Abstract
Among the diverse alkaliphilic Bacillus strains, only a little have been reported to be genetically transformed. In this study, an efficient protoplast transformation procedure was developed for recalcitrant alkaliphilic Bacillus sp. N16-5. The procedure involved polyethylene glycol-induced DNA uptake by the protoplasts and subsequent protoplast regeneration with a developed hard agar regeneration medium. An in vivo methylation strategy was introduced to methylate the exogenous plasmid DNA for improving the transformation efficiency. The transformation efficiency reached to 1.1×105 transformants per µg plasmid DNA with methylated plasmid pHCMC04 and the developed hard agar regeneration medium. This procedure might also be applicable to the genetic transformation of other Bacillus strains.
Highlights
Alkaliphiles are defined as microorganisms that grow optimally at pH of over 9 [1]
Since the diversity of alkaliphilic Bacillus strains are very rich [13] and different strains may adopt distinct strategies to adapt to the external alkaline environment [8,14], the establishment of genetic transformation systems in other alkaliphilic Bacillus strains may be helpful to broaden the study on the physiological alkaline adaptation mechanisms of alkaliphiles
Protoplast regeneration and transformation with a hard gelatin regeneration medium Succinate medium solidified with 1.2% agar (SA1.2 medium), a soft agar regeneration medium modified from DM3 medium [29], was firstly used to test the protoplast regeneration of Bacillus sp
Summary
Alkaliphilic Bacillus strains are of industrial importance because they produce various alkaline extracellular enzymes exhibiting great values in industrial application [2,3,4]. For in-depth study of the metabolic mechanisms and physiological alkaline adaptation mechanisms of the alkaliphilic Bacillus strains at the molecular level, the development of genetic transformation systems is indispensable. Most investigations on physiological alkaline adaptation mechanisms of alkaliphiles are limited in the two facultatively alkaliphilic strains, Bacillus pseudofirmus OF4 and Bacillus halodurans C-125 [8,9], which can be genetically transformed [10,11,12]. N16-5 is a recalcitrant facultatively alkaliphilic strain, producing multiple extracellular hydrolases and organic acids.
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