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Identification of Extracytoplasmic Proteins in Bradyrhizobium japonicum Using Phage Display

Rosander, Anna, Frykberg, Lars, Ausmees, Nora, Müller, Peter
Molecular plant-microbe interactions 2003 v.16 no.8 pp. 727-737
Bradyrhizobium japonicum, DNA, bacteriophages, cysteine, gene banks, genes, monoclonal antibodies, nucleotide sequences, protein secretion, recombinant fusion proteins, signal peptide
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 In addition, the N-terminal half of protein III has been replaced by a short linker region (the E-tag) that is recognized by a monoclonal antibody, which enables isolation of phages displaying a fusion protein.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.