title: Disulfide cross-linking influences symbiotic activities of nodule peptide NCR247.
abstract: Interactions of rhizobia with legumes establish the chronic intracellular infection that underlies symbiosis. Within nodules of inverted repeat-lacking clade (IRLC) legumes, rhizobia differentiate into nitrogen-fixing bacteroids. This terminal differentiation is driven by host nodule-specific cysteine-rich (NCR) peptides that orchestrate the adaptation of free-living bacteria into intracellular residents. Medicago truncatula encodes a family of >700 NCR peptides that have conserved cysteine motifs. NCR247 is a cationic peptide with four cysteines that can form two intramolecular disulfide bonds in the oxidized forms. This peptide affects Sinorhizobium meliloti transcription, translation, and cell division at low concentrations and is antimicrobial at higher concentrations. By preparing the three possible disulfide-cross-linked NCR247 regioisomers, the reduced peptide, and a variant lacking cysteines, we performed a systematic study of the effects of intramolecular disulfide cross-linking and cysteines on the activities of an NCR peptide. The relative activities of the five NCR247 variants differed strikingly among the various bioassays, suggesting that the NCR peptide-based language used by plants to control the development of their bacterial partners during symbiosis is even greater than previously recognized. These patterns indicate that certain NCR bioactivities require cysteines whereas others do not. The results also suggest that NCR247 may exert some of its effects within the cell envelope whereas other activities occur in the cytoplasm. BacA, a membrane protein that is critical for symbiosis, provides protection against all bactericidal forms of NCR247. Oxidative folding protects NCR247 from degradation by the symbiotically relevant metalloprotease HrrP (host range restriction peptidase), suggesting that disulfide bond formation may additionally stabilize NCR peptides during symbiosis.
authors.affiliation: Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139;
authors.forename: Mohammed
authors.initials: M
authors.lastname: Shabab
authors.affiliation: Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139;
authors.forename: Markus F F
authors.initials: MF
authors.lastname: Arnold
authors.affiliation: Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139;
authors.forename: Jon
authors.initials: J
authors.lastname: Penterman
authors.affiliation: Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA 02139;
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Wommack
authors.affiliation: Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139;
authors.forename: Hartmut T
authors.initials: HT
authors.lastname: Bocker
authors.affiliation: Department of Microbiology and Molecular Biology, Brigham Young University, Provo, UT 84602.
authors.forename: Paul A
authors.initials: PA
authors.lastname: Price
authors.affiliation: Department of Microbiology and Molecular Biology, Brigham Young University, Provo, UT 84602.
authors.forename: Joel S
authors.initials: JS
authors.lastname: Griffitts
authors.affiliation: Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA 02139; gwalker@mit.edu lnolan@mit.edu.
authors.forename: Elizabeth M
authors.initials: EM
authors.lastname: Nolan
authors.affiliation: Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139; gwalker@mit.edu lnolan@mit.edu.
authors.forename: Graham C
authors.initials: GC
authors.lastname: Walker
mesh_terms: D020816:Amino Acid Motifs; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D003545:Cysteine / Q000737:chemistry; D004220:Disulfides / Q000737:chemistry; D015964:Gene Expression Regulation, Bacterial; D018506:Gene Expression Regulation, Plant; D058507:Host Specificity; D046913:Medicago truncatula / Q000235:genetics / Q000378:metabolism / Q000382:microbiology*; D026901:Membrane Transport Proteins / Q000235:genetics* / Q000378:metabolism; D009586:Nitrogen Fixation; D010455:Peptides / Q000235:genetics* / Q000378:metabolism / Q000494:pharmacology; D010940:Plant Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000494:pharmacology; D053204:Root Nodules, Plant / Q000235:genetics / Q000378:metabolism / Q000382:microbiology; D015398:Signal Transduction; D016962:Sinorhizobium meliloti / Q000187:drug effects* / Q000235:genetics / Q000254:growth & development / Q000378:metabolism; D013329:Structure-Activity Relationship; D013559:Symbiosis / Q000235:genetics*
chemical_list: D001426:Bacterial Proteins; D004220:Disulfides; D026901:Membrane Transport Proteins; D010455:Peptides; D010940:Plant Proteins; C082397:bacA protein, Rhizobium meliloti; D003545:Cysteine
keywords: host peptides; host--microbe interactions; oxidative folding; regioisomers; signaling peptides