title: Isolation of a strong promoter fragment from endophytic Enterobacter cloacae and verification of its promoter activity when its host strain colonizes banana plants.
abstract: To engineer endophytic Enterobacter cloacae as a biocontrol agent against banana fusarium wilt, a promoter-probe plasmid pUCK was constructed to identify a strong promoter to express disease resistance genes. Using a kanamycin resistance gene for selection, 10 fragments with strong promoter activity were identified from the genome of the E. cloacae KKWB-10 strain. The regions of these 10 fragments that were the primary contributors to the promoter function were identified, and their promoter activities were further evaluated using green fluorescent protein (GFP) as a reporter gene. Fragment 132a″ drove the highest level of GFP activity when the bacteria bearing the fragments were cultured in Luria-Bertani and banana stem extract media. The GFP-expressing strain harboring fragment 132a″ (K-pUCK7-132a″-GT) was then inoculated into banana plantlets (about 1 × 10(7) CFU per plant) to verify the activity of fragment 132a″ in planta. Ten days after inoculation, tissue sections of these banana plantlets were observed by laser confocal scanning microscope. Green fluorescence was observed in the tissues of banana plantlets inoculated with K-pUCK7-132a″-GT but not in uninoculated controls. These results suggest that fragment 132a″ possesses strong promoter activity when its host strain colonizes the banana plants and can be used to engineer endophytic E. cloacae KKWB-10 for biocontrol.
authors.affiliation: Key Laboratory of Tropical Crop Biotechnology, Ministry of Agriculture, Institute of Tropical Bioscience and Biotechnology, Chinese Academy of Tropical Agricultural Sciences, Haikou, People's Republic of China.
authors.forename: Yu Guang
authors.initials: YG
authors.lastname: Wang
authors.forename: Qi Yu
authors.initials: QY
authors.lastname: Xia
authors.forename: Wen Liang
authors.initials: WL
authors.lastname: Gu
authors.forename: Jian Bo
authors.initials: JB
authors.lastname: Sun
authors.forename: He
authors.initials: H
authors.lastname: Zhang
authors.forename: Xue Hua
authors.initials: XH
authors.lastname: Lu
authors.forename: Juan
authors.initials: J
authors.lastname: Lu
authors.forename: Ming
authors.initials: M
authors.lastname: Peng
authors.forename: Xin
authors.initials: X
authors.lastname: Zhang
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology; D019677:Artificial Gene Fusion; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D060026:Endophytes / Q000235:genetics; D016972:Enterobacter cloacae / Q000235:genetics*; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000235:genetics; D007612:Kanamycin / Q000494:pharmacology; D008969:Molecular Sequence Data; D028521:Musa / Q000382:microbiology*; D011401:Promoter Regions, Genetic; D012641:Selection, Genetic; D017422:Sequence Analysis, DNA
chemical_list: D000900:Anti-Bacterial Agents; D004269:DNA, Bacterial; D049452:Green Fluorescent Proteins; D007612:Kanamycin