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.
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.
Sutton
J M
JM
John Innes Centre, Colney, Norwich, U.K.
Peart
J
J
Dean
G
G
Downie
J A
JA
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
D000911:Antibodies, Monoclonal; D001426:Bacterial Proteins; D002135:Calcium-Binding Proteins; C062095:NodO protein, Rhizobium leguminosarum; D021382:Protein Sorting Signals; D011994:Recombinant Proteins