“Direct cloning in Lactobacillus plantarum: Electroporation with non-methylated plasmid DNA enhances transformation efficiency and makes shuttle vectors obsolete”
© Spath et al.; licensee BioMed Central Ltd. 2012
Received: 3 July 2012
Accepted: 21 October 2012
Published: 25 October 2012
Lactic 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.
Best 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.
Transformation 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.
Lactobacillus plantarum and many other lactic acid bacteria (LAB) are “generally regarded as safe” (GRAS) organisms and possess the ability to efficiently secrete recombinant proteins directly into the culture medium. Thus, they are recognized as emerging candidates for the expression of recombinant proteins as well as for genetic and metabolic cell engineering, both in the fields of medical and industrial biotechnology[1, 2]. Lactococcus lactis is to date the most widely used LAB strain for recombinant protein expression and was engineered to express cytokines, bacterial and viral antigens[4, 5], membrane proteins and enzymes. L. plantarum plays an important role in many processes of animal feed industry. For example, preservation of crop silage is often improved by adding starter cultures containing L. plantarum and other LAB. During the past decade the interest in developing new and genetically engineered LAB strains with improved properties has been continuously growing[9–13], e.g. to produce cellulose degrading enzyme activity. L. plantarum has also been proved being a feasible expression host, recently expressing a ß-galactosidase from Lactobacillus delbrueckii and a chitinase from Bacillus licheniformis.
Several vector systems exist[17–19], and different plasmid backbones and promoters are available for cloning and protein expression in LAB. Unlike for recombinant protein expression in Escherichia coli, the availability of different engineered LAB expression strains is limited. Often protein yields are very low, constraining the system to applications where small amounts are sufficient, e.g. to produce cellulose degrading enzyme activity. In order to adapt a host’s metabolic capacities to the needs in biotechnology, the approach to use genetic engineering has been shown to successfully enhance product yield and quality in E. coli, Saccharomyces cerevisiae and Pichia pastoris. Also for L. plantarum, genetic engineering was shown to improve the metabolic performance, when mutations in sigma-factor (rpoD) conferred resistance against low pH conditions. Often, these approaches are based on the generation of a great variety of genetically different clones and the subsequent screening for the desired phenotype. The strategy of cell engineering on a systems molecular level requires the possibility of high-throughput screening of a heterogenic pool of mutants. Thus, the generation of diverse genetic libraries is a prerequisite for fast and efficient host engineering. Also for protein engineering such as the adaptation of enzymatic activities to environmental conditions within a certain cell system, requires library based systems. While gene libraries can be generated in the size of 1010 in E. coli, and in the range of 107 in S. cerevisiae, for LAB low transformation efficiencies are often a limiting factor. During the last decades many transformation protocols for LAB strains have been published. The successful introduction of plasmid DNA into LAB is dependent on strain specific features such as cell wall structure and composition plasmid size and the origin of replication.
In some LAB, the low number or even lack of transformants obtained after electroporation, may be attributed to various restriction modification (RM) systems encoded by the host. RM systems are widely spread in bacteria and serve the protection of invading DNA such as foreign plasmids or the DNA of bacteriophages. Most of these systems consist of a restriction enzyme and a corresponding methyltransferase that blocks the restriction activity, thus, protects the genome from self-cleavage (type I and III RM systems). 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].
Since gene manipulation of expression vectors is much easier in E. coli than in LAB, and in order to gain sufficient amounts of plasmid DNA, normally, shuttle vectors are used to first build and propagate the final plasmid in E. coli. After subsequent purification the desired plasmid is then transformed into LAB. Many shuttle vectors are based on cryptic plasmids derived from LAB strains, which have been modified to contain both, the LAB specific and E. coli specific replicative elements. Often these shuttle vectors are structurally unstable either in E. coli or in the LAB-expression hosts, maybe due to their size or their chimeric nature, e.g. differences in GC-content (50% GC for E. coli versus 30 – 40% GC for LAB). Savijoki et al. used L. lactis MG1363 as an intermediate host to circumvent such problems. However, this approach is limited to origins of replication functional in L. lactis. Another strategy is to design shuttle vectors that contain replicons which replicate in Gram (+) as well as in Gram (–) bacteria, e.g. based on the origin of replication of pWV01 or pSH71[29, 30]. Yet, due to their rolling circle-replication mechanism these plasmids tend to suffer from structural and segregational instability. Often, plasmids containing large DNA inserts cannot stably be maintained[31, 32], and thus, rolling circle-replicating plasmids are only suitable for small genes. Therefore, cloning procedures would substantially improve by having plasmids available that are devoid of any E. coli derived sequences and are based on a stable origin of replication. We have previously compared several LAB strains in terms of transformation efficiency and plasmid stability. One of the tested L. plantarum (CD033) strains showed unexpectedly high transfection yields (6 x 105 colony forming units (cfu)/μg DNA). Furthermore, this strain was found to be transformable with unmethylated DNA and therefore, became an interesting organism for further examinations regarding the influence of DNA methylation on transformation efficiency.
