Study On Biological Functions Of Crystalline Inclusion Proteins Of Entomopathogenic Bacterium Photorhabdus

Entomopathogenic bacteria Photorhabdus, symbiotically associated with nematodes of the family Heterorhabditidae, exist as the key role in the mass production of the nematodes. Two types of proteinaceous crystalline inclusion proteins designated CipA and CipB are found in the phase I cells of Photorhabdus spp. The crystal proteins, whose amino acid composition is similar with nutrient requirements of nematodes S. glaseri, are not energy reserves for bacterial cells. It is believed that the proteins are involved in the symbiosis. The association between bacterium-nematode is a complex interaction and the biological significance for the proteins is undefined. The research in the important proteins produced by symbiotic bacterium will be helpful to explore nematode-bacterium symbiotic mechanism, but also promote the commercialization of nematode products.To explore the function of crystalline inclusion proteins in the symbiotic system, the genes encoding CipA and CipB from P. luminescens H06 were obtained by amplification and expressed in protocaryotic (Escherichia coli) and eukaryotic (Saccharomyces cerevisiae) expression system, respectively. Co-culture systems of recombinant bacteria or yeasts and three Entomopathogenic nematode strains [Steinernema longicaudum X-7 (X-7), S. carpocapsae All (All) and Steinernema sp. Sy-5 (Sy-5)], or Panagrellus redivivus, were set up to determine the influence of expressed proteins on the development of target nematodes.A BLAST analysis of the cipA and cipB genes of H06 revealed 93% identity to the corresponding genes of P. luminescens Hm that had been reported in GenBank.Based on the construction pCR4-TOPO-cipA and pCR-TOPO-cipB clone vectors for identification and conservation, pET expression system was applied to construct two high level protocaryotic expression vectors, namely pET-15b-cipA and pET-15b-cipB, which were transformed respectively into protease-deficient E. coli BL21 (DE3) host cells by conventional chemical transformation protocol. Positive clones were screened on Carbenicillin selection plates. After over 100 passages, both recombinant strains showed stable plasmid inheritance and consistence. Meanwhile, the Cip expression level remained 27%-35% of the total bacterial protein. When recombinant E. coli were induced with 1 mmol/L IPTG added at the early exponential phase for 8 h at 37°C, CipA and CipB accumulated as inclusion bodies constituted 30.9% and 32.6% of the total bacterial protein, respectively. There was little leakage expression when induced. With a warm extract method, recombinant...