AnaToc75 (Alr2269) mediates calcium uptake across the outer membrane in <i>Anabaena</i> sp. PCC 7120

It has been a long-standing notion that cyanobacterial Omp85 proteins are essential because of their role in insertion of β-barrel proteins into the outer membrane (OM). Alr2269 from <i>Anabaena</i> sp. PCC 7120 is such an Omp85 protein, structurally related to the Toc75 channel of the translocon at the outer envelope membrane of chloroplasts but is actually non-essential to <i>Anabaena</i> under calcium-replete conditions. In a completely segregated <i>alr2269</i>-null mutant, a predicted S-layer protein, All3983, and many β-barrel proteins in the OM were upregulated compared with the wild type. By removal of each component from the medium, Ca²⁺ deficiency was identified to be the key environmental signal for upregulation of P<i>_all3983-luxAB</i> in the mutant. Assays of ⁴⁵Ca²⁺ uptake in the wild type and the mutant indicated that Alr2269 is a relatively specific channel for transport of Ca²⁺ across the OM in low-Ca²⁺ medium. Accordingly, inactivation of <i>alr2269</i> greatly reduced the growth under Ca²⁺-limiting conditions, while the complemented strain grew as the wild type. In low-Mg²⁺ medium, however, the <i>alr2269</i> mutant showed only a slight difference in growth from the wild type. These results establish Alr2269 (AnaToc75) as the main channel for Ca²⁺ uptake across the OM of <i>Anabaena</i> in low-Ca²⁺ environments.IMPORTANCECa²⁺ is required for photosynthesis and various cellular activities in cyanobacteria, but Ca²⁺ uptake, in particular how Ca²⁺ is transported across the outer membrane, has been barely investigated in cyanobacterial species. In this study, we found that a cyanobacterial Toc75 homolog is not necessarily essential for protein integration into the outer membrane as was thought before but instead is required for Ca²⁺ uptake in low-Ca²⁺ environments. This finding not only establishes a Ca²⁺ channel across the outer membrane of cyanobacteria but also provides an example of additional functions for Omp85 proteins.