Lu LinHouhui SongYuetong JiZhili HeYunting PuJizhong ZhouJian Xu2015-01-232016-03-302015-01-232016-03-302010-09-04Lin L, Song H, Ji Y, He Z, Pu Y, et al. (2010) Ultrasound-Mediated DNA Transformation in Thermophilic Gram-Positive Anaerobes. PLoS ONE 5(9): e12582. doi:10.1371/journal.pone.0012582http://hdl.handle.net/11244/14064We thank Yunfeng Yang for helpful discussion and Christoper L. Hemme, Jason Huff and Aifen Zhou for critical reading of the manuscript.Conceived and designed the experiments: HS. Performed the experiments: LL YJ YP. Analyzed the data: LL HS. Contributed reagents/materials/analysis tools: ZH JZ JX. Wrote the paper: LL HS JX.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.en-USAttribution 3.0 United StatesPLOSPublic Library of ScienceOpen AccessOpen-AccessScienceMedicineBiologyResearchPeer-reviewInclusiveInterdisciplinaryAnte-disciplinaryPhysicsChemistryEngineeringResearch Article10.1371/journal.pone.0012582false