BacA(SbmA) importer of legume symbiotic NCR peptides: Protein architecture, function, and evolutionary implications

Some legumes encode families of NCR (Nodule-Cysteine-Rich) peptides that cause their rhizobial partners to terminally differentiate during the development of a nitrogen-fixing symbiosis. <i>Sinorhizobium meliloti,</i> whose plant hosts <i>Medicago truncatula</i> and <i>Medicago sativa</i> express <i>ca.</i> 600 NCR peptides during root nodule development, possesses a symbiotically essential BacA<i>_Sm</i> protein that imports certain NCR peptides into the cytoplasm. This import permits proteolytic degradation of the NCR peptides, thereby protecting the endocytosed bacteria from their antimicrobial peptide-like lethality, while also allowing certain NCR peptides to undergo their symbiotically critical interactions with cytoplasmic components, for example heme-sequestration in the case of NCR247. Our study employed 54 <i>S. meliloti bacA</i>_<i>Sm</i> missense mutants (35 to cysteine and 19 to glycine) that we tested for protein production, ability to establish a nitrogen-fixing symbiosis, and their susceptibility to killing by higher levels of the NCR247 and the Bac7(1-35) peptides. We also used the Single Cysteine Accessibility Method to make topological inferences. Our detailed genetic, biochemical, structural, and physiological analyses have revealed that BacA_<i>Sm</i> and SbmAhomodimers function as finely tuned transporters, whose structures can be relatively easily disrupted by single amino acid changes. Our finding that several mutations that differentially separate nitrogen-fixation, NCR247 import, and Bac7(1-35) import map to the lining of the peptide-binding cavity suggests a molecular explanation underlying the paradoxical observation that SbmA/BacAs from pathogens can fully replace BacA<i>_Sm</i>, whereas BacAs from other rhizobia cannot.