Indian Journal of Biotechnology Vol. 17, July 2018, pp 416-421

Application of mobilization gene promoter for heterologous expression and curing of plasmid pSMA23 Sudhamani M1*, Batish V K2 and Heller K J3

1*Department of Biotechnology, K L University, Vaddeswaram, Guntur 522502, India 2Department of Dairy Microbiology, National Dairy Research Institute, Karnal 132001, India

3Department of Microbiology and Biotechnology, Max Rubner-Institut (Federal Research Institute of Nutrition and Food), Hermann-Weigmann-Straße1, D-24103 Kiel, Germany

Received 3 June 2015; revised 18 November 2016; accepted 17 March 2017

Lactobacilli have been recognized as key members of the group of probiotic bacteria by virtue of expressing a wide spectrum of physiological functions beneficial to human health. Genetic modification of this genus requires the availability of tools for gene expression like promoter and suitable host vector systems. In the context, plasmid pSMA23 was cured from its native host Lactobacillus casei A23 by electroporation with a construct based on the pSMA23 replicon. Cured strain A23 can be employed as host for vectors or constructs based on the replicon of pSMA23 and for heterologous expression of genes. The promoter less gene cat194 coding for chloramphenicol resistance was cloned under the control of the putative mobilisation gene promoter present on the native plasmid pSMA23 of Lactobacillus casei A23. Constructs were electroporated into Lactobacillus casei LK1 and transformants were obtained on media containing chloramphenicol, indicating expression of the cat194 gene. The putative promoter is thus active and can be recruited for gene expression.

Keywords: Lactobacillus, promoter, mobilization, plasmid curing, incompatibility

Introduction The therapeutic role of fermented milks being

consumed by man since more than 5,000 years has been well documented and chiefly attributed to lactobacilli. Moreover, lactobacilli are also used extensively as starters in food and feed industry. They are the potential hosts for expression of heterologous proteins like antigens, enzymes, bacteriocins etc. This requires the development of appropriate expression systems. Many expression elements have been used, most notably the nisin promoter-based expression system1. Curing of plasmids is an essential step to decipher their function. Also, host cells should be devoid of native plasmids if to be used for cloning studies or as industrial recombinant hosts. Curing is done by growing host strain with agents like novobiocin, and intercalating agents2-6. Incompatibility of plasmid replicons was used to cure pSMA23 from Lactobacillus casei A23 by electroporation of a construct based on the replicon of pSMA23. A previous study7 provided indirect proof for the promoter of mobilization gene present on

plasmid pSMA23 to be functional. This study confirms the suitability of the mobilization gene promoter for heterologous expression through expression of a reporter devoid of its native promoter. Material and Methods

Bacterial Strains and Media Strains of Escherichia coli and L. casei used

in this study are listed in Table 1. Strain L. casei A23 was isolated from the dairy environment2. Strains of E. coli were routinely grown in LB medium at 37°C, whereas strains of Lactobacillus were grown at 37°C in Modified Rogosa Sharpe (MRS) medium8, supplemented with glucose or lactose. For preservation of all strains, one milliliter of overnight culture was mixed with 300 µl of 87% sterile glycerol. Recombinant strains were propagated and maintained as above but with addition of appropriate antibiotics in the medium. For propagating clones of E. coli, ampicillin (100 g/ml), erythromycin (100 g/ml) or chloramphenicol (10 g/ml) were used in LB broth or agar. Recombinant lactobacilli were grown in MRS medium, supplemented with chloramphenicol (10 g/ ml) and erythromycin (10 g /ml), respectively.

—————— *Author for correspondence: Tel: 91-9703470598 sudhamani1@rediffmail.com

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DNA Manipulations and Analysis Plasmids from strain A23 were isolated on a large

scale by alkali lysis method9 or by a modified small scale preparation procedure using the Nucleo-Spin Plasmid Kit (Macherey-Nagel, Germany). Plasmid DNA from E. coli was purified by use of different plasmid isolation kits (Macherey-Nagel). DNA was extracted from agarose gels using kits MinElute or QiaQuick (Qiagen GmbH, Hilden, Germany). Restriction enzymes, T4 DNA ligase, Klenow fragment and T4 polymerase were obtained from commercial providers and used as per manufacturer’s instructions. Lambda DNA digest with StyI with fragments sizes 19,329 bp; 7,743 bp; 6,223 bp; 4,254 bp; 3,472 bp; 2,690 bp; 1,882 bp, 1,489 bp; 925 bp; 421 bp; was used as ladder. Agarose gel electrophoresis of plasmids, restriction fragments, and PCR products were carried out as described10. PCR Amplification of the Promoter less cat Gene from pC194

