The BacA(SbmA) Importer of Symbiotically Important Legume Nodule Cysteine-Rich Peptides: Insights into Protein Architecture, Function, and Evolutionary Implications

Abstract

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. Sinorhizobium meliloti, whose plant hosts Medicago truncatula and M. sativa express ca. 600 NCR peptides during root nodule development, possesses a symbiotically essential BacASm 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. BacAs Escherichia coli ortholog SbmAEc can restore a wildtype phenotype to a{Delta} bacASm mutant. Our study employed 54 S. meliloti bacASm 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 BacASm and SbmA homodimers function as finely tuned import machines, whose structures can be relatively easily disrupted by single amino acid changes. Our discovery that several mutations that differentially separate nitrogen-fixation, NCR247 import, and Bac7(1-35) import map to the lining of the peptide-binding cavity in the outward-open SbmA/BacA conformation suggests a molecular explanation the other otherwise paradoxical observation that SbmA/BacAs from pathogens can fully replace BacASm, whereas BacAs from other rhizobia cannot. Significance StatementSinorhizobium meliloti BacASm and Escherichia coli SbmAEc are closely related proteins that function as homodimeric transporters to import peptides and other cargos through the cytoplasmic membrane into the cytoplasm. BacA is critical for S. meliloti to establish a nitrogen-fixing symbiosis with its legume hosts because of its ability to import Nodule Cysteine-Rich (NCR) nodule-specific plant peptides. This import protects the bacteria inside the nodule from the potentially lethal effects of these NCR peptides while also enabling NCRs to make their intracellular interactions that are necessary for symbiosis. Our extensive multidisciplinary studies offer new insights into function of BacA/SbmA transporters and provide a molecular explanation for why BacA/SbmA orthologs from mammalian pathogens can replace BacASm but those from other rhizobia cannot.