Abstract
BackgroundThermophilic, Gram-positive, anaerobic bacteria (TGPAs) are generally recalcitrant to chemical and electrotransformation due to their special cell-wall structure and the low intrinsic permeability of plasma membranes.Methodology/Principal FindingsHere we established for any Gram-positive or thermophiles an ultrasound-based sonoporation as a simple, rapid, and minimally invasive method to genetically transform TGPAs. We showed that by applying a 40 kHz ultrasound frequency over a 20-second exposure, Texas red-conjugated dextran was delivered with 27% efficiency into Thermoanaerobacter sp. X514, a TGPA that can utilize both pentose and hexose for ethanol production. Experiments that delivered plasmids showed that host-cell viability and plasmid DNA integrity were not compromised. Via sonoporation, shuttle vectors pHL015 harboring a jellyfish gfp gene and pIKM2 encoding a Clostridium thermocellum β-1,4-glucanase gene were delivered into X514 with an efficiency of 6×102 transformants/µg of methylated DNA. Delivery into X514 cells was confirmed via detecting the kanamycin-resistance gene for pIKM2, while confirmation of pHL015 was detected by visualization of fluorescence signals of secondary host-cells following a plasmid-rescue experiment. Furthermore, the foreign β-1,4-glucanase gene was functionally expressed in X514, converting the host into a prototypic thermophilic consolidated bioprocessing organism that is not only ethanologenic but cellulolytic.Conclusions/SignificanceIn this study, we developed an ultrasound-based sonoporation method in TGPAs. This new DNA-delivery method could significantly improve the throughput in developing genetic systems for TGPAs, many of which are of industrial interest yet remain difficult to manipulate genetically.
Highlights
There have been relatively few reported successes in the transformation of thermophilic, Gram-positive, anaerobes (TGPAs) [1,2,3]
Some thermophilic bacteria are difficult to transform due to the unique features of their cell envelope, the formation of endospore and the low permeability of plasma membrane [4]
To develop and optimize the sonoporation method for TGPAs, we first tested the delivery of fluorescent Texas-red conjugated dextran into native Thermoanaerobacter sp
Summary
There have been relatively few reported successes in the transformation of thermophilic, Gram-positive, anaerobes (TGPAs) [1,2,3]. There is a tremendous need for developing a simple, rapid and effective method for genetically transforming TGPAs. Thermoanaerobacter are a group of gram-positive anaerobes with optimal growth temperature of 60–65uC. Thermoanaerobacter are a group of gram-positive anaerobes with optimal growth temperature of 60–65uC They have attracted growing attention over the past few years due to their rare capability utilizing both hexose and pentose at high temperature for ethanol production [7,8,9,10,11]. The advantages of sonoporation-based transformation, the ease and simplicity of procedure, the preservation of cells in indigenous medium and the minimal perturbation to cell viability, suggested this method could be suitable for high throughput development of genetic systems
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