A system for heterologous expression of bacteriocins inLactobacillus sake

Lars Axelsson a;*, Tone Katla a, Merete BjÖrnslett 1;a, Vincent G.H. Eijsink b,Askild Holck a

a MATFORSK, Norwegian Food Research Institute, Osloveien 1, N-1430 Aî s, Norwayb Department of Biotechnological Sciences, Agricultural University of Norway, N-1432 Aî s, Norway

Received 21 July 1998; received in revised form 15 September 1998; accepted 16 September 1998

Abstract

A system for efficient heterologous expression of class II bacteriocins is described that is based on introducing two plasmidsin a bacteriocin-negative Lactobacillus sake strain. The first plasmid (pSAK20) contains the genes necessary for transcriptionalactivation of the Sakacin A promoter as well as export and processing of bacteriocin precursors. The second plasmid (apLPV111 derivative) contains the structural and immunity genes for the bacteriocin of interest fused to the sakacin Apromoter. Using this system, various bacteriocins were produced at levels equal to or higher than those obtained with thecorresponding wild-type producer strains. z 1998 Published by Elsevier Science B.V. All rights reserved.

Keywords: Bacteriocin; Lactobacillus sake ; Sakacin; Expression

1. Introduction

Bacteriocins and bacteriocin producing strains ofthe lactic acid bacteria (LAB) have been the subjectsof extensive research in recent years due to theirpotential as biopreservatives [1]. The developmentof e¤cient methods for heterologous expression isan important goal in bacteriocin research. Heterolo-gous expression of bacteriocins may be used to con-struct new LAB strains with improved protective

properties. It may also be used to construct isogenicLAB strains that di¡er only in the ability to producea certain bacteriocin, thus facilitating the scienti¢cevaluation of the e¡ect of this bacteriocin in food.Finally, the availability of e¤cient, simple-to-use ex-pression systems is of utmost importance for basicresearch on bacteriocins, such as structure^functionstudies using site-directed mutagenesis [2,3].

Several studies of heterologous expression of LABbacteriocins have been described [4^9]. One conclu-sion to be drawn from these studies is that the trans-port machineries mediating secretion and processingof class II pre-bacteriocins with so-called double-gly-cine leader peptides [2,10] are frequently, but notalways, interchangeable. The heterologous expres-sion systems described so far are not optimally suited

0378-1097 / 98 / $19.00 ß 1998 Published by Elsevier Science B.V. All rights reserved.PII: S 0 3 7 8 - 1 0 9 7 ( 9 8 ) 0 0 4 3 1 - 5

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* Corresponding author. Tel. : +47 6497-0100;Fax: +47 6497-0333; E-mail: lars.axelsson@matforsk.no

1 Present address: Biotechnology Centre of Oslo, University ofOslo, Norway.

FEMS Microbiology Letters 168 (1998) 137^143

for the above-mentioned aspects of bacteriocin re-search because: (1) production levels are low; (2)the host strains used are not bacteriocin-negative;(3) the genetic constructs used are not easy to usein site-directed mutagenesis; (4) the bacteriocin isproduced as a chimeric protein (e.g. fused to themaltose-binding protein); and (5) the systems arenot suitable for use in meat-associated lactobacillisuch as Lactobacillus sake.

Sakacin A is a class II bacteriocin whose secretionis directed by a typical double-glycine leader peptide[11]. It was shown that the genes necessary for pro-duction of sakacin A can be divided into two oper-ons that complement each other in trans. One oper-on contains the genes necessary for transcriptionalregulation and secretion of the pre-bacteriocin,whereas the other contains the structural and im-munity genes for sakacin A [11]. Here we haveused these results to establish a standardized, easy-to-manipulate system for heterologous expression ofclass II bacteriocins in L. sake. This system is basedon exchanging the sakacin A structural and immun-ity gene with corresponding genes for other bacter-iocins. E¤cient heterologous expression of sakacin P[12], pediocin PA-1 [13], and piscicolin 61 [14] wasobtained.

