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Scientific
Publications - Work Done by Microbiology Reader
Applied Microbiology and Biotechnology, Volume
59, Number 4-5, January 2002,
pp. 467-471 Original Paper A food - grade cloning vector for lactic acid bacteria based on the nisin immunity gene nisIT. M. Takala1 and P. E. J. Saris1 (1)
Received: 30 January 2002 / Accepted: 19 April 2002 / Published online: 4 June 2002
ABSTRACT A new food-grade cloning vector for lactic acid bacteria was constructed using the nisin immunity gene nisI as a selection marker. The food-grade plasmid, pLEB590, was constructed entirely of lactococcal DNA: the pSH71 replicon, the nisI gene, and the constitutive promoter P45 for nisI expression. Electroporation into Lactococcus lactis MG1614 with 60 international units (IU) nisin/ml selection yielded approximately 105 transformants/µg DNA. MG1614 carrying pLEB590 was shown to be able to grow in medium containing a maximum of 250 IU nisin/ml. Plasmid pLEB590 was succesfully transformed into an industrial L. lactis cheese starter carrying multiple cryptic plasmids. Suitability for molecular cloning was confirmed by cloning and expressing the proline iminopeptidase gene pepI from Lactobacillus helveticus in L. lactis and Lb. plantarum. These results show that the food-grade expression system reported in this paper has potential for expression of foreign genes in lactic acid bacteria in order to construct improved starter bacteria for food applications.
INTRODUCTION The genetic manipulation of lactic acid bacteria (LAB) has many potential applications in food safety and in the development of improved food products. The safe use of genetically modified LAB requires the development of cloning systems that are composed solely of DNA from food-grade organisms. Different food-grade cloning systems published during the past decade have been reviewed by de Vos (1999). Closely related to the antibiotic resistance selection markers are dominant food-grade bacteriocin-resistance markers. Among bacteriocins, nisin is the most studied. Unlike other bacteriocins, nisin is commercially available and its use as a food preservative is accepted in more than 50 countries (Delves-Broughton et al. 1996). Thus, the usage of a nisin resistance selection marker for developing bacteriocin-resistant food-grade starters for the food industry is favorable. The first food-grade systems based on bacteriocin resistance were plasmids pVS40, pFM011, and pFK012 containing the nisin resistance marker nsr from Lactococcus lactis (Froseth and McKay 1991; Hughes and McKay 1991; von Wright et al. 1990). The nisin resistance marker nsr in previously published nisin resistance plasmids is not present in nisin-producing L. lactis strains. For nisin production, eleven nis genes in two operons, nisZ/ABTCIPRK and nisFEG, are needed (McAuliffe etal. 2001). Nisin-producing lactococci protect their own cell membrane against the pore-forming activity of nisin with the immunity lipoprotein NisI and a putative ABC translocator complex NisF/E/G (Engelke et al. 1994; Immonen and Saris 1998; Kuipers et al. 1993; Siegers and Entian 1995). NisI seems to play a more crucial role in nisin immunity than the NisF/E/G-complex, because disruption of the nisI gene renders the cells more sensitive to nisin than disruption of the nisFEG genes (Ra et al. 1999). However, L. lactis cells with the wild-type level of NisI and containing no other nisin genes showed a nisin immunity level of only 1-4% (10-40 international units (IU) nisin/ml) of that of the wild-type L. lactis nisin-producer strain (1,000 IU nisin/ml; Kuipers et al. 1993; Qiao et al. 1995). Thus, it seems obvious that the NisF/E/G proteins are needed to achieve the wild-type nisin immunity level, with the potential co-operation of NisI. Although the nisI-mediated level of nisin immunity is considerably lower than the immunity level of nisin producers, it has not been shown whether increasing the expression level of the nisI gene could yield a higher nisin resistance. In such a case, the nisin immunity gene nisI could be potentially used as a dominant food-grade selection marker for L. lactis and maybe also for other LAB. No studies concerning nisin resistance in lactobacilli have yet been published. In this study, we describe a new food-grade plasmid vector for LAB. The selection marker, the nisin immunity gene nisI, originates from the nisin Z-producing strain of L. lactis. This plasmid, pLEB590, is composed of lactococcal DNA and it can be efficiently transformed into nisin-sensitive L. lactis and Lactobacillus plantarum strains.
