The Study Of Prodigiosin Synthesis And Regulation Related Proteins

Prodigiosin is a red-pigmented secondary metabolite produced by actinomycetas,Serratia marcescens and some other bacteria.Prodigiosin is synthesized through a bifurcated biosynthesis pathway,MBC(4-methoxy-2,2’-bipyrrole-5-carbaldehyde)and MAP(2-methyl-3-n-amyl-pyrrole).PigG is an Acyl carrier protein(ACP)and have been shown to be involved in the first steps of MBC biosynthesis(incorporation of proline).The phosphopantetheinylated form of PigG can covalently tethered to the L-prolyl-AMP which was converted from L-proline by PigI for next two steps dehydrogenation catalized by PigA to form the A ring in tripyrrole structure.To elucidate the catalytic mechanism of PigG,the gene of PigG from S.marcescens FS14 was cloned and expressed in E.coli C43.The purifed protein was then used for crystallization.The best crystal was diffracted to a resolution of 2.1 A.The structure of the PigG was solved by molecular replacement.The monomeric structure of PigG has a helical bundle fold containing four helices.Helix al runs approximately antiparallel to helix α2,which is the longest helix of the bundle(14 residues).A short helix α3 lies nearly perpendicular to helices α1 and α2.Helix α4 runs antiparallelly to helix α2 at an angle about 45°.Structural based sequence alignment showed that a conserved amino acid Ser36 is located at the beginning of α2 in all aligned acyl carrier proteins.Further study showed that no prodigiosin was produced after the in-frame deletion mutation of PigG gene or mutated Ser36 to Ala.These results indicate that PigG and the conserved Ser36 are important in the prodigiosin biosynthesis,and Ser36 in PigG may be involved in ligand binding.CpxRA is an important two-component regulatory system including histidine kinase CpxA and a response regulator CpxR.CpxA is reported to mainly involve in response to the pressure changes of outer envelope.The CpxA accepts signals from the environment,and phosphorylates the CpxR,the phosphorylates CpxR then functions as a transcription factor to regulate expression of target genes and helps bacteria respond to environmental changes.In this study,It was found that the in-frame cpxA deletion mutation of FS14 affected the prodigiosin biosynthesis,bacterial swimming,swarming motility,antibiotic resistance,and secretion of 50kDa proteases.In order to study the regulatory mechanism of CpxRA,the promoter of pigA-O was fused to reporter lacZ gene,and galactosidase activities were measured in different mutants and wild type strain FS14 respectively.The galactosidase activitiy of FS14△CpxApigA-lacZ decreased dramatically compared to FS14pigA-lacZ,while almost no differences could be found among FS14pigA-lacZ,FS14ACpxRpigA-lacZ and FS14△CpxR+ApigA-lacZ This indicates that the CpxA may regulate the synthesis of prodigiosin by regulating the transcription of pigA-O.