Transformation of<i>Lactobacillus plantarum</i>by electroporation with in vitro modified plasmid DNA

Alegre, M. Teresa; Rodríguez, Marianela; Mesas, Juan M.

doi:10.1016/j.femsle.2004.10.006

Cited by 71 publications

(26 citation statements)

References 32 publications

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“…In contrast, the type IV RM systems produce restriction enzymes which cleave solely methylated DNA. There are several reports, mostly referring to DNA adenine methylation ( dam )/ DNA cytosine methylation ( dcm ) that methylation pattern of plasmid DNA has a major impact on transformation efficiency and allows plasmid DNA to circumvent host restriction mechanisms [24-26]. …”

Section: Introductionmentioning

confidence: 99%

“Direct cloning in Lactobacillus plantarum: Electroporation with non-methylated plasmid DNA enhances transformation efficiency and makes shuttle vectors obsolete”

2012

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BackgroundLactic acid bacteria (LAB) play an important role in agricultural as well as industrial biotechnology. Development of improved LAB strains using e.g. library approaches is often limited by low transformation efficiencies wherefore one reason could be differences in the DNA methylation patterns between the Escherichia coli intermediate host for plasmid amplification and the final LAB host. In the present study, we examined the influence of DNA methylation on transformation efficiency in LAB and developed a direct cloning approach for Lactobacillus plantarum CD033. Therefore, we propagated plasmid pCD256 in E. coli strains with different dam/dcm-methylation properties. The obtained plasmid DNA was purified and transformed into three different L. plantarum strains and a selection of other LAB species.ResultsBest transformation efficiencies were obtained using the strain L. plantarum CD033 and non-methylated plasmid DNA. Thereby we achieved transformation efficiencies of ~ 109 colony forming units/μg DNA in L. plantarum CD033 which is in the range of transformation efficiencies reached with E. coli. Based on these results, we directly transformed recombinant expression vectors received from PCR/ligation reactions into L. plantarum CD033, omitting plasmid amplification in E. coli. Also this approach was successful and yielded a sufficient number of recombinant clones.ConclusionsTransformation efficiency of L. plantarum CD033 was drastically increased when non-methylated plasmid DNA was used, providing the possibility to generate expression libraries in this organism. A direct cloning approach, whereby ligated PCR-products where successfully transformed directly into L. plantarum CD033, obviates the construction of shuttle vectors containing E. coli-specific sequences, as e.g. a ColEI origin of replication, and makes amplification of these vectors in E. coli obsolete. Thus, plasmid constructs become much smaller and occasional structural instability or mutagenesis during E. coli propagation is excluded. The results of our study provide new genetic tools for L. plantarum which will allow fast, forward and systems based genetic engineering of this species.

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Section: Introductionmentioning

confidence: 99%

“Direct cloning in Lactobacillus plantarum: Electroporation with non-methylated plasmid DNA enhances transformation efficiency and makes shuttle vectors obsolete”

2012

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“…Taken together, it is concluded that the adenine methylation modification system in Thermoanaerobacter sp. X514 was active and is likely the major restriction modification system in this organism, suggesting that methylation of foreign DNA by specific methylases or cell extracts [22] is essential prior to introducing into X514 cells.…”

Section: Resultsmentioning

confidence: 99%

“…X514 were prepared from stationary-phase cells as described previously with minor modifications [22]. Briefly, cells from 100 ml stationary-phase culture of X514 were collected by centrifugation (3,500 g for 10 min), washed once with 100 ml chilled PENP buffer (10 mM KH 2 PO 4 -K 2 HPO 4 , 10 mM EDTA, 50 mM NaCl and 0.2 mM PMSF, pH 7.0), and then resuspended in 4 ml of the same buffer.…”

Section: Methodsmentioning

confidence: 99%

“…The DNA modification assay was performed as described elsewhere [22] by combining 53 µl TNE buffer (50 mM Tris pH 7.5, 50 mM NaCl, 10 mM EDTA), 10 µl SAM (0.8 mM), 1 µl BSA (10 mg/ml), 25 µl X514 cell extract and 3 µg plasmid DNA (pHL015) prepared from E. coli DH5α (Dam + /Dcm + ) or JM110 (Dam − /Dcm − ) in a final volume of 100 µl. The mixture was incubated at 60°C for 16 h. As a control for methylation experiments, a commercial Dam methyltransferase was purchased from New England Biolabs.…”

Section: Methodsmentioning

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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“…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).…”

Section: Methodsmentioning

confidence: 99%

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

Zhou

et al. 2015

Appl Environ Microbiol

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bMethanosaeta spp. are widely distributed in natural environments, and their filamentous cells contribute significantly to sludge granulation and the good performance of anaerobic reactors. A previous study indicated that Methanosaeta harundinacea 6Ac displays a quorum sensing-regulated morphological transition from short to long filaments, and more acetate is channeled into methane production in long filaments, whereas more is channeled into biomass synthesis in short filaments. Here, we performed transcriptomic and physiological analysis to gain insights into active methanogenesis in long filaments of M. harundinacea 6Ac. Both RNA sequencing (RNA-seq) and quantitative reverse transcription-PCR indicated that transcription of the genes involved in aceticlastic methanogenesis and energy metabolism was upregulated 1.2-to 10.3-fold in long filaments, while transcription of the genes for the methyl oxidative shunt was upregulated in short filaments. [2-13 C]acetate trace experiments demonstrated that a relatively higher portion of the acetate methyl group was oxidized to CO 2 in short filaments than in long filaments. The long filaments exhibited higher catalase activity and oxygen tolerance than the short ones, which is consistent with increased transcription of the oxidant-scavenging genes. Moreover, transcription of genes for cell surface structures was upregulated in the long filaments, and transmission electron microscopy revealed a thicker cell envelope in the filaments. RNA-seq determined a >2-fold upregulation of a variety of antistress genes in short filaments, like those encoding chaperones and DNA repair systems, which implies that the short filaments can be stressed. This study reveals the genetic basis for the prevalence of the long filamentous morphology of M. harundinacea cells in upflow anaerobic sludge blanket granules. Methanogenic degradation of organic complexes is a widely used approach in wastewater treatment; it promotes the development of upflow anaerobic sludge blanket (UASB) reactors (1). Efficient mineralization of organic complexes to CH 4 and CO 2 in the UASB reactor is implemented by diverse microbes through a food chain mode, where methanogens implement the final chemical reaction producing CH 4 (2, 3). In UASB reactors and other ecosystems, acetate-derived methane (aceticlastic methanogenesis) contributes about 70% of the methane produced by either the generalist aceticlastic methanogens of the species Methanosarcina or the obligate aceticlastic methanogens of the species Methanosaeta (4, 5). Methanosaeta species are believed to be the key components in anaerobic digesters not only because of their ability to use very low concentrations of acetate (threshold, 5 to 20 M) (6) but also because of their fiber-like cells that serve as a scaffold for the attachment of other organisms to promote the formation of UASB granules (7), an essential self-immobilized organization of the functional microbes. It has been reported that Methanosaeta cells comprise one-third of the anaerobic migrating ...

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Transformation ofLactobacillus plantarumby electroporation with in vitro modified plasmid DNA

Cited by 71 publications

References 32 publications

“Direct cloning in Lactobacillus plantarum: Electroporation with non-methylated plasmid DNA enhances transformation efficiency and makes shuttle vectors obsolete”

“Direct cloning in Lactobacillus plantarum: Electroporation with non-methylated plasmid DNA enhances transformation efficiency and makes shuttle vectors obsolete”

Ultrasound-Mediated DNA Transformation in Thermophilic Gram-Positive Anaerobes

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

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