DABs are inorganic carbon pumps found in chemolithoautotrophs and heterotrophic human pathogens
The full complement of genes important for CO2 concentration in chemolithoautotrophs was unknown. Using a genetic screen we identified CO2 concentrating mechanism genes. We then characterized an inorganic carbon pump from chemolithoautotrophs and identified functional homologs from human pathogens.
Nearly all biological CO2 fixation is due to the Calvin-Benson-Bassham cycle and, in particular, to the enzyme Rubisco. Rubisco evolved early in Earth's history when CO2 was abundant and O2 was virtually absent from the atmosphere. Today the opposite is true: today’s atmosphere is about 21% O2 and 0.04% CO2. This is a problem for organisms that depend on Rubisco today because Rubisco can react with O2 instead of CO2. Many autotrophic organisms have evolved CO2 concentrating mechanisms - CCMs - that increase the CO2 concentration near Rubisco to inhibit the reaction with O2 and saturate CO2 fixation.
The bacterial CCM relies on the activity of an enormous and impressively complex protein organelle called the carboxysome that our lab has been studying for almost a decade. However, around the time Jack Desmarais joined the lab it was still unclear whether we had a complete list of CCM genes. We were curious about the role of two classes of CCM genes: inorganic carbon transporters and Rubisco chaperones. Rubisco often depends chaperones to become fully active, so we were curious if these chaperones are required for the CCM. Similarly, all bacterial CCMs require Ci transport activity, but the proteobacterial Ci transporter was unknown!
We were happy to discover that our neighbors in the Arkin and Deutschbauer lab had just developed a new and much-simplified method for doing whole-genome screens in batch. After a summer of optimization, we had a library of ~100,000 mutants in the high CO2 incubator. This library enabled us to map essential genes and search for genes with CCM phenotypes. We were able to identify new CCM components including a Rubisco chaperone, and two small operons DAB1 and DAB2. Kathleen Scott’s lab had recently shown similar operons act as Ci transporters in deep-sea thermal-vent bacteria. Using an E. coli reporter strain, we showed that two genes from the DAB2 cluster were necessary and sufficient to drive HCO3- uptake in E. coli. A series of follow up experiments suggested that this uptake activity is due to a “vectorial carbonic anhydrase” at the cell membrane. It's pretty hard to imagine a biochemical mechanism that coupled a carbonic anhydrase to energy transduction at a membrane, but this sort of activity has been implicated in the CCM for 20+ years. We think the DAB-type transporters might hold the key to understanding vectorial carbonic anhydrases because they are relatively simple two-component systems that are found throughout the prokaryotic tree of life. We also found DAB operons in a large number of heterotrophic organisms including a pair of functioning operons in the notable human pathogens Anthrax and Cholera. Interestingly, just 3 days ago an article came out linking a homologous operon to growth and virulence in Staphylococcus aureus (https://www.nature.com/articles/s41467-019-11547-5).
We think the DABs are a great model for biological processes that involve the coupling of energy to catalysis at membranes and we are super curious about what they are doing in heterotrophs and pathogens. Studying these operons in diverse heterotrophs may shed light on the role of inorganic carbon in heterotrophic metabolism.
Image credit: Rachel Shipps
Our full paper is available here: https://www.nature.com/articles/s41564-019-0520-8