Primer pairs Hcat/ Revcat and Lcat/ Revcat (Table 2) were used separately to amplify the cat gene from pC194 (Fig. 2). The program for PCR

amplification of the cat gene comprised initial denaturation at 94°C for 2 min followed by 30 cycles each of denaturation at 94°C for 30 sec, annealing at 65°C for 30 sec, and extension at 72°C for 1 min, with final extension at 72°C for 10 min. The PCR reaction after completion was held at 4°C until further use. Both sets of primer pairs resulted in PCR products (800 bp).

Electroporation of Lactobacilli The protocol of B. Henrich (personal communication)

was followed for electroporation of lactobacilli. An overnight culture was diluted 1 : 10 in MRS and incubated at 37°C until an OD600 of 2.2 to 2.8 was obtained. Cells were centrifuged at 4000 x g at 4°C. The supernatant fraction was drained and the cells were washed at least twice with electroporation buffer (EPB; 0.05 M sucrose, 1 mM MgCl2 and 7 mM potassium phosphate buffer, pH 7.2). Cell pellet was suspended in 1/6 volume EPB and left for 5 min on ice or shock-frozen in liquid nitrogen and stored at -70°C until further use.

To an aliquot of 200 µl competent LK1 cells, 50 µl of plasmid DNA in TE was mixed and kept in a pre-cooled cuvette with a gap of 0.2 cm. Cells were electroporated with a pulse of 1400 V at 25 µF. After electroporation, cells were incubated in 50 µl of 10X MRS at 37°C for 2 h in case of selection on erythromycin. However, in case of chloramphenicol, cells were plated directly on agar medium containing chloramphenicol.

Results

Curing of Plasmid pSMA23 The wild host strain A23 was electroporated with

construct pL81 (Fig. 1 and Table 1a). Transformants (clones) were obtained by selecting for erythromycin resistance. The minpreps of the clones were compared with that of wild host A23. The clones (Fig. 3a, lane 3) showed disappearance of the band corresponding to pSMA23 (Fig. 3a; lane 1; Fig. 3b, lane 6) and the appearance of the band corresponding to pL81 (Fig. 3a; lane1; Fig. 3b, lane 6). This suggested that the pSMA23 was replaced by pL81 in the clones.

The clones were grown in medium devoid of erythromycin for four propagations and plated on

Table 2 — Primers used in this study

Primer DNA sequence

Hcat 5'-CGTATCGGATCCTGACTAAGAAAGCAGACAAGTAAGCC-3' Lcat 5'-CATTAGGATCCTGATTGATTGAATTTAGGAGGCATATCAAATGAACTT-3' Revcat 5'-AAAAAGGAGAAGTCGGTTCAGAAAAAGAAGGATATGGATCTGGA-3'

Table 1 — List of strains used

Strain Source

Escherichia coli Easypore Eurogentec, Cologne, Germany Lactobacillus caseiLk1 Kindly provided by B. Henrich,

University of Kaiserslautern, Germany

Lactobacillus casei A23 Sudhamani et al, 2008

Plasmid Description Source

pSMA23 Resident plasmid from Lactobacillus isolate A23; 3.4 kb

Sudhamani et al, 2008

pL93.2 Recombinant construct containing pSMA23, Ampr; 6.5 kb

Sudhamani et al, 2008

PL155 pL93.2 containing the cat194 gene devoid of the native promoter (obtained using primer pair Hcat/Revcat) cloned downstream of putative mob promoter; Ampr; Cmr; 7.5 kb

This study

PL156 pL93.2 containing the cat194 gene devoid of the native promoter (obtained using primer pair Lcat/Revcat) cloned downstream of putative mob promoter; Ampr; Cmr; 7.5 kb

This study

pL81 pLME.19 (2) with Emr as ClaI fragment from pBCE1; 7.5 kb

This study

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medium without erythromycin. Colonies obtained showed the disappearance of bands corresponding to pL81. However, miniprep still had other bands (Fig. 3b, Lanes 2-5). It can be concluded that wild host A23 was cured of pSMA23 by first replacing it with pL81 under erythromycin selection and then losing pL81 by removing the selection pressure. Development of Expression System

Both sets of primer pairs Hcat/Revcat, and Lcat/Revcat resulted in PCR products (800 bp). The PCR products obtained were purified and used seperately for cloning into pGEM T-Easy vectors. PCR product was ligated with linearised pGEMT-Easy vector, transformed into the E. coli and plated on agar plates containing ampicillin.

