Construction of an expression vector for Lactococcus lactis based on an indigenous cryptic plasmid

To construct an expression vector for Lactococcus lactis, the EmPMT fragment which contained the erythromycin resistance gene, P32 promoter, multiple cloning site (MCS) and terminator (T) was subcloned into the small cryptic plasmid pAR141. The resulting vector, designated as pAR1411, was found to be stably maintained in L. lactis MG1363 after transformation for at least 100 generations under non-selective conditions. The vector was also demonstrated to be able to express the gene coding for chloramphenicol acetyltransferase (cat) in L. lactis.


INTRODUCTION
Lactococcus lactis is one of the best characterized lactic acid bacteria (LAB) and is widely used as a dairy starter culture traditionally.The extensive knowledge and the advancement of molecular biology techniques for this bacterium have not only enhanced its performance in dairy production, but also expanded its potential application in other areas (Konings et al., 2000).Due to its generally regarded as safe (GRAS) stature, this Grampositive food-grade bacterium has been used as cell factories for the production of various chemicals for food industry (Luoma et al., 2001), pharmaceutical (Bermúdez-Humarán et al., 2003) and neutraceutical (Hugenholtz and Smid, 2002), in addition to being developed for vaccine delivery (Ramasamy et al., 2006;van Roosmalen et al., 2006;Mercenier et al., 2000).Recombinant L. lactis has also been used for the expression of several viral and eukaryotic proteins (Kunji et al., 2003;Madsen et al., 1999).Recently, the potential of L. lactis in nanobiotechnology has been exploited (Novotny et al., 2005).
Most of these applications of L. lactis require the use of cloning and expression vectors for the introduction of foreign genes or genetic manipulations of the hosts.These vectors were generally derived from cryptic plasmids (Shareck et al., 2004;de Vos and Simons, 1994).Cryptic plasmids are extrachromosomal DNA elements that encode no recognizable phenotype besides their replication functions.Selectable markers are then incorporated into these plasmids to enable recombinant bacteria carrying them to be scored easily.The present study reported the construction of a constitutive expression vector for L. lactis based on an indigenous small cryptic plasmid.

Bacterial strains, plasmids and growth conditions
Bacterial strains and plasmids used in this study are listed in Table 1.L. lactis and Escherichia coli strains were cultured in GM17 (Terzaghi and Sandine, 1975) and LB (Sambrook and Russell, 2001) at 30 and 37°C, respectively.For L. lactis, erythromycin and chloramphenicol were used at a final concentration of 5 and 7.5 µg ml -1.For E. coli, erythromycin at a concentration of 150 µg ml -1 was used.
Plasmids were isolated from Lactococcal strains using alkaline  polymerase (Fermentas).The PCR conditions were: initial denaturation at 95°C for 5 min, followed by 25 cycles of denaturation at 95°C for 1 min, annealing at 53°C for 1 min, extension at 72°C for 3 min, and ended by final extension at 72°C for 10 min.The PCR product was then analysed with agarose gel electrophoresis.The EmPMT fragment was cloned into the unique FspI site of pAR141 (Raha et al., 2006) with T4 DNA ligase (Fermentas).The ligation mix was transformed into competent L. lactis MG1363 by electroporation as described by Holo and Nes (1989) with minor modification.The electroporation was carried out with GenePulser (Bio-Rad; 2.3 kV, 25 µF and 200 Ω) and the transformants were screened on SGM17 (GM17 supplemented with 0.5 M sucrose) containing erythromycin after incubation at 30°C for 24 to 48 h.The recombinant plasmid was then isolated and verified by restriction enzyme digestion analysis.The stability of the recombinant plasmid was tested as described previously (Raha et al. 2006).

Transformation into E. coli
The newly constructed plasmid was then transformed into E. coli TOP10 and XL1-Blue competent cells using standard heat-shock method as described by Sambrook and Russell (2001).A widely used lactococcal shuttle vector pMG36e was used as the positive control for transformation.Transformants were selected from LB agar plates containing 150 µg/ml of erythromycin after overnight incubation at 37°C.Erythromycin resistant transformants were subjected to plasmid extraction followed by RE digestion analysis and PCR screening.

Cloning and expression of heterologous gene in pAR1411
Primers were designed to isolate the chloramphenicol acetyltransferase cat gene from a lactococcal vector pNZ8048.The open reading frame (ORF) of chloramphenicol acetyltransferase (cat) gene was PCR amplified from pNZ8048 using CmF (5′-GGC TCT AGA TAT GAA CTT TAA TAA AAT TGA TTT AG-3′) and CmR (5′-AAT CTG CAG TTA TAA AAG CCA GTC ATT AGG-3′) primers that contain Xba I and Pst I site at their respective 5′-end.The PCR conditions used were: initial denaturation at 95°C for 5 min, 25 cycles of denaturation at 95°C for 1 min, annealing at 60°C for 1 min, extension at 72°C for 1 min, and final extension at 72°C for 10 min.The PCR product was analysed by agarose gel electrophoresis, cloned into pAR1411 and transformed into L. lactis MG1363 as described above.The transformants were selected on SGM17 agar containing erythromycin, chloramphenicol or both.The recombinant plasmids were then isolated from the transformants and verified.

