Abstract
In order to develop a method for electroporation of environmental Bacillus mycoides strains, we optimized several conditions that affect the electroporation efficiency of this bacterium. By combining the optimized conditions, the electroporation efficiency of strain EC18 was improved to (1.3±0.6)×105cfu/μg DNA, which is about 103-fold increase in comparison with a previously reported value. The method was further validated on various B. mycoides strains, yielding reasonable transformation efficiencies. Furthermore, we confirmed that restriction/modification is the main barrier for electroporation of this bacterium. To the best of our knowledge, this is the first systematic investigation of various parameters of electroporation of B. mycoides. The electroporation method reported will allow for efficient genetic manipulation of this bacterium.
Highlights
Bacillus mycoides is a spore-forming Gram-positive bacterium that is commonly found in soil and the rhizosphere
Our preliminary experiments show that by applying the method reported by Ehling-Schulz et al (2005), which was originally developed for B. cereus, the B. mycoides strain EC18 could be transformed, albeit at a low efficiency
EC18 cells were first grown in BHIS medium, and when the optical density at 600 nm (OD600) reached about 0.85, glycine and threonine were added at different concentrations (0%, 1%, 2%, 3%, 4% and 5%)
Summary
Bacillus mycoides is a spore-forming Gram-positive bacterium that is commonly found in soil and the rhizosphere. It belongs to the B. cereus sensu lato group, which includes B. cereus, B. thuringiensis, and B. anthracis. B. mycoides has received the least attention among this group because it is not a human pathogen as B. anthracis and B. cereus. The B. mycoides species features a unique filamentous growth pattern, either being rotated clockwise or counterclockwise (Di Franco et al, 2002). More and more studies on B. mycoides are focusing on their plant-growth promoting activities (Ambrosini et al, 2016; Bargabus et al, 2002; Neher et al, 2009)
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