2. Materials and methods

2.1. Strains, vectors and growth conditions

Bacterial strains and plasmid vectors are listed inTable 1. All strains except Escherichia coli DH5Kwere routinely grown in still, non-aerated culturesat 25³C. MRS (Oxoid, Basingstoke, UK) was usedfor Lactobacillus and Pediococcus strains, cMRS [15]for Carnobacterium piscicola LV61 and Listeria en-richment broth (Oxoid) containing 0.1% Tween 80for Listeria ivanovii Li4(pVS2). E. coli DH5K wasgrown in BHI (Oxoid) with vigorous shaking at37³C. Agar plates contained 1.5% (w/v) agar. Softagar MRS was made with 0.8% (w/v) agar. B-MRS[11] was used for plating L. sake transformants andfor bacteriocin spot tests. Antibiotics were used inthe following concentrations: chloramphenicol, 10Wg ml31 ; erythromycin, 10 Wg ml31 for LAB andLi. ivanovii and 200 Wg ml31 for E. coli.

2.2. DNA manipulations, transformations and plasmidconstructions

Plasmids from E. coli were prepared with the Wiz-ard system (Promega, Madison, WI) according tothe manufacturers directions. Methods for molecularcloning techniques, transformation of E. coli and L.sake, DNA sequencing and PCR reactions were asdescribed previously [11].

pSAK20 is a pVS2 derived plasmid containing thegenes necessary for activation of transcription of thesakacin A structural gene, as well as genes encodingthe proteins needed for export and processing of pre-sakacin A (Fig. 1A; [11]). pLPV111 derivativespSPP1, pPED1 and pPSC1 (Fig. 1B) were con-structed by cloning PCR fragments containing thestructural and (putative) immunity genes of sakacinP (sppA, spiA) [16], pediocin PA-1 (pedA, pedB) [13]and piscicolin 61 (psc61, orfX) [14], respectively. Thefragments contained the native promoters as well asapproximately 100 basepairs upstream comprisingidenti¢ed or possible regulatory regions [17].pLPV111 derivatives pSPP2, pPED2 and pPSC2(Fig. 1B) were constructed in a similar way, exceptthat the PCR fragments contained the structural andimmunity genes of the respective bacteriocins fusedto the sakacin A promoter. The fusions were madeby the overlap extension (one-sided SOEing) techni-que [18] and the fusion point was at the ATG startcodon of the bacteriocin structural gene. The pro-moter part included the transcriptional activation re-gion of the sakacin A promoter described previously[11]. pSPP3 is a variant of pSPP2, in which the fu-sion point was at the codons encoding the conserveddouble glycine at the end of the leader peptide. AllpLPV111-derived plasmids were ¢rst made in E. coliDH5K and the DNA sequence of the PCR-derivedinserts was con¢rmed before transfer to L. sakeLb790(pSAK20).

2.3. Bacteriocin production, quanti¢cation andpuri¢cation

Bacteriocin production was tested qualitatively onagar plates by a spot test as described previously[11]. For quanti¢cation of bacteriocin productionduring growth, 100 ml medium was inoculated toan OD600 of 0.1. This culture was divided into iden-

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tical portions which were incubated at 25³C. At var-ious time points one portion was taken from theincubator for measurement of OD600. Subsequently,the sample was heated to 80³C for 15 min and cen-trifuged. Supernatants were frozen or analyzed di-rectly for bacteriocin activity. Bacteriocin activitywas quanti¢ed with a microtiter plate assay [19],modi¢ed to improve sensitivity and reproducibilityby: (1) careful standardization of the inoculum ofthe indicator strain with regard to OD600 and stateof growth; (2) using a bacteriocin preparation with aset concentration as a standard on each plate; and(3) using linear regression of plots from the spectro-photometric readings to calculate bacteriocin activ-ity. Listeria ivanovii Li4(pVS2) was used as indicatorfor sakacin P and pediocin PA-1 and L. sakeDSM20017(pVS2) for piscicolin 61 in this assay.One arbitrary unit (AU) was de¢ned as the amountof bacteriocin inhibiting growth of the indicator by50% (50% of the turbidity of the control culture

without bacteriocin). The bacteriocins producedwere puri¢ed and subjected to N-terminal aminoacid sequencing as described previously [14,20].

