Isolation of a strong promoter fragment from endophytic Enterobacter cloacae and verification of its promoter activity when its host strain colonizes banana plants

• Published in: Applied microbiology and biotechnology Vol. 93; no. 4; pp. 1585 - 1599
• Main Authors: Wang, Yu Guang, Xia, Qi Yu, Gu, Wen Liang, Sun, Jian Bo, Zhang, He, Lu, Xue Hua, Lu, Juan, Peng, Ming, Zhang, Xin
• Format: Journal Article
• Language: English
• Published: Berlin/Heidelberg Springer-Verlag 01.02.2012; Springer Nature B.V
• Subjects: Agriculture; Analysis; Anti-Bacterial Agents - pharmacology; Applied Genetics and Molecular Biotechnology; Artificial Gene Fusion; Bacteria; Bananas; Biological control; Biomedical and Life Sciences; Biotechnology; Cloning; DNA, Bacterial - chemistry; DNA, Bacterial - genetics; E coli; Endophytes - genetics; Enterobacter cloacae; Enterobacter cloacae - genetics; Genes; Genes, Reporter; Genetic engineering; Genomes; Green Fluorescent Proteins - analysis; Green Fluorescent Proteins - genetics; Kanamycin - pharmacology; Laboratories; Life Sciences; Microbial Genetics and Genomics; Microbiology; Molecular Sequence Data; Musa - microbiology; Plant diseases; Plasmids; Promoter Regions, Genetic; Selection, Genetic; Sequence Analysis, DNA; Studies; GFP; Endophyte; Fluorescence observation; Promoter cloning
• ISSN: 0175-7598; 1432-0614
• DOI: 10.1007/s00253-011-3684-6

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.