Two putative phosphate binding proteins of Bradyrhizobium japonicum, detected by phage display

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

Department of Microbiology, SLU, P.O. Box 7025, SE-750 07 UPPSALA, SWEDEN, Philipps University Marburg, Cell Biology and Applied Botany, Karl-von-Frisch-Str., 35032 MARBURG, GERMANY

A novel gene bank of Bradyrhizobium japonicum USDA110 was constructed using pG3DSS, a phagemid vector designed for detecting genes encoding secreted proteins. This phagemid contains the truncated gene of phage protein III (C-terminus) which normally is translocated into the cytoplasmic membrane and finally to envelop the phage DNA molecule. However, its 5'-gene region encoding the N-terminus has been replaced by a short linker region (E-tag protein) which is recognized by a monoclonal antibody. In pG3DSS the phage protein III lacks its indigenous leader sequence required for protein secretion, thus recombinant protein fusions are displayed only if functional signal sequences are provided by suitable genomic DNA fragments. This expression library therefore provides a powerful means to screen for B. japonicum genes encoding sec-dependent periplasmic proteins and proteins of the inner and outer membranes. Among 200 clones, analysed by DNA sequencing, two clones were found to be highly similar to phosphate binding proteins. Currently some cloning strategies are developped to create defined B. japonicum mutants and to analyse the functional importance of these new loci for the uptake of phosphates and the symbiotic interaction with soybeans.