MATERIALS AND METHODS Bacterial strains, plasmids, and growth conditionsThe bacterial strains and plasmids used in this study are listed in Table 1 . Escherichia coli strains were grown in LB medium at 37 °C. L. lactis strains were grown in M17 medium (Oxoid) containing 0.5% glucose (GM17) at 30 °C. After electrotransformation, L. lactis cells were plated onto GM17 agar plates containing 0.5 M sucrose. Lb. plantarum strains were grown in MRS medium (Difco). Erythromycin was used at a final concentration of 300 µg/ml for E. coli and 5 µg/ml for L. lactis. Food-grade transformants were selected by the pour-plate method with nisin (Sigma) at a final concentration of 60 IU/ml. Table 1. Bacterial strains and plasmids
DNA isolations and manipulationsE. coli plasmid DNA was isolated with the Wizard DNA miniprep
purification kit (Promega, Madison, Wis.). Plasmids from L. lactis and
Lb. plantarum strains were isolated according to O'Sullivan and Klaenhammer
(1993). Standard molecular cloning techniques were performed as described by
Sambrook et al. (1989). Eppendorf Mastercycler apparatus was used for polymerase
chain reactions (PCR). L. lactis, Lb. plantarum, and E. coli were transformed by
electroporation as described previously (Holo and Nes 1989; Luchansky et al.
1989; Zabarovsky and Winberg 1990). For Lb. plantarum, 3.5 Segregational stabilityThe stability of maintenance of the food-grade plasmid pLEB590 in L. lactis under non-selective conditions was determined using the method described by Wessels (1993); and the percentage plasmid loss per cell per generation was calculated as described by Roberts et al. (1990). Proline iminopeptidase activity assayFor the determination of proline iminopeptidase (PepI) activity, cell suspensions were lysed as described by Varmanen et al. (1998). PepI activities were determined from cell-free extracts in 50 mM potassium phosphate buffer (pH 7) by measuring spectrometrically the liberation of para-nitroanilide from the substrate L-proline-para-nitroanilide (Sigma) at 410 nm. Cuvettes containing 21 µg of protein extract and 0.55 mM substrate were incubated for 5 min at 40 °C before measurement. Nisin resistance assayTo determine the resistance to nisin of L. lactis strains, 3 µl of overnight cultures were added to 300 µl of GM17 medium containing different concentrations of nisin (0-500 IU/ml) in Bioscreen microtiter plates (100 wells). The plates were grown in the Bioscreen C apparatus (Labsystems, Helsinki, Finland) at 30 °C for 15 h. Every 20 min the plates were mixed by moderate shaking and then the optical density was measured with a wideband filter (420-580 nm).
