Rickettsial ompB Promoter Regulated Expression of GFPuv in Transformed Rickettsia montanensis 英文参考文献.docVIP

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Rickettsial ompB Promoter Regulated Expression of GFPuv in Transformed Rickettsia montanensis 英文参考文献.doc

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Rickettsial ompB Promoter Regulated Expression of GFPuv in Transformed Rickettsia montanensis 英文参考文献

RickettsialompBPromoterRegulatedExpressionof GFPuv inTransformedRickettsiamontanensis GeraldD.Baldridge*,NicoleY.Burkhardt,AdelaS.Oliva,TimothyJ.Kurtti,UlrikeG.Munderloh DepartmentofEntomology,UniversityofMinnesota,St.Paul,Minnesota,UnitedStatesofAmerica Abstract Background: Rickettsia spp. (Rickettsiales: Rickettsiaceae) are Gram-negative, obligate intracellular, a-proteobacteria that have historically been associated with blood-feeding arthropods. Certain species cause typhus and spotted fevers in humans, but others are of uncertain pathogenicity or may be strict arthropod endosymbionts. Genetic manipulation of rickettsiaeshouldfacilitateabetterunderstandingoftheirinteractionswithhosts. Methodology/PrincipalFindings:Wetransformedaspeciesneverassociatedwithhumandisease,Rickettsiamontanensis, byelectroporationwithaTN5transposon(pMOD700)containinggreenfluorescentprotein(GFPuv)andchloramphenicol acetyltransferase(CAT)genesunderregulationofpromotersclonedfromtheRickettsiarickettsiiompBgene,andisolateda Chloramphenicol-resistant GFP-fluorescent rickettsiae population (Rmontanensis700). The Rmontanensis700 rickettsiae containedasingletransposonintegratednearanacetyl-CoAacetyltransferasegeneintherickettsialchromosome.Northern blots showed that GFPuv and CAT mRNAs were both expressed as two transcripts of larger and smaller than predicted length. Western immunoblots showed that Rmontanensis700 and E. coli transformed with a plasmid containing the pMOD700transposonbothexpressedGFPuvproteinsofthepredictedmolecularweight. Conclusions/Significance:Long-standingbarrierstotransformationofrickettsiaehavebeenovercomebydevelopmentof transposon-based rickettsial transformation vectors. The ompB promoter may be the most problematic of the four promoterssofaremployedinthosevectors. Citation: Baldridge GD, Burkhardt NY, Oliva AS, Kurtti TJ, Munderloh UG (2010) Rickettsial ompB Promoter Regulated Expression of GFPuv in Transformed Rickettsiamontanensis.PLoSONE5(1):e8965.doi:10.1371/jo