Transformation of Thermoanaerobacterium sp. strain JW/SL-YS485 with plasmid pIKM1 conferring kanamycin resistance

Mai, Volker

doi:10.1016/s0378-1097(97)00028-1

Cited by 15 publications

(25 citation statements)

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“…It was widely found in higher organisms, but varies in frequency in thermophilic bacteria. For instance, foreign DNA must be methylated before it can be introduced into C. thermocellum [2] via electroporation, while foreign-DNA methylation was not required for electro-transforming Thermoanaerobacterium saccharolyticum JW/SL-YS485 [1].…”

Section: Resultsmentioning

confidence: 99%

“…However, the lack of suitable donor strains makes conjugation difficult for thermophilic low G/C organisms such as Thermoanaerobacter . Thus most of the described gene transfer protocols for these types of organisms are based on electroporation [1], [2], [5], [24]. However, success with these methods was limited to only a few laboratories, which used custom-built cuvettes and pulse generator [2], [3] that are not accessible to most laboratories.…”

Section: Discussionmentioning

confidence: 99%

“…There have been relatively few reported successes in the transformation of thermophilic, Gram-positive, anaerobes (TGPAs) [1], [2], [3]. Firstly, 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].…”

Section: Introductionmentioning

confidence: 99%

“…Firstly, 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]. Secondly, most of the reported gene transfer protocols for these organisms were based on electroporation [1], [2], [5], where higher electroporation efficiency was limited to only a few laboratories that used sophisticated customer-built cuvettes and pulse generator [2]. Key drawbacks of electroporation include the need for an ion-free condition and the requirement of contact between electrodes and solution.…”

Section: Introductionmentioning

confidence: 99%

“…A few exceptions reported have involved custom-made electroporation-apparatus that are not accessible to most laboratories [1], [3]. Thus, it is of paramount importance that quick and efficient transformation systems be developed for these organisms.…”

Section: Introductionmentioning

confidence: 99%

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Ultrasound-Mediated DNA Transformation in Thermophilic Gram-Positive Anaerobes

Lin

Song

et al. 2010

PLoS ONE

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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.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Ultrasound-Mediated DNA Transformation in Thermophilic Gram-Positive Anaerobes

Lin

Song

et al. 2010

PLoS ONE

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Promiscuous plasmid replication in thermophiles: Use of a novel hyperthermophilic replicon for genetic manipulation of Clostridium thermocellum at its optimum growth temperature

Groom

Chung

Olson

et al. 2016

Metabolic Engineering Communications

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Clostridium thermocellum is a leading candidate for the consolidated bioprocessing of lignocellulosic biomass for the production of fuels and chemicals. A limitation to the engineering of this strain is the availability of stable replicating plasmid vectors for homologous and heterologous expression of genes that provide improved and/or novel pathways for fuel production. Current vectors relay on replicons from mesophilic bacteria and are not stable at the optimum growth temperature of C. thermocellum. To develop more thermostable genetic tools for C. thermocellum, we constructed vectors based on the hyperthermophilic Caldicellulosiruptor bescii replicon pBAS2. Autonomously replicating shuttle vectors based on pBAS2 reproducibly transformed C. thermocellum at 60 °C and were maintained in multiple copy. Promoters, selectable markers and plasmid replication proteins from C. bescii were functional in C. thermocellum. Phylogenetic analyses of the proteins contained on pBAS2 revealed that the replication initiation protein RepL is unique among thermophiles. These results suggest that pBAS2 may be a broadly useful replicon for other thermophilic Firmicutes.

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Engineering of a functional thermostable kanamycin resistance marker for use inMoorella thermoaceticaATCC39073

Iwasaki

Kita

Sakai

et al. 2013

FEMS Microbiol Lett

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A transformation system for Moorella thermoacetica ATCC39073 was developed using thermostable kanamycin resistant gene (kanR) derived from the plasmid pJH1 that Streptococcus faecalis harbored. When kanR with its native promoter was introduced into uracil auxotrophic mutant of M. thermoacetica ATCC39073 together with a gene to complement the uracil auxotrophy as a selection marker, it did not give kanamycin resistance due to poor transcription level of kanR. However, the use of glyceraldehyde-3-phosphate dehydrogenase promoter cloned from M. thermoacetica ATCC39073 significantly improved transcription level of kanR and resulted in the cell growth in the presence of more than 150 μg mL(-1) kanamycin. It was also demonstrated that kanR with G3PD promoter can be used as a selection marker for transformation of wild-type strain of M. thermoacetica ATCC39073.

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Transformation of Thermoanaerobacterium sp. strain JW/SL-YS485 with plasmid pIKM1 conferring kanamycin resistance

Cited by 15 publications

References 0 publications

Ultrasound-Mediated DNA Transformation in Thermophilic Gram-Positive Anaerobes

Ultrasound-Mediated DNA Transformation in Thermophilic Gram-Positive Anaerobes

Promiscuous plasmid replication in thermophiles: Use of a novel hyperthermophilic replicon for genetic manipulation of Clostridium thermocellum at its optimum growth temperature

Engineering of a functional thermostable kanamycin resistance marker for use inMoorella thermoaceticaATCC39073

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