title: Analysis of the C-terminal secretion signal of the Rhizobium leguminosarum nodulation protein NodO; a potential system for the secretion of heterologous proteins during nodule invasion.
abstract: We used deletions to analyze the domains required for secretion of the Rhizobium leguminosarum bv. viciae nodulation protein, NodO, by the sec-independent pathway. Deletion of the C-terminal 24 amino-acids (residues 261 to 284) reduced secretion by at least 95%. A monoclonal antibody that recognizes the C-terminal domain of NodO was used to identify four nested deletions that retained the C-terminal 24 residues of NodO but had lost up to 133 residues (amino acids 128 to 259); all four proteins were secreted into the growth medium with an efficiency between 50 and 90% of normal. A deleted derivative of NodO that retained residues 1 to 21 and 167 to 284 (and therefore lacked most of the N-terminal Ca(2+)-binding domain) was secreted at around 80% of normal efficiency. Taken together, these observations indicate that the C-terminal 24 amino acids are sufficient for NodO secretion although the region adjacent to this domain appears to affect secretion efficiency. A derivative of the Escherichia coli alkaline phosphatase (phoA) gene was cloned into two derivatives of nodO such that PhoA (lacking the N-terminal transit peptide) was in-frame at both ends, with the C terminus fused to either the last 24 or 50 amino acids of NodO. These fusion proteins were secreted at 40 and 80% of the wild-type level, respectively, and the larger of the two retained alkaline phosphatase activity. A hybrid protein, containing E. coli beta-glucuronidase (GUS) fused to the N terminus of NodO, was not secreted, and it reduced the levels of wild-type NodO secreted by R. leguminosarum bv. viciae. The nature of the NodO C-terminal secretion signal is discussed with regard to its use as a delivery system for heterologous proteins useful for investigating the Rhizobium-legume interaction.
authors.affiliation: John Innes Centre, Colney, Norwich, U.K.
authors.forename: J M
authors.initials: JM
authors.lastname: Sutton
authors.forename: J
authors.initials: J
authors.lastname: Peart
authors.forename: G
authors.initials: G
authors.lastname: Dean
authors.forename: J A
authors.initials: JA
authors.lastname: Downie
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D002135:Calcium-Binding Proteins; D003001:Cloning, Molecular; D016384:Consensus Sequence; D004926:Escherichia coli; D005260:Female; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010944:Plants / Q000382:microbiology; D021382:Protein Sorting Signals / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D051381:Rats; D011919:Rats, Inbred Strains; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry; D015183:Restriction Mapping; D016961:Rhizobium leguminosarum / Q000502:physiology*; D017384:Sequence Deletion; D017386:Sequence Homology, Amino Acid
chemical_list: D000911:Antibodies, Monoclonal; D001426:Bacterial Proteins; D002135:Calcium-Binding Proteins; C062095:NodO protein, Rhizobium leguminosarum; D021382:Protein Sorting Signals; D011994:Recombinant Proteins