Proteomics Researches On Metabolic Pathway Of Daptomycin In Streptomyces Roseosporus And Copper-resistance Of Proteus Hauseri ZMd44

Proteome and proteomics have developed quickly and applied in many researches in recent years. In this study, proteomics technology was used as the core method in the following researches:metabolic pathway of daptomycin in Streptomyces roseosporus and copper-resistant mechanism of Proteus hauseri ZMd44. Attempts were made to decipher the mechanisms at protein levels in Streptomyces roseosporus and Proteus hauseri.Precursor is necessary for daptomycin production. Herein, decanoic acid and sodium decanoate were chosen as the precursor. The former one was the most popular precursor regarding to other reports and the latter one has never been reported before. As a result, sodium decanoate was more preferred for daptomycin producing as it was water-soluble substance. Thus, it can be absorbed by cells easily.As precursor played an important role in daptomycin producing, further explaination for mechanism of daptomycin producing was expected. This study tried to explore the mechanism at protein level through proteomics techniques. Hence,1D-SDS-PAGE, MALDI-TOF-MS/MS and LC-MS/MS were used to analyze the differential proteins between precursor group and non-precursor group, respectively. As a result, five functional proteins which were closely related to daptomycin producing were identified, including putative regulatory protein, guanosine pentaphosphate synthetase, polyribonucleotide nucleotidyltransferase phosphorylase (PNPase), putative secreted tripeptidylamino peptidase and putative two-component system histidine kinase. Subsequently, whole proteome analysis was carried out with LC-MS/MS. As a result,601and935proteins were identified in precursor group and non-precursor group, including357and691different proteins respectively. The distribution of pI, molecular weight (Mw) and2D maps were then calculated and simulated. Based on their functions, differential proteins were classified into14groups. The "transport/membrane function" was the most different group, which occupancy of12.4%was in precursor group while that of5.2%was in non-precursor group.Finally, gene cluster of daptomycin synthesis was used to explain the mechanism. From the differential proteins which divided into five groups were corresponding to daptomycin synthesis. They were resistance protein, ATP-binding cassette transporter (ABC transporter), acyl carrier protein, methyltransferase and regulator. Furthermore, more daptomycin synthesis related proteins were expressed in the precursor group. However, peptide synthetases which directly related to daptomycin were never found in all the identified proteins. So it was supposed that precursor mainly affected the regulating genes of daptomycin synthesis to improve the production.Another part in this study was copper resistance of Proteus hauseri ZMd44. Through the one-dimensional and two-dimensional gel electrophoresis, MALDI-TOF-MS/MS and LC-MS/MS, it was found that more membrane proteins were induced under copper stress. They are supposed to the similar mechanism of RND system which was very important in copper resistance. The disulfide isomerase was also found in the identified protein. It can repair the copper-damaged disulfide bond, thus the cells can survived from the high level of copper. In addition, expression of outer membrane protein was inhibited so that the transport of copper into cells was decreased. The copper concentration will retain at a normal level in the cells making Proteus hauseri ZMd44can survive in a high copper concentration. The resistance of copper in Proteus hauseri could also apply in the prohibition of pathogen as the swarming motility was repressed.