3. Results

3.1. Heterologous expression of bacteriocins

More than 95% of the colonies obtained aftertransforming L. sake Lb790(pSAK20) with pSPP2,pSPP3, pPED2, pPSC1 and pPSC2 gave inhibitionzones in a direct agar overlay screening for bacter-iocin activity. Such zones were not observed aftertransforming L. sake Lb790(pSAK20) with pSPP1,pPED1 and pLPV111, nor after transforming L.sake Lb790 without pSAK20 with any of thepLPV111 derivatives. Transformation e¤ciencieswere in the range of 104 transformants per WgDNA in all cases. The L. sake Lb790 double trans-

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Table 1Plasmids and strains

Plasmid/strain Relevant characteristicsa Reference/source

PlasmidsPSAK20 11.8 kb; contains the operon RIRorf4sapKRTE, necessary for transcriptional

activation and processing/transport of sakacin A; Cmr

[11]

pVS2 5.0 kb; cloning vector, used here for creating double-resistant indicator strains;Emr Cmr

[25]

PLPV111b 4.2 kb; E. coli/L. plantarum/L. sake shuttle vector; Emr [11]

StrainsLactobacillus sake

DSM20017(pVS2) L. sake type strain (obtained from DSMc) transformed with pVS2, indicator strain;bac3 Emr Cmr

This study

Lb790 Wild-type strain; bac3 ; sensitive to sakacin A, sakacin P, pediocin PA-1 and piscicolin 61 [11]Lb790(pVS2) Lb790 transformed with pVS2, indicator strain; bac3 Emr Cmr [11]Lb790(pSAK20) Lb790 containing pSAK20, host strain; bac3 Cmr [11]Lb674 Wild-type sakacin P (sakacin 674) producer [20]

Escherichia coli DH5K Host strain; F3 vlacU169(x80dlacZvM15) recA1 end A1 hsdR17 supE44 thi-1gyrA96 relA1 V3

Gibco-BRL

Listeria ivanovii Li4(pVS2) Strain Li4 (kind gift from L. Kroëckel) containing pVS2, indicator strain; Emr Cmr This studyPediococcus acidilacticiPAC1.0

Wild-type pediocin PA-1producer

[13]

Carnobacteriumpiscicola LV61

Wild-type piscicolin 61 producer [14]

aEmr and Cmr, erythromycin and chloramphenicol resistant, respectively; bac3, bacteriocin non-producing.bpLPV111 derived plasmids made in the present study are described in Section 2.2 and shown in Fig. 1.cDSM, Deutsche Sammlung von Mikroorganizmen.

L. Axelsson et al. / FEMS Microbiology Letters 168 (1998) 137^143 139

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Fig. 1. Schematic representation of the relevant regions in the plasmids used in this work. (A) Insert in pSAK20, containing the genesnecessary for transcriptional activation and processing/transport [11]. (B) PCR derived inserts in pLPV111 derivatives with bacteriocinstructural and immunity genes. Filled regions, derived from the sakacin A (sap) gene cluster [11] ; open regions, derived from the sakacinP (spp-spi) gene cluster [16]; shaded regions, derived from the pediocin PA-1 (ped) genes; striped regions, derived from the piscicolin 61(psc) gene cluster [14]. The `P' pre¢x denotes promoter including upstream regulatory regions. The scale bars are approximate: note thedi¡erence between A and B.

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formants will hereafter be referred to as 20/SPP1, 20/SPP2, 20/SPP3, 20/PED1, 20/PED2, 20/PSC1, 20/PSC2 and 20/111. N-terminal sequencing of the pu-ri¢ed bacteriocins from strains 20/SPP2, 20/SPP3, 20/PED2 and 20/PSC2 con¢rmed that all had been cor-rectly processed by the sakacin A secretion machi-nery despite di¡erences in the leader peptides (Fig.1).

3.2. Quanti¢cation of bacteriocin production

In liquid cultures of the transformants and thewild-type producer strains, bacteriocin productionfollowed growth (Fig. 2) and the relative di¡erencesbetween the strains were essentially the samethroughout the sampling period. Maximum activitywas obtained in the early stationary phase, followedby a decrease (10^50%) during the stationary phase.The only exception was pediocin PA-1 production byP. acidilactici PAC1.0. For this strain, productionwas essentially zero until the late log phase where asudden burst of bacteriocin activity occurred (Fig.2). Maximum bacteriocin levels are shown in Table2. This shows that 20/SPP2, 20/SPP3, 20/PED2, 20/PSC1, and 20/PSC2 produced bacteriocins in

amounts that were similar to or higher than theamounts produced by the corresponding wild-typestrains. A low amount of bacteriocin activity wasdetectable in culture supernatants of 20/SPP1 and20/PED1. Taken together, the data show that e¤-cient production of sakacin P and pediocin PA-1was only obtained when the sakacin A promoterwas used, whereas piscicolin 61 was produced in ap-preciable amounts, regardless of the promoter used.