Verification of insertion of cat gene in pAR1411
Single colonies of transformants were subcultured in SGM17 broth standing culture containing erythromycin and chloramphenicol, or only chloramphenicol at 30°C.Plasmid DNA was then extracted from the culture.The insertion of cat gene in pAR1411 in the putative recombinant plasmid was verified through RE digestion analysis with PstI and XbaI.The RE digests were then subjected to agarose gel electrophoresis.was found to contain a single plasmid that was larger than pAR141.The calculated size of the recombinant plasmid was about 3.2 kbp.Restriction enzyme analysis of these plasmids recovered from erythromycin resistant L. lactis transformants showed that the 1.6 kbp bluntended EmPMT fragment has been successfully cloned into pAR141.Digestion with HindIII yielded two visible fragments of ~0.7 and ~2.5 kb, which indicated that the ermC gene was in the same orientation with the repA and repB genes of pAR141 (Figures 1 and 2).Constructs with resistance and replication genes in opposite direction, which would have given rise to two detectable fragments of ~1.4 and ~1.8 kb, were not obtained.This newly constructed plasmid vector was designated as pAR1411.

Each of the erythromycin resistant transformants selected
Plasmid stability test showed that pAR1411 was highly stable (~100%) in L. lactis for at least 100 generations under non-selective conditions.However, after several attempts, pAR1411 could not be recovered from the E. coli erythromycin resistant transformants.
In order to examine the usefulness of pAR1411 as a cloning and expression vector, a gene coding for chloramphenicol was selected as the reporter marker.The cat gene was isolated from pNZ8048 by PCR amplification, devoid of its transcriptional and translational signals.Both the amplicon and pAR1411 were digested with PstI and XbaI.The open reading frame of the cat gene (~0.7 kb) was then cloned in-frame with the P 32 promoter in pAR1411, producing the contstruct pAR1411- cat, and transformed into L. lactis MG1363.Initially, a total of 28 transformants were recovered from SGM17 agar plate containing erythromycin (5 µg/ml), but not on the SGM17 with erythromycin (5 µg/ml) and chloramphenicol (7.5 µg/ml).Interestingly, after two subsequent transfers, one colony grew in the broth medium containing both antibiotics.Plasmid extraction revealed that this erythromycin and chloramphenicol-double resistant strain contained a plasmid bearing the expected molecular size.The putative recombinant plasmid pAR1411cat was further verified through RE digestion analysis Digestion analyses indicated that the plasmid extracted from the transformant harbored the expected fragment (Figure 3).

DISCUSSION
A lactococcal cloning and expression vector was constructed based on the indigenous plasmid pAR141.pAR 141 is a small plasmid isolated from L. lactis subsp.lactis M14 (Raha et al., 2006).Detailed examination of the plasmid sequence revealed that this cryptic plasmid only carried genes essential for its own replication.There are no selection markers which would enable the strains harboring it to be scored.In addition, pAR141 also contained limited unique RE sites for use in cloning.These features of pAR141 restricted its use as a cloning vector.Therefore, in this study a fragment from the L. lactis expression vector pMG36e was introduced into pAR141 to produce a cloning and expression vector, pAR 1411.Based on the sequence of pMG36e, primers were carefully designed to amplify the whole fragment of complete erythromycin resistance gene (ermC) including its expression signals, and the multiple cloning sites together with the promoter P 32 and terminator (T) sequences.The other nonessential sequence was minimized to keep the newly constructed plasmid as small as possible.
Antibiotic resistance is generally used as a selection marker due to its easy selection.However, antibiotic resistance genes are not commonly present in lactococcal plasmids.Although several plasmids in L. lactis had been studied, many of these plasmids turned out to be cryptic (McKay, 1983), including pWV01 (Leenhouts et al., 1991), pSH1 (Gasson, 1983) and pAR141 (Raha et al., 2006).Only a limited number of the identified plasmids such as pAJ01 (Raha et al., 2002) encoded antibiotic resistances.Therefore, the erythromycin resistance marker, ermC which originated from Staphylococcus aureus plasmid, pE194, was selected because it has been shown to express in L. lactis, Bacillus.subtilis and E. coli efficiently and utilized in various constructions of lactococcal plasmid vectors (de Vos and Simons, 1994).
Since pAR141 possess limited number of unique RE sites, a fragment constituting several restriction enzyme recognition sites was added to enhance cloning.With the addition of the multiple cloning sites, cloning of genes into the vector would be made easier as there are more choices of RE sites.Directional cloning could also be performed where two different REs are used to orientate the insertion and the problem of inappropriate insertions can also be avoided.
In addition to the selection marker and MCS, the constitutive P 32 promoter and transcriptional terminator of pMG36e (van de Guchte et al., 1989) were also incurporated into pAR1411.These two gene expression signals flanked the MCS of pMG36e.Although useful, the constitutive promoter P 32 which originated from the chromosome of L. lactis subsp.cremoris Wg2 is not a very strong promoter (van der Vossen et al., 1987).This may be caused by the difference in two nucleotides in the sequence of P 32 from the consensus in the -35 region (van der Vossen et al., 1987).The fragment also encompassed the RBS sequence (GGAGG) and the 5′-end of the orf32 gene.
The transcriptional terminator used in pAR1411 originated from the proteinase gene of plasmid pWV05 of L. lactis Wg2 (Kok et al., 1988).The terminator consisted of two complementary IRs which could form a stem of 15 bp followed by a stretch of T's, which are the features of a rho-independent terminator.It is beneficial to have a transcriptional terminator downstream of the site in which to clone foreign genes (Stueber and Bujard, 1982).This could prevent read-through of the promoter to the replication region and affect the plasmid replication and its stability.
During the construction of the recombinant vector from the cryptic plasmid pAR141, care was taken that the replication function of the plasmid was not disturbed.The repA, repB genes and the sequence encoding dso are essential features for plasmid replication and maintenance.The successful transformation and recovery of pAR1411 in L. lactis indicated that the unique FspI site used for the insertion of pMG36e fragment is not located within the region important for replication of the plasmid.This observation also appeared as evidence that plasmids from L. lactis subsp.lactis could replicate in subsp.