Colonies obtained after 24 - 48 hrs incubation at 37oC were tested for the presence of vector carrying inserts by double-digest with BamHI and NotI. The BamHI–NotI fragment (800 bp) was extracted from the gel and ligated to pL93.2, digested with the same enzymes. The ligation mixture was transformed into E. coli. Transformants were obtained on LB medium containing ampicillin. In this way, two different constructs pL155 and pL156 were obtained. The construct pL155 was obtained from the PCR product of primer pair Hcat/Revcat while pL156 was obtained using PCR product of primer pair Lcat/Revcat (Table 1a; Fig. 2).

Both constructs pL155 and PL156 were transformed into L. casei LK1 by electroporation. Clones were obtained on plates containing chloramphenicol. Discussion

Curing of Plasmid pSMA23 The wild host strain A23 was cured of pSMA23 by

electroporating with construct pL81, derived from pSMA23 and harboring an erythromycin resistant (Emr) gene. However, presence of further bands in the cured host indicates that strain A23 contains resident plasmids other than pSMA23 (Fig. 3b; Lanes 2-5). These results, besides providing confirmation that strain A23 harbors plasmids in addition to pSMA23, show that curing is specific for plasmids carrying the same origins of replication, i.e. being of the same incompatibility group. The cured strain can be used as host for vectors derived from pSMA23 or similar plasmids, and used for heterologous expression of genes.

Curing of endogenous cryptic plasmids using stress agents like heat, antibiotics, dyes etc is laborious, and uncertain. On the contrary, they are prone to contamination. In fact, Posno et al11 reported that endogenous plasmids in a strain of L. pentosus could not be cured by any of the conventional techniques of cultivation of the cells at elevated, sub lethal temperature or in the presence of curing agents such as ethidium bromide, acriflavine and novobiocin. However, transformation with constructs resulted in curing the endogenous plasmids, reportedly due to incompatibility of the constructs with the endogenous plasmids. Curing of plasmids by transformation has been earlier reported in E. coli or B. thuringenesis12,13. Recently, Liu et al14 used plasmid incompatibility to eliminate pXO1 from B. anthracis. Use of plasmid

Fig. 1 — Constructs used in this study: Dotted line: backbone ofpbluescript; dark arrows represent mob gene; open arrowrepresents rep gene; coloured arrow indicates promoter ofrespective genes; pin head represents terminator; dso: doublestranded origin of replication; erm: erythromycin resistancemarker from pE194; cat 194: chloramphenicol resistance markerfrom PC194. Plasmid pSMA23 is shown linearized at EcoRV site.See Table 1 and /or Sudhamani et al, 2008 for details.

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incompatibility may be a more directed and easier approach to cure plasmids. Also, it is safer as it does not use any mutagenic or carcinogenic chemicals, which may result in mutations in the host chromosome in the process of curing the plasmid. Also, it is highly specific to the plasmid to be cured unlike conventional methods, where other resident plasmids may be also lost. Such arguments were also made earlier14. Development of Expression System

Plasmid pSMA23 has a potential promoter and ribosomal binding site (RBS) upstream of predicted orfM (mob gene). Construct pL93.2 (Fig. 1) contains pSMA23 cloned as an EcoRV fragment in pBluescriptskII+15. The multiple cloning site of pBluescriptskiII+ is downstream of the putative expression signals of mob. Assuming that the expression

signals of mob are functional, cloning a promoter less reporter downstream will lead to its expression.

Hence, primers were designed to obtain the promoter less cat gene (Fig. 2). Primers Hcat and Lcat containing stop codons in all the reading frames (to prevent formation of protein fusion with the cat gene product) were designed with BamHI sites at their 5’ ends (Table 1). Both primers were used separately with reverse primer Revcat to amplify the cat gene without promoter but with RBS. The cat gene from pC194 has the RBS sequestered in a stem-loop structure16-20 (Fig. 2b). This structure is responsible for the inducible nature of cat and strong promoters are required to cause expression sufficient for growth on chloramphenicol21. Without any knowledge on the strength of the putative mob promoter, we decided to modify the cat regulatory region so as to detect sufficient expression even from weak promoters.

