Abstract
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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Data availability
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.
Code availability
All custom code is available on GitHub at https://github.com/jackdesmarais/DabTransporterPaper.
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Acknowledgements
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).
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J.J.D., A.I.F. and D.F.S. conceived, designed and supervised this study, and wrote the final manuscript with input from all of the authors; J.J.D. performed and analysed most of the biochemical experiments. C.B. performed CA activity assays. J.J.D., E.J.D. and K.W. generated biochemical reagents and strains. T.G.L. performed size-exclusion chromatography. J.J.D., T.G.L., L.M.O. and J.Y.W. developed the purification strategy. J.J.D., T.G.L. and L.M.O. developed the X-ray fluorescence assays. J.J.D. and A.W.C. generated the RB-TnSeq library. J.J.D. and S.D. generated the phylogenetic trees. All of the authors reviewed and approved the final manuscript.
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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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Supplementary Information
Legends for Supplementary Files 1–5, Supplementary Table 1 and Supplementary Figs. 1–10.
Supplementary File 1
Information on important strains and reagents used in the study.
Supplementary File 2
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.
Supplementary File 3
Fitness effects and HCR phenotypes broken down by gene. Data are from two replicates of the competitive growth assay.
Supplementary File 4
FASTA file containing the genes used to generate Supplementary Fig. 4a.
Supplementary File 5
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
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DOI: https://doi.org/10.1038/s41564-019-0520-8
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