlorococcus carboxysome reveal the presence of the novel shell protein CsoS1D. J. Bacteriol. 194, 787–795 (2012).
Wheatley, N. M., Sundberg, C. D., Gidaniyan, S. D., Cascio, D. & Yeates, T. O. Structure and identification of a pterin dehydratase-like protein as a ribulose-bisphosphate carboxylase/oxygenase (RuBisCO) assembly factor in the α-carboxysome. J. Biol. Chem. 289, 7973–7981 (2014).
Aigner, H. et al. Plant RuBisCo assembly in E. coli with five chloroplast chaperones including BSD2. Science 358, 1272–1278 (2017).
Mueller-Cajar, O. The diverse AAA+ machines that repair inhibited Rubisco active sites. Front. Mol. Biosci. 4, 31 (2017).
Krulwich, T. A., Hicks, D. B. & Ito, M. Cation/proton antiporter complements of bacteria: why so large and diverse? Mol. Microbiol. 74, 257–260 (2009).
Merlin, C., Masters, M., McAteer, S. & Coulson, A. Why is carbonic anhydrase essential to Escherichia coli? J. Bacteriol. 185, 6415–6424 (2003).
Du, J., Förster, B., Rourke, L., Howitt, S. M. & Price, G. D. Characterisation of cyanobacterial bicarbonate transporters in E. coli shows that SbtA homologs are functional in this heterologous expression system. PLoS ONE 9, e115905 (2014).
Cronk, J. D., Endrizzi, J. A., Cronk, M. R., O’Neill, J. W. & Zhang, K. Y. Crystal structure of E. coli β-carbonic anhydrase, an enzyme with an unusual pH-dependent activity. Protein Sci. 10, 911–922 (2001).
Krishnamurthy, V. M. et al. Carbonic anhydrase as a model for biophysical and physical-organic studies of proteins and protein–ligand binding. Chem. Rev. 108, 946–1051 (2008).
Cronk, J. D. et al. Identification of a novel noncatalytic bicarbonate binding site in eubacterial β-carbonic anhydrase. Biochemistry 45, 4351–4361 (2006).
Shibata, M. et al. Distinct constitutive and low-CO2-induced CO2 uptake systems in cyanobacteria: genes involved and their phylogenetic relationship with homologous genes in other organisms. Proc. Natl Acad. Sci. USA 98, 11789–11794 (2001).
Maeda, S.-I., Badger, M. R. & Price, G. D. Novel gene products associated with NdhD3/D4-containing NDH-1 complexes are involved in photosynthetic CO2 hydration in the cyanobacterium, Synechococcus sp. PCC7942. Mol. Microbiol. 43, 425–435 (2002).
Battchikova, N., Eisenhut, M. & Aro, E. M. Cyanobacterial NDH-1 complexes: novel insights and remaining puzzles. Biochim. Biophys. Acta 1807, 935–944 (2011).
Antonovsky, N. et al. Sugar synthesis from CO2 in Escherichia coli. Cell 166, 115–125 (2016).
Aguilera, J., Van Dijken, J. P., De Winde, J. H. & Pronk, J. T. Carbonic anhydrase (Nce103p): an essential biosynthetic enzyme for growth of Saccharomyces cerevisiae at atmospheric carbon dioxide pressure. Biochem. J. 391, 311–316 (2005).
Sirard, J. C., Mock, M. & Fouet, A. The three Bacillus anthracis toxin genes are coordinately regulated by bicarbonate and temperature. J. Bacteriol. 176, 5188–5192 (1994).
Abuaita, B. H. & Withey, J. H. Bicarbonate induces Vibrio cholerae virulence gene expression by enhancing ToxT activity. Infect. Immun. 77, 4111–4120 (2009).
Baba, T. et al. Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection. Mol. Syst. Biol. 2, 2006.0008 (2006).
Rubin, B. E. et al. The essential gene set of a photosynthetic organism. Proc. Natl Acad. Sci. USA 112, E6634–E6643 (2015).
Oakes, B. L., Nadler, D. C. & Savage, D. F. in Methods in Enzymology, Vol. 546 (eds Doudna, J. A. & Sontheimer, E. J.) Ch. 23, 491–511 (Academic, 2014).
Dobrinski, K. P., Longo, D. L. & Scott, K. M. The carbon-concentrating mechanism of the hydrothermal vent chemolithoautotroph Thiomicrospira crunogena. J. Bacteriol. 187, 5761–5766 (2005).