RESULTS Construction of the food-grade vectorThe replicon for the food-grade vector was produced by PCR from plasmid pVS2. The erythromycin resistance gene ermC was amplified in the same 2.9-kb fragment to facilitate the first cloning steps. The 1.4-kb fragment containing the nisI gene with the constitutive lactococcal promoter P45 (Koivula et al. 1991; Sibakov et al. 1991) was restricted from a previously constructed nisI expression plasmid pLEB415 with ClaI and SmaI. The fragments were made blunt-ended, ligated, and transformed into E. coli TG1 with erythromycin selection, resulting in plasmid pLEB580. The ermC gene was removed from pLEB580 as a ClaI fragment and
the resulting plasmid was transformed into L. lactis MG1614 with nisin
selection (60 IU/ml). Transformant colonies exhibited an Erms Nisr
phenotype and the transformed plasmid was stably maintained under nisin
selection pressure. The construction of the food-grade plasmid pLEB590 is shown
in Fig. 1. It is constructed entirely of lactococcal DNA, except for a multiple
cloning site of synthetic DNA. It consists of a replicon and the nisI
gene as the selectable marker with a constitutive promoter. The transformation
frequency of pLEB590 for L. lactis was determined to be approximately 3.6
Fig. 1. Schematic representation of the food-grade plasmid pLEB590 and the pepI expression plasmid pLEB605
The segregational and structural stability of pLEB590 were then evaluated. Plasmid loss per cell per generation was calculated to be approximately 0.1% in L. lactis. After 18 generations in non-selective medium, 97.8% of cells retained plasmid and showed nisin-resistant phenotype on nisin-containing media. Resistance to nisinTo determine the maximum resistance to nisin, L. lactis MG1614 carrying the food-grade plasmid pLEB590 (strain LAC233) was grown in media containing different concentrations of nisin (0-500 IU/ml) and compared with the nisin producer strain N8, the host strain MG1614, and the nisI expression strain LAC34 (MG1614 carrying pLEB415). The growth curves of the strains are shown in Fig. 2. The nisin producer strain N8 exhibits the wild-type level of resistance to nisin; and the host strain MG1614 displays the sensitivity of a nisin-negative strain. The food-grade strain LAC233 grew well with 250 IU nisin/ml. Its growth was clearly weakened at concentrations of 250-350 IU/ml; and no growth was observed at 400 IU/ml (data not shown). However, strain LAC34, in which expression of nisI is under the control of the same P45 promoter as in LAC233, grew well with 20 IU nisin/ml, but was strongly inhibited with 150 IU/ml.
Fig. 2. Growth curves of Lactococcus lactis strains in medium containing 0, 20, 150, and 250 IU nisin/ml. L. lactis N8 (white circles) exhibits the wild-type nisin producer nisin resistance level (1,000 IU/ml = 100%). MG1614 (black triangles) is a nisin-sensitive strain, with a nisin resistance level lower than 2%. Plasmid pLEB415 (white triangles) is the nisI expression plasmid, with a nisin resistance level lower than 5%. Plasmid pLEB590 (black circles) is the nisI food-grade plasmid, with a nisin resistance level of 25%. IU International units of activity, OD optical density (wide band filter, 420-580 nm)
Food-grade vector for Lb. plantarum and an industrial L. lactis starterTo investigate the functionality of the nisin immunity gene nisI in other LAB, pLEB590 was electroporated into nisin-sensitive Lb. plantarum strain 755 with 60 IU nisin/ml. Transformants obtained showed a stable Nisr phenotype under nisin selection pressure. Plasmid content was verified by plasmid isolation, restriction analysis, and agarose gel electrophoresis. Expression of the nisI gene was confirmed by Western blotting with the NisI antibody (data not shown). The expression of nisI in Lb. plantarum was assessed to be at the same level as in L. lactis. Plasmid pLEB590 was also transformed into the industrial cheese starter strain L. lactis SSL110, which contains several indigenous cryptic plasmids. Plasmid DNA from transformant colonies was isolated and separated in agarose gel. The transformed pLEB590 displaced the smallest cryptic plasmid (approximately 2 kb), suggesting that they belonged to the same compatibility group. No phenotypic effect was observed resulting from loss of the cryptic plasmid. Expression of proline iminopeptidase gene pepIThe applicability of pLEB590 as a vector for expression of heterologous genes in LAB was evaluated by cloning the proline iminopeptidase gene pepI from Lb. helveticus. The pepI gene was amplified by PCR from Lb. helveticus chromosomal DNA, cloned as a BamHI-XhoI fragment into pLEB590, transformed into L. lactis MG1614, and further into Lb. plantarum 755. The construction of this plasmid pLEB605 is shown in Fig. 1. The activity of PepI was determined from L. lactis and Lb. plantarum transformants and compared with the plasmid-free host strains and the pepI origin strain of Lb. helveticus. The host L. lactis and Lb. plantarum strains were almost deficient in PepI activity. Expression of pepI from pLEB605 resulted in approximately 200-fold higher PepI activity in L. lactis and Lb. plantarum, compared with the host strains, and approximately 30- to 40-fold higher activity than in Lb. helveticus. The marker gene nisI in pLEB590 and pLEB605 lacks a transcription terminator, allowing a read-through expression of the promoterless pepI gene. Thus, pLEB590 can be regarded as a food-grade expression vector for LAB.