cremoris.
The replication of the recombinant plasmid pAR1411 in E. coli strains was tested.However, pAR1411 failed to be retrieved from the transformants.In some instances, plasmids isolated from the transformants showed differences in sizes and RE patterns as compared to the pAR1411 obtained from L. lactis.Most of the time, no plasmid could be extracted from the cells.This was Yeng et al. 5625 unexpected because based on sequence similarity to pWV01 (Leenhouts et al., 1991), pAR141 and its derivative should be functional in a wide range of bacteria.These may indicate the structural and segregational instability of the construct in E. coli.The functionality of the replication system of this plasmid might be another point for these results.The possibility of contamination of the transformants was low as the control reaction with pMG36e was successful.
To test the functionality of the construct pAR1411 as cloning and expression vector in L. lactis, the cat gene conferring chloramphenicol resistantance was used.The use of antibiotic resistance gene as the reporter provided an easy selection of recombinants expressing the inframe inserted DNA.The cat gene has been used as a reporter gene in B. subtilis and L. lactis for the screening of promoters and transcriptional terminators (van der Vossen et al., 1985).The problems of low yield of transformants with double resistance encountered during the attempts of expression might be caused by several factors.The higher yield on the agar plates with erythromycin selection alone indicated that the electrotransformation processes were successful.However, a majority of these transformants were erythromycin sensitive indicating that the plasmid might have undergone selfligation.This might be improved by using higher ratio of insert to vector in the ligation, or by dephosphorylation of the vector with calf/shrimp alkaline phosphatase to reduce self-ligation.At the same time, the failure to recover transformants on the agar plates supplemented with both antibiotics might be due to the high concentrations of the antibiotics.Kok et al. (1984) used only 5 and 1 µg/ml of chloramphenicol and erythromycin, respectively, while in this study, the concentrations were 7.5 and 5 µg/ml.These researchers also showed that reduced concentrations of antibiotics were required during selection in E. coli when plasmid with these two resistant markers was used.
A recombinant plasmid pAR1411-cat containing cat gene was successfully recovered from the L. lactis transformants.Although SDS-polyacrimide gel electrophoresis was not carried out to demonstrate the production of the CAT protein, the ability of the transformants to grow on the chloramphenicol-containing agar plate was a strong evidence that a functional substance conferring resistance to the antibiotic was present and expressed in the strain.
In conclusion, a recombinant vector pAR1411 was constructed by subcloning the EmPMT fragment from pMG36e into pAR141.This construct could replicate in L. lactis, but was found to be unable to transform the E. coli strains tested.pAR1411 could be used to clone and express catalase acetyltransferase, a heterologous reporter protein, in L. lactis.The expression vector could be further developed and improved to serve different purpose.

Figure 2 .
Figure 2. Analysis of putative recombinant plasmids digested with HindIII and Eco321.Lane M: GeneRuler™ DNA Ladder Mix (Fermentas); Lanes 1 and 2: putative pAR1411 from L. lactis; Lane a: HindIII-digested samples; Lane b: Eco321digested samples; Lane c: undigested samples.The arrows showed the expected bands of HindIII digest.The yield of two visible bands of ~0.7 and ~2.5 kbp reveals that the ermC was in the same orientation with the repA and repB genes.

Table 1 .
Bacterial strains and plasmids used in this study.