A Preliminary Study Of The Biological Function And The Transcriptional Regulation Of The Phosphate Specific Transport System(pstSCAB)in Amycolatopsis Mediterranei U32

Phosphorus is one of the key elements for biological productivity,and the research of its metabolism has an important position in both industrial and agricultural fields.In comparision with the nitrogen and carbon metabolisms,fewer studies were carried out in the phosphate metabolism.Although a lot of researchers are studying the phosphate transportation system to uncover the mechanism of phosphate metabolism,major studies are limited in Escherichia coli and Bacillus subtilis,and little is known in actinomycetes that are widely used in industry.is relatively The balance of carbon,nitrogen and phosphorus nutrition was often mentioned in agricultural and industry.Therefore,the study of phosphate metabolism and its regulation can help better understand the whole metabolic networks and their coordinatory regulation.Amycolatopsis mediterranei belongs to the actinomycete family,and is widely used for production of rifamycin SV and B in industry.Optimization of the fermentation medium has been proved to be an effective method to improve the rifamycin yield.For example,addition of nitrate into the medium can significantly enhance the rifamycin production.However,besides the nitrogen sources,it is still unclear whether phosphate supply can affect the rifamycin production.Therefore,this study mainly focused on the study of the relationship between phosphate metabolism and its relationship with rifamycin production.As a rare actinomycete,A.mediterranei U32 does not produce spores during the growth,which is probably caused by continuous mutagenesis,making it is impossible to introduce foreign DNA into the strain via conjugation.Therefore,the electroporation method has been established in our lab to introduce foreign DNA into U32.However,because of the relatively low efficiency,it is difficult to obtain double cross-over mutants.Therefore,in this study,we first improved the electroporation efficiency from optimizing the following conditions,including cell age,protectant,voltage,resistance and capacity.Then,orthogonal experiment was employed to obtain the best combination,e.g.cell age 12 h,voltage 1.76 KV,resistance 900 ?and capacity 50 ?F.After electroporation,cells were restored in liquid Bennet medium for 4 h and then plated on Bennet plate supplemented with 0.25 ?l/ml soybean oil and appropriate antibiotics.With this condition,the electroporation efficiency was largely improved by 100 times,reaching 3.6*104 transformants per microgram plasmids.The phosphate specific transporter?pstSCAB?was knocked out with the improved electroporation system,obtaining the pstS null mutant ?pstS.Then,pstS was introduced back into to generate its complementation strain,pstS+.Different growth conditions were used to study the influence of pstS mutation on the bacterial growth and phosphate metabolism.Moreover,scanning electron microscope and confocal microscope were used to study the mycellium of the mutant,which was found to be aggregated.Pro-Q Diamond Phosphoprotein Gel Staining was also employed to study the changes of the phosphorylation level of the proteome after knock out of pstS in both U32 and E.coli BW25113 and distinct differences were observed.With mass spectrum,proteins with significant changes in phosphorylation were identified and the phosphorylation of most proteins decreased in U32 except AMED₃228.The protein was then expressed in E.coli and purified for characterization of its biochemical properties.In this study,the transcription of pstSCAB was also studied,and the four genes in this cluster were proved to be cotranscribed,forming an operon.With RT-PCR technology,we found that the transcription of the operon was positively regulated by GlnR.With EMSA,GlnR was found to directly bind the promoter of pstS,and the protection sites were further characterized by DNase I Footprinting assay,which were from-97 to-106 bp and from-112 to-123 bp relative to the translation start site of pstS.In both GlnR protected regions,two GlnR binding sites of "cctac" were identified,which are typically recognized by GlnR.Therefore,GlnR positively regulates the transportation of phosphate via directly regulating the transcription of pstSCAB operon.Taken together,above evidences obviously showed that both nitrogen and phosphate metabolisms are closely related in A.mediterranei U32.