Identification of extracytoplasmic proteins in Bradyrhizobium japonicum using phage display.
A novel gene bank of Bradyrhizobium japonicum USDA110spc4 was constructed using pG3DSS, a phagemid vector designed for detecting genes encoding secreted proteins. In this phagemid, the phage protein III lacks its indigenous signal peptide required for protein secretion, thus recombinant fusion proteins are displayed on the phage surface only if a functional signal peptide is provided by an inserted DNA fragment. In addition, the N-terminal half of protein III has been replaced by a short linker region (the E-tag) that is recognized by a monoclonal antibody, which enables isolation of phages displaying a fusion protein. The expression library described here, therefore, provides a powerful means to affinity select for B. japonicum genes encoding extracytoplasmic proteins. In total, 182 DNA sequences were analyzed, among which 132 different putative extracytoplasmic proteins could be identified. The function of most proteins could be predicted and support an extracytoplasmic localization. In addition, genes encoding novel extracytoplasmic proteins were found. In particular, a novel family of small proteins has been identified that is characterized by a conserved pattern of four cysteine residues.
Rosander
Anna
A
SLU, Department of Microbiology, P.O. Box 7025, SE-750 07 Uppsala, Sweden.
Frykberg
Lars
L
Ausmees
Nora
N
Müller
Peter
P
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics*; D020369:Bradyrhizobium / Q000235:genetics*; D008969:Molecular Sequence Data; D019151:Peptide Library; D021382:Protein Sorting Signals / Q000235:genetics*; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
D001426:Bacterial Proteins; D019151:Peptide Library; D021382:Protein Sorting Signals