Sequencing And Analysing The Genes Of Stenotrophomonas Maltophilia D2Genaral Secretory Pathway Proteins

Objective We obtained an protein-hyperproducing Stenotrophomonas maltophilia strain D2from the digestive tract of Perinereis aibuhitensis Grube. Based on the sequenced genes, we found the gene of this protein was clustered by genes of general secretory pathway proteins. So we speculated the type Ⅱ secretion system may be responsible for the the hyperproducion of SMP in D2. We expect to know more about the high level protein secretion mechanism of D2according to exploring the type II machinery. Methods The genes of SMP was blasted and it had a high identity with AKP gene sequences of Stenotrophomonas maltophilia K279a and R551-3correspondingly was discovered. The forward primers were designed according to the high conserved regions of K279a and R551-3, the reward primers were designed based on the sequenced genes by the methord of gene moving. Then the PCR amplified products were connected to pMD18-T-vector and transformed into Escherichia coli JM109by TSS. After sceened by white and blue clonies and identificated by double enzymes cutting, the positive clonies with the genes of interest were sequenced. The obtained gene fragments were spliced and blasted. The homology of the obtained ORFs were analysed by Clustalx and the phylogenetic relationships by MEGA3.0. Results Seven gene framents were obtained and eleven ORFs were found and named GspF, GspE, GspD, GspM, GspL, GspK, GspG, GspJ, Gspl, GspH, GspC, and their GeneBank numbers were GU377211、HM151387、JQ070336、JQ070343、JQ070342、 JQ07034、JQ070337、JQ070340、JQ070339、JQ070338、JQ070335successively. The translted amino acid sequences of the former ORFs had high identity with the corresponding proteins of both Stenotrophomonas maltophilia R551-3and K279a. The phylogenetic analyse indicated the sequenced nine proteins were homologous with the correspongding proteins of the same kind bacteria. Conclusion We obtain the complete primary structure of general secretory pathway gene cluster. The results of homologous and phylogenetic analysis lay the foundation for exploiting protein-hyperproducing mechanism of D2.