Isolation And Identification Of Pseudomonas G4 And The Study Of Its Antibiotic Secondary Metabolic Components

Fluorescent Pseudomonas refer to the pseudomonas strains which could produce soluble fluorescent pigment,including Pseudomonas fluorescens,Pseudomonas putida,Pseudomonas choloeaphtis and so on,which belong to the family of Pseudomonadaceae.Fluorescent Pseudomonas strains are the most important biocontrol bacterium.The previous studies have shown fluorescent Peudonos could produce many kinds of antibiotic secondary metabolites such as pyoluteorin,2,4-diacetylphlo-roglucinol,pyrrolnitrin,phenazine-1-carboxyl acid,coronatine,siderophore,hydrogen cyanide and surfactant.Here,we isolated a strain named G4 from tobacco rhizosphere soil.Interestingly,it could produce surfactant and other metabolites,which inhibited some pathogens such as Staphylococcus aureus,Rhodococcus thodochrous and so on.The good inhibitory activity of G4 prompted us to study the main bioactive substances.The main contents and results were as follows:(1)426 fluorescent Pseudomonas strains were isolated from the different rhizosphere soils planting cucumber,leaf lettuce,leek,lettuce,brassica oleracea and tobacco.By oil displacement activity assay,we confirmed 51 strains could produce surfactant.Furthermore,the surfactant could be classified into different families analyzed by HPLC.A strain named G4 was selected for further study as it could suppress the growth of Staphylococcus aureus,Rhodococcus rhodochrous,Micrococcus luteus and so on.(2)Based on the 16S rDNA gene and rpoB gene sequences analysis,strain G4 should be positioned within the genus Pseudomonas.The 16S rDNA phylogentic analysis showed that strain G4 formed a cluster with Pseudomonas helmanticensis OHA11(T),while the rpoB gene tree showed strain G4 formed an out group to the clade containing Pseudomonas sp.46-1 and Pseudomonas sp.46-2.G4 was different from P.fluorescens as it couldn't hydrolyze starch.The fatty acids components assay indicated that strain G4 had a 71.8%match with Pseudomonas chlororaphis,Pseudomonas aureofaciens,Pseudomonas aurantiaca in the MIDI database.While,P.chlororaphis,PRaureofaciens,P.aurantiaca can produce pigment.On the basis of the data presented,strain G4 was named Pseudomonas sp.G4.(3)To provide more means for isolation and identification the bioactive substances,the metabolites of strain G4 was predicted and analyzed at first.PCR was carried out to amplify the part of the functional genes of Plt,Prn,PCA,Phl,NRPS and PKS in Pseudomonas.The result showed that G4 might have two sets of NRPS and one set of PKS biosynthetic gene cluster at least.The common antibiotic secondary metabolites produced by Pseudomonas was detected such as phosphatide,glycolipid,lipopeptid,siderophore and hydrogen cyanide by using different methods.The result showed that G4 might produce lipopeptide surfactant and a kind of volatile antibiotic named hydrogen cyanide.(4)Acid precipitation was employed to obtain the crude bioactive substances of G4 secondary metabolites from the fermentation centrifugal supernatant by using the 6mol/L HCl adjusting the pH to 2.0.Then the crude was extracted by methanol and the centrifugal supernatant was evaporated.The crude extract was further purified by flash chromatography employing the 300-400 mesh silica gel while collected fractions were appraised with the biological activity assays.According to the antibacterial test,three active components named Z2,Z4,Z5 was determined.By ESI-MS,Z2 had the molecular weight of 506.3Da.There were two kinds of components in Z4,the molecular weight was 562.3 Da and 592.3Da respectively.Z4 was also surface active.Thus,it was speculated that Z4 may be a mixture of two kinds of lipopeptide biosurfactants which was consistented with the genetic prediction result.Z5 had the molecular weight of 319.1Da.It was speculated that Z5 was a kind of antibiotic secondary metabolite produced by Pseudomonas named coronatine based on the result of the thin-layer chromatography and the physicochemical properties of Z5.(5)To examine the antibacterial activity of Z2,Z4,Z5,an expanded indicator panel composed of 30 strains of clinically-relevant pathogens S.aureus and 6 pathogenic bacteria strains were tested.The result indicated Z2 had a relative narrow antibacterial spectrum as it only showed slight inhibition to Psychrobacter faecalis,Micrococcus luteus,Rhodococcus rhodochrous,Bacillus subtilis A47,Staphylococcus aureus and two strains of clinically-relevant pathogens S.aureus.However,Z4 and Z5 exhibited evident inhibitory activities to almost all kinds of pathogens.Strain G4 could produce biosurfactant,hydrogen cyanide and other kinds of antibiotic secondary metabolites.The good biological activity of strain G4 has potential application value in biological degradation,food and drug processing,biological control and therapeutic fields.For Strain G4,further researches were required to optimize the fermentation conditions to increase the yield,and then optimize the separation conditions of the crude to obtain a good purity component for the structure elucidation.