Results and discussion
Role of plasmid methylation in transformation of L. plantarum CD033
Transformation efficiency of L. plantarum CD033 with pCD256 isolated from different E. coli strains
Transformation efficiency [cfu/μg DNA]
2.5 x 105
3.0 x 105
5.0 x 105
3.0 x 105
4.1 x 105
4.7 x 105
1.6 x 105
2.3 x 105
3.0 x 105
5.0 x 108
8.7 x 108
9.7 x 108
Transformation efficiency of LAB strains transformed with variously methylated plasmid pCD256
Transfromation efficiency [cfu/μg DNA] of the following LAB strains
L. plantarum CD032
L. plantarum CD033
L. plantarum DSM20174
L. buchneri CD034
E. faecium CD036
L. lactis MG1363*
dam + dcm +
4.0 x 102
3.0 x 105
8.0 x 104
2.0 x 105
dam - dcm -
6.0 x 103
8.7 x 108
1.8 x 102
Furthermore, three other LAB-species were tested for their susceptibility to be transformed by unmethylated plasmid DNA. Lactobacillus buchneri CD034 and Enterococcus faecium CD036 were shown previously to maintain the origin of replication from plasmid p256, whereas for L. lactis MG1363 plasmid pCDWV01, containing the origin of replication from the lactococcal plasmid pWV01, had to be used in order to provide replication, which has been shown to have similar stability properties as compared to pCD256. Results showed that for none of the performed electroporations colonies were received, possibly indicating restriction of non-methylated DNA (Table2). RM systems have been described for different L. lactis strains and appropriate genes have also been identified in the chromosome of L. buchneri CD034, supporting this hypothesis. Furthermore, restriction analysis of plasmids obtained from L. plantarum CD033 indicated that no dam/dcm-methylation is present in this strain. Hence, we failed to introduce plasmid DNA deriving from L. plantarum CD033 into L. buchneri CD034 (data not shown). The suggestion arises, that insights in methylation patterns of other species might serve to in vitro-methylate ligation reactions or unmethylated plasmids in order to overcome the bottle neck of a specific host’s restriction system.
Direct cloning in L. plantarum CD033
In the present study, we examined the influence of DNA methylation patterns on transformation efficiency in LAB and developed a direct cloning approach for L. plantarum CD033. Therefore, we transformed various LAB strains with plasmid DNA exhibiting different dam/dcm-methylation patterns. Best results were obtained using non-methylated DNA, resulting in transformation efficiencies of ~ 109 cfu/μg DNA in L. plantarum CD033. Thereby, it becomes feasible to generate expression libraries, promoter libraries, etc. of sufficient size in this strain. We demonstrated direct transformation of recombinant expression vectors received from PCR/ligation reactions into L. plantarum CD033 to be feasible, making the construction of shuttle vectors obsolete. This new approach allowed the construction of minimal plasmids consisting exclusively of a LAB-origin of replication and a selection marker. Besides providing smaller expression vectors, this method excludes any structural instability or mutagenesis which is occasionally associated with plasmid propagation in E. coli. The results of our study provide new genetic tools for L. plantarum which allow faster and facilitated cloning procedures as well as systems based genetic engineering based on library techniques.
Materials and methods
Bacterial strains and growth conditions
Bacterial strains used in this study
New England Biolabs GmbH
New England Biolabs GmbH
M. G. Marinus
New England Biolabs GmbH
Lactic acid bacteria
L. plantarum CD032
wilde type strain
stable grass silage accession number BT6146*
L. plantarum CD033
wilde type strain
stable grass silage accession number BT6326*
L. buchneri CD034
wilde type strain
stable grass silage accession number BT6327*
E. faecium CD036
wilde type strain
stable grass silage accession number BT6329*
L. lactis MG1363
Plasmid free derivative of SH4109
L. plantarum DSM20174
Plasmids, primers and synthetic expression cassette
Plasmids used in this study
pUC19 containing origin from p256, AmpR, CmR
pUC19 containing origin from pWV01, AmpR, CmR
pCD256 containing hTFF1 expression cassette
pCD256 lacking E. coli sequences
pCD256ΔEc containing hTFF1 expression cassette
Primers used for cloning
PCR, restriction digestion and ligation of DNA fragments
Unless otherwise stated, DNA fragments were amplified using the Phusion High-Fidelity DNA Polymerase in HF-buffer (New England Biolabs, NEB, USA). PCRs were performed as follows: initial denaturation for 30 s at 98°C, followed by 30 cycles of 10 s at 98°C, annealing for 20 s at a melting temperature (Tm) +3°C of the lower Tm primer and extension for 25 s/kb at 72°C. Amplification was concluded with a final extension step at 72°C for 6 min. All PCRs were carried out with a T3 Thermocycler (Biometra, Germany). All restriction enzymes were purchased from NEB, restriction digests were performed according to the manufacturer’s recommendations. DNA fragments were purified from PCRs, enzyme reactions or agarose gels using the NucleoSpin Gel and PCR Clean-up Kit (Macherey-Nagel, Germany). Ligation reactions were performed using T4 DNA Ligase (NEB, USA). For a 20 μl ligation reaction 250 ng digested and purified plasmid DNA was mixed on ice with 1 μl T4 DNA ligase, 2 μl of 10x T4 ligase buffer and with a 5-fold molar excess of digested and purified insert DNA. The ligation reaction was incubated at 16°C over night precipitated with isopropanol, washed with 70% v/v ethanol, air dried for 15 min, solved in sterile ddH20 and used for transformation.