Katoh, K. & Standley, D. M. MAFFT multiple sequence alignment software version 7: improvements in performance and usability. Mol. Biol. Evol. 30, 772–780 (2013).
Price, M. N., Dehal, P. S. & Arkin, A. P. FastTree: computing large minimum evolution trees with profiles instead of a distance matrix. Mol. Biol. Evol. 26, 1641–1650 (2009).
Zallot, R., Oberg, N. O. & Gerlt, J. A. ‘Democratized’ genomic enzymology web tools for functional assignment. Curr. Opin. Chem. Biol. 47, 77–85 (2018).
Dehal, P. S. et al. MicrobesOnline: an integrated portal for comparative and functional genomics. Nucleic Acids Res. 38, D396–D400 (2010).
Sievers, F. & Higgins, D. G. Clustal Omega for making accurate alignments of many protein sequences. Protein Sci. 27, 135–145 (2018).
Letunic, I. & Bork, P. Interactive tree of life (iTOL) v3: an online tool for the display and annotation of phylogenetic and other trees. Nucleic Acids Res. 44, W242–W245 (2016).
Slabinski, L. et al. XtalPred: a web server for prediction of protein crystallizability. Bioinformatics 23, 3403–3405 (2007).
Kelley, L. A., Mezulis, S., Yates, C. M., Wass, M. N. & Sternberg, M. J. E. The Phyre2 web portal for protein modeling, prediction and analysis. Nat. Protoc. 10, 845–858 (2015).
Roy, A., Kucukural, A. & Zhang, Y. I-TASSER: a unified platform for automated protein structure and function prediction. Nat. Protoc. 5, 725–738 (2010).
Newby, Z. E. R. et al. A general protocol for the crystallization of membrane proteins for X-ray structural investigation. Nat. Protoc. 4, 619–637 (2009).
Khalifah, R. G. The carbon dioxide hydration activity of carbonic anhydrase. J. Biol. Chem. 246, 2561–2573 (1971).
We thank A. Deutschbauer and M. Price for assistance with RB-TnSeq experiments and analysis, respectively; Z. Netter and K. Seed (V. cholerae), and D. Portnoy and R. Calendar (B. anthracis Sterne) for providing genomic DNA samples; A. Martin and J. Bard for assistance with stopped-flow experiments- E. Charles, W. Fischer, B. Forster, B. Long, R. Nichols, D. Price and P. Shih for conversations and comments on the manuscript. X-ray-based experiments were performed at the Lawrence Berkeley National Laboratory Advanced Light Source Beamline 8.3.1. J.J.D. was supported by National Institute of General Medical Sciences grant (T32GM066698). A.I.F. and T.G.L. were supported by a National Science Foundation Graduate Research Fellowship. C.B. was supported by an International Postdoctoral grant from the Swedish Research Council (637-2014-6914). D.F.S. was supported by a National Science Foundation grant (MCB-1818377; for genetic screen) and by a US Department of Energy Grant (DE-SC00016240; for DAB characterization).
The Regents of the University of California have filed a patent related to this work that lists J.J.D., A.I.F. and D.F.S. as inventors. D.F.S. is a co-founder of Scribe Therapeutics and a scientific advisory board member of Scribe Therapeutics and Mammoth Biosciences. All other authors declare no competing interests.
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Legends for Supplementary Files 1–5, Supplementary Table 1 and Supplementary Figs. 1–10.
Information on important strains and reagents used in the study.
Transposon insertion information and essentiality determinations broken down by gene. Data are from two technical replicates of the library mapping experiment. P values were calculated using a one-tailed binomial test as defined in the methods. P values are provided both before and after a Bonferroni correction. The numbers of transposon insertions seen for each gene in each replicate are also provided.
Fitness effects and HCR phenotypes broken down by gene. Data are from two replicates of the competitive growth assay.
FASTA file containing the genes used to generate Supplementary Fig. 4a.
FASTA file containing the genes used to generate Fig. 5a.
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Desmarais, J.J., Flamholz, A.I., Blikstad, C. et al. DABs are inorganic carbon pumps found throughout prokaryotic phyla. Nat Microbiol 4, 2204–2215 (2019). https://doi.org/10.1038/s41564-019-0520-8
Nature Microbiology (2019)