Isolation and characterization of active promoters from Gluconacetobacter diazotrophicus strain PAL5 using a promoter-trapping plasmid

Abstract

Gluconacetobacter diazotrophicus is a nitrogen-fixing, endophytic bacterium that has the potential to promote plant growth and increase yield. Genetically modified strains might get more benefits to host plants, including through expression of useful proteins, such as Cry toxins from B. thuringiensis, or enzymes involved in phytohormone production, proteins with antagonistic activity for phytopathogens, or that improve nutrient utilization by the plant. For that, expression systems for G. diazotrophicus are needed, which requires active promoters fused to foreign (or innate) genes. This article describes the construction of a G. diazotrophicus PAL5 promoter library using a promoter-less lacZ-bearing vector, and the identification of six active promoters through β-galactosidase activity assays, sequencing and localization in the bacterial genome. The characterized promoters, which are located on distinct regions of the bacterial genome and encoding either sense or antisense transcripts, present variable expression strengths and might be used in the future for expressing useful proteins.

Enhanced extracellular ammonium release in the plant endophyte Gluconacetobacter diazotrophicus through genome editing

“…Primers required for the construction of the strains are listed in Table 3 . Plasmids were transformed into G. diazotrophicus through electroporation as described previously ( 53 , 54 ). DNA transfer was accomplished by growing G. diazotrophicus in liquid GADN medium until it had reached an OD 600 of approximately 1.0, then 1 mL of cells were spun down at 7,000 × g and washed two times with 10% glycerol in distilled water and resuspended in 100 µL.…”

On the way toward regulatable expression systems in acetic acid bacteria: target gene expression and use cases

“…Promoter screening in Ga. diazotrophicus For Ga. diazotrophicus, a promoter library was constructed using the promoter probe vector pPW452, a derivative of the broad-host-range vector pMP220 obtained from pTJS75 with an RK2 replicon and containing a multiple cloning site upstream of a promoterless lacZ reporter gene (Schwab et al 2016). With the library prepared from total Ga. diazotrophicus DNA, 480 clones were screened and six promoters were isolated and characterized further.…”