DISCUSSION In this research, a new food-grade plasmid vector was constructed using the lactococcal nisin immunity gene nisI from a nisin producer strain as a selectable marker. The NisI-mediated immunity level has been described to be approximately 1-4% of that of the nisin producer strain (Kuipers et al. 1993; Qiao et al. 1995). However, the food-grade plasmid pLEB590 reported in this paper resulted in at least 25% immunity level; and transformants were easily selected on nisin plates. This phenomenon is most probably explained by a higher copy number of the plasmid and a higher expression level of the nisI gene than the previously constructed NisI expression plasmids. Despite the relatively high expression level, pLEB590 was maintained stably under both nisin selection and non-selective conditions. High transformation frequency is a desirable property for cloning vectors as is an easy, cheap, and tight selection without background problems. Plasmid pLEB590 meets all these requirements. The transformation frequency was one magnitude higher than that of the previously published nisin resistance plasmid pVS40 (105 vs 104) and the concentration of nisin used for selection was nearly nine-fold (60 IU/ml vs 7 IU/ml) that described for pVS40 (von Wright et al. 1990). A lower nisin concentration in selection might increase transformation frequencies, but it also increases background problems and thus makes selection much more difficult and unreliable. Nisin resistance markers have not previously been transformed into LAB other than L. lactis, despite the fact that nisin-sensitive species occur commonly in the genera Lactobacillus, Leuconostoc, and Pediococcus (Chung and Hancock 2000; Radler 1990) and thus are potential hosts for nisin-resistance vectors. Electroporation of pLEB590 into Lactobacillus plantarum showed that NisI functions as an immunity protein in lactobacilli, enabling nisin selection. Thus, pLEB590 can also be used as a food-grade vector in LAB other than lactococci. Furthermore, pLEB590 can be transformed into industrial starter strains. Plasmid incompatibility is a common phenomenon due to the indigenous plasmids of the wild-type strains but, under nisin selection pressure, the transformed plasmid pLEB590 is more competitive and displaces the indigenous plasmids of the same compatibility group. Expression of pepI in L. lactis and Lb. plantarum from the plasmid pLEB605 resulted in approximately 30- to 40-fold higher PepI activity, compared with the activity of a swiss-type cheese starter, Lb. helveticus. The high activity of proline-specific peptidases in cheese-making decreases the amount of bitter-tasting proline-rich oligopeptides, while the amount of sweet-tasting free proline increases, thus having a favorable effect to cheese flavor formation (Ishibashi et al. 1988; Sullivan and Jago 1972). We thus believe that the food-grade cloning vector pLEB590 has a high potential for use in dairy processes, in order to construct improved LAB starter strains. Acknowledgements. This work was supported by The Academy of Finland and Valio Ltd. We thank Shea Beasley for Lb. plantarum NisI Western analysis.
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SMEB electroporation buffer was used. Electroporation was performed with a Gene
pulser apparatus (Bio-Rad Laboratories, Richmond, Calif.).![[Figure]](../../images/b_images/img_abstr/s00253-002-1034-4flb1.gif)
![[Figure]](../../images/b_images/img_abstr/s00253-002-1034-4flb2.gif)
