title: Construction of a native promoter-containing transposon vector for the stable monitoring of the denitrifying bacterium Pseudomonas stutzeri LYS-86 by chromosomal-integrated gfp.
abstract: Green fluorescent protein (GFP) is the most potential useful marker for the in situ monitoring of biofilm microbes. The objective of this study was to construct and compare the efficacy of transposon vectors containing native and foreign promoters in monitoring the denitrifying bacterium Pseudomonas stutzeri LYS-86 by chromosomal-integrated gfp. The promoter of nitrite reductase (Pnir) was cloned from LYS-86 and utilized to construct the transposon vector pUT/mini-Tn5-km2-Pnir-gfp. Another transposon vector, pUT/mini-Tn5-km2-Plac-gfp, containing the lactose promoter Plac was also constructed. These two transposon vectors and pUT-luxAB-gfp containing the promoter PpsbA were individually inserted into the chromosome of P. stutzeri LYS-86 by conjugation. Three GFP-tagged recombinant strains, LYS-Plac-gfp, LYS-Pnir-gfp, and LYS-PpsbA-gfp, were selected from the conjugants. Green fluorescence was observed only in LYS-Pnir-gfp, suggesting that the native promoter Pnir may be more suitable for GFP expression in P. stutzeri than the foreign promoters Plac and PpsbA. Indeed, LYS-Pnir-gfp maintained stable GFP fluorescence over 16 subcultures without significant changes in the denitrifying capacity.
authors.affiliation: College of Biological and Environmental Engineering, Zhejiang University of Technology, Hangzhou 310032, PR China.
authors.forename: Min
authors.initials: M
authors.lastname: Wei
authors.forename: Zhuoyi
authors.initials: Z
authors.lastname: Kong
authors.forename: Li
authors.initials: L
authors.lastname: Zhong
authors.forename: Lequan
authors.initials: L
authors.lastname: Qiu
authors.forename: Yansheng
authors.initials: Y
authors.lastname: Li
authors.forename: Lei
authors.initials: L
authors.lastname: Zhao
authors.forename: Xuanzhen
authors.initials: X
authors.lastname: Li
authors.forename: Weihong
authors.initials: W
authors.lastname: Zhong
mesh_terms: D001483:Base Sequence; D018441:Biofilms; D002876:Chromosomes, Bacterial; D003001:Cloning, Molecular; D004251:DNA Transposable Elements; D058440:Denitrification; D005786:Gene Expression Regulation; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000235:genetics* / Q000378:metabolism; D007785:Lactose / Q000235:genetics; D008969:Molecular Sequence Data; D009572:Nitrite Reductases / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic; D044223:Pseudomonas stutzeri / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D004251:DNA Transposable Elements; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins; D009572:Nitrite Reductases; D007785:Lactose