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Identification of Extracytoplasmic Proteins in Bradyrhizobium japonicum Using Phage Display

August 2003 , Volume 16 , Number  8
Pages  727 - 737

Anna Rosander , 1 Lars Frykberg , 1 Nora Ausmees , 1 and Peter Müller 2

1SLU, Department of Microbiology, P.O. Box 7025, SE-750 07 Uppsala, Sweden; 2Philipps University Marburg, Cell Biology and Applied Botany, Karl-von-Frisch-Str. 8, D-35032 Marburg, Germany

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Accepted 4 April 2003.

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 In addition, the N-terminal half of protein III has been replaced by a short linker region (the E-tag) that is recognized by a monoclonal antibody, which enables isolation of phages displaying a fusion protein.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.

© 2003 The American Phytopathological Society