Ultrasound-Mediated DNA Transformation in Thermophilic Gram-Positive Anaerobes

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.

Establishment of an efficient transformation protocol and its application in marine-derived Bacillus strain

“…p3101GFP, which harbors a green fluorescent protein (GFP) gene gfp under the control of the promoter of glyceraldehyde-3-phosphate dehydrogenase gene gapDH (gapDHp), was constructed as follows: the gapDHp promoter was amplified from the genome of B. marinus B-9987 by using primer pair of gapDHp-FP/gapDHp-RP: 5′-GGAATTCT-GGAATACGAGCTGAGTG-3′ (EcoR I site underlined)/5-ACGCGTCGACCATTCTTCCGATTCTAC-3′ (Sal I site underlined); the gfp gene was amplified from plasmid pHL015 [28] by using primer pair of gfp-FP/gfp-RP: 5′-ACGCGTCGACATGTCGAAGGGCGAGGAGC-3 (Sal I site underlined)/5′-GCTCTAGATTATCCTTACTACTTG-3′ (Xba I site underlined); the above DNA fragments were cloned into the EcoR I and Xba I sites of pHT3101 to yield p3101GFP. p3101GFP isolated from E. coli ET12567 was introduced into B. marinus B-9987.…”

Increased expression of β-glucosidase A in Clostridium thermocellum 27405 significantly increases cellulase activity

“…14 More recently in 2010, Lin et al developed a minimally invasive ultrasound-based sonoporation method for simple and rapid transformation of thermophilic Gram positive anaerobes. 18 In doing so, they transformed Thermoanaerobacterium sp strain X514 with pIKM2 harboring a C. thermocellum β-1-4-glucanase gene, endoglucanase activity was observed in both electroporated and sonoporated X514 samples. Now for one of the first times reported, we expressed a functional gene (β-glucosidase A) from a Dam methylated plasmid pIBglA (constructed from pIKM1) in C. thermocellum 27405 using a modified electroporation protocol.…”

Transcriptomic and Physiological Insights into the Robustness of Long Filamentous Cells of Methanosaeta harundinacea, Prevalent in Upflow Anaerobic Sludge Blanket Granules

“…Long-and short-filament cell extracts (CEs) were prepared as described previously, with modifications (18,19). Briefly, cultures were harvested from mid-log-phase cells by centrifugation at 5,000 ϫ g at 4°C for 15 min, and the cell pellets were washed twice with chilled 0.1 M PBS (pH 7.2).…”