Dedection of the mrr gene in L. plantarum CD033
The presence of an mrr gene was confirmed by colony PCR using the Phusion High-Fidelity DNA Polymerase using GC-buffer (NEB, USA) and the primers mrr_Lp_F / mrr_Lp_R. PCR was conducted as follows: initial denaturation at 98°C for 30 s, followed by 30 cycles of denaturation at 98°C for 10 s, annealing at 60°C for 20 s and elongation at 72°C for 30 s. Cycling was completed with a final extension step at 72°C for 6 min. The amplicon was sequenced (GATC Biotech, Germany) and confirmed by BLAST analysis.
Construction of pCD256_hTFF1, pCD256ΔEc and pCD256ΔEc _hTFF1
The synthetic hTFF1 expression cassette was amplified by PCR using the primers hTFF1_SacI_F / Tldh_amp_R and cloned Sac I/Sal I into pCD256 resulting in the plasmid pCD256_hTFF1.
For the construction of pCD256ΔEc, a DNA fragment comprising only lactobacillus specific elements (CAT, miniori256) was amplified from pCD256 by PCR using the primers M13_R_NheI / Cat_F_NheI, thereby introducing a novel Nhe I restriction site. The obtained fragment was digested with Nhe I and subsequently self-ligated.
For the ligation of two PCR products, pCD256ΔEc and the hTFF1 expression cassette were amplified by PCR using the primers M13_R_XhoI / Cat_F_NheI and hTFF1_F_NheI / Tldh_amp_R_XhoI. The two PCR products were Xho I/Nhe I-digested and ligated one with each other, resulting in the plasmid pCD256ΔEc _hTFF1. DNA-cloning techniques in E. coli were performed according to Sambrook and Russell. All vector constructs were confirmed by DNA sequencing (GATC Biotech, Germany).
Electroporation of LAB
Electroporation was done using an ECM 630 Precision Pulse (BTX Harvard apparatus, USA). L. plantarum CD033 and L. buchneri CD034 were transformed according to, L. plantarum DSM20174 was transformed using the same protocol as for L. plantarum CD033, L. lactis MG1363 was transformed according to, E. faecium CD036 and L. plantarum CD032 were transformed as follows. An overnight culture was diluted with MRS broth to an optical density at 600 nm wavelength (OD600) of 0.2 and incubated at the appropriate temperature until an OD600 of 0.5 was reached. The cells were harvested by centrifugation at 10,000 rcf for 6 min at 4°C. The pellets were washed three times with ice cold 0.3 M sucrose. The pellet was resuspended to one hundredth of initial volume in 0.3 M sucrose and kept on ice until electroporation. Plasmid DNA (0.25-1 μg) was mixed with 40 μl cell suspension in a ice-cold electroporation cuvette (0.2 cm gap, Sigma Aldrich, Germany) and electroporated at 2.5 kV, 200 Ω and 25 μF. After the pulse, cells were resupended in 360 μl MRS broth for 2 h at the appropriate temperature. The bacteria were spread on MRS plates containing chloramphenicol and incubated for 3 days at the appropriate conditions. Proper assemble of recombinant plasmids was confirmed by colony PCR and supsequent sequencing of the obtained amplicons using the primers CAT_seq_back/p256miniori_for. In the case of pCD256ΔEc _hTFF1 which was transformed directly with a purified ligation reaction, structural integrity was proven by PCR using the primers CAT_seq_R/Tldh_amp_R and CAT_seq_back/GG_hTFF1_sense yielding overlapping DNA fragments and subsequent DNA sequencing of the obtained fragments.
This work was supported by the Christian Doppler Research Association, Vienna, Austria.
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