Fig. 2 — (a) The 5’ Utr and 3’Utr region of cat gene mRNA on pC194. The sequence in red shows possibility of forming a stem loopstructure sequestering the RBS (double arrow). The stem is represented as dotted arrows. Note the position of the primers Hcat, Lcat andRevcat indicated by dark arrows. The stem loop structure is represented above the mRNA; (b) PCR product obtained using the primerpair Hcat-Revcat and Lcat-Revcat. A PCR product of around 900 bp was obtained. Lanes: 1-4) PCR product obtained using primer pair Hcat-Revcat; 5-7) PCR product product obtained using primer pair Lcat-Revcat; (c) Confirmation of pL156 using restriction digestion.The results indicate that pL156 contains cat gene as a BamHI-Not I insert in pL93. Lane: 1) PCR product obtained using primer pair Hcat–Revcat; 2) PCR product obtained using primer pair Lcat-Revcat; 3) PL156 restricted with BamHI and Not I; 4) pL93 restricted with BamHI and Not I; (d) Confirmation of pL156 using restriction digestion. Lanes: 1) pL155 linearised with BamHI; 4) pL93 linearised with BamHI; and (e) Confirmation of pL811 (approx. 1 kb insert) for presence of Emr. Lane 1: pL811 digested with ClaI Lane 2: pLME.19 linearised ; Lane 3: pL811 digested with SacI; The ‘*’ indicates that two similar sized bands appear as a single band. The construct pL811was obtained by cloning 1 kb Emr gene from pBCE as a ClaI fragment at similar site of pL93.

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Primer Lcat was designed to prevent formation of the stem-loop structure by removing the complementarity required for such a formation (Fig. 2). This should facilitate constitutive expression of the cat gene. A similar approach had been made previously in order to screen for weak promoters, using reporter gene cat86 but providing an additional RBS in presence of an intact stem loop structure in pBV50321. Cloning of the PCR products in pL93.2 positioned the promoter less cat gene under the control of the promoter of the mob gene of pL93.2 (as in pL155 and pL156). Strain L. casei LK1 could be successfully transformed with pL155 and pL156 by selecting for chloramphenicol resistance, indicating that the cat reporter gene was expressed from the promoter of the mob gene. The cat194 gene was previously used as reporter by Constable and Mollet (1994)22.

Expression of promoter less cat194 under the mob promoter could be obtained in both cases, with and

without destabilizing the leader stem loop structure trapping the RBS. The results support our previous work15. The cbh gene with native promoter was cloned in either orientation with respect to the putative mob promoter in pL120 resulting in constructs pS3B and pS3A. However, expression was detected only in pS3B containing cbh aligned in the same direction as the mob promoter, suggesting that expression was indeed driven by the mob promoter. This study confirms that the mob promoter of pSMA23 is functional and can be recruited for gene expression. This is only second report of heterologous expression using mob promoter. Conclusions

Although various expression elements have been reported, they do not perform uniformly across all the species of the large (more than 200 species/subspecies) and diverse genus of Lactobacillus23-32. In this context, the mob promoter and A23, definitely add to the choice of expression tool/hosts available for heterologous expression of beneficial genes in this large and diverse Lactobacillus genus. Acknowledgement

M. Sudhamani gratefully acknowledges funding by a fellowship from Deutscher Akademischer Austausch Dienst (DAAD). References 1 Fang F & O 'Toole P W, Genetic tools for investigating the

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Fig. 3 — (a) Curing of pSMA23 from strain A23. Strain A23(lane 2) was transformed with pL81 (lane 3) and transformantsselected on erythromycin (lane 1). Observe the loss of bandcorresponding to pSMA23 in (lane 1) and appearance of bandcorresponding to pL81. λ+ StyI is ladder, and (b) Curing ofpSMA23 from A23. The miniprep of colonies thus selected(lane 2-5) showed the loss of band corresponding to pL81. Thus,the strain A23 (lane 1) was finally cured of pSMA23 throughincompatibility with pL81. λ+ StyI is ladder.

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