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Table 2Maximum bacteriocin activity in liquid culturesa

Strain Bacteriocin activity (kAU ml31)b

Sakacin P producers20/SPP1 2420/SPP2 2.7U103

20/SPP3 3.0U103

Lb674 1.8U103

Pediocin PA-1 producers20/PED1 2.020/PED2 32PAC1.0 14

Piscicolin 61 producers20/PSC1 2.420/PSC2 3.9LV61 3.5

a Measurements were made on three independent cultures and themean is reported. The coe¤cient of variation was in no case morethan 20%. Bacteriocin activity was not detected in supernatantsfrom L. sake Lb790, Lb790(pSAK20), Lb790(pSAK20+pLPV111)or Lb790 not containing pSAK20 transformed with any pLPV111derivative.b kAU ml31 = 1000 AU ml31.

Fig. 2. Growth and pediocin PA-1 production as a function oftime for strain 20/PED2 (A) and P. acidilactici PAC1.0 (B). R,growth measured as OD600 ; b, pediocin PA-1 production meas-ured as kAU ml31 (1000 AU ml31). For all other producerstrains tested in this study, bacteriocin production followedgrowth in a manner similar to that displayed for 20/PED2.

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4. Discussion

The present system for heterologous expression ofbacteriocins has several advantages: (1) thepLPV111 based plasmids are small, stable, and easilypropagated in E. coli with high copy numbers; (2)the host strain L. sake Lb790(pSAK20) is bacterio-cin-negative, fast growing, and relatively easy totransform; and (3) production levels obtained forsakacin P, pediocin PA-1 and piscicolin 61 are inthe same range or higher as those obtained thewild-type producer strains. It should be noted thatthe production levels for one particular bacteriocinin a wild-type producer are easily over-estimated be-cause there may be several bacteriocins present in theculture medium [7,14,17,21].

An additional advantage of the present system isthat the bacteriocins are easy to purify from the cul-ture medium of L. sake Lb790, using standard puri-¢cation schemes. For example, during site-directedmutagenesis studies of sakacin P, we routinely ob-tained 0.3^0.5 mg highly pure bacteriocin with the20/SPP2 system. Such levels are di¤cult to obtainwith wild-type producer strains (G. Fimland, M.Skeie, L. Axelsson and V.G.H. Eijsink, unpublishedobservations). The system has also been used inmodel studies of the in£uence of food componentson the activity of a number of bacteriocins [22], withthe advantage that an accurate comparison betweenbacteriocins could be made since the background(i.e. the production strain) was the same. The poten-tial of constructing strains with preferred character-istics to wild-type producers in food applications,e.g. continuous production during growth, is shownby pediocin PA-1 production by strain 20/PED2compared to the wild-type strain (Fig. 2).

E¤cient production of sakacin P and pediocinPA-1 required the structural and immunity genes tobe under control of the sakacin A promoter. Appa-rently, the native promoters, including (putative) up-stream regulatory elements, were not recognized bythe pSAK20-encoded machinery for activation of thesakacin A promoter. Production of piscicolin 61(which is identical to carnobacteriocin A [23]) waslargely independent of the promoter used, probablybecause of the high similarity between the regulatoryelements upstream of the sakacin A promoter andthe native piscicolin 61 (= carnobacteriocin A) pro-

moter [17]. It has recently been shown that L. sakeLb790(pSAK20) containing the sakacin P structuraland immunity genes under control of the knownstrong lactococcal promoter P32 [24] produces ap-proximately ¢ve times less sakacin P than 20/SPP2(M.B. Brurberg, M. Skeie, I.F. Nes, and V.G.H.Eijsink, unpublished observations). This illustratesthe e¡ectiveness of the sakacin A promoter forover-production of bacteriocins.

The fact that 20/SPP2 and 20/SPP3 produced sim-ilar amounts of correctly processed bacteriocin (Ta-ble 2) indicates that the sakacin A and sakacin Pleader peptides are equally e¤ciently recognizedand processed by the (pSAK20-encoded) sakacin Asecretion machinery. This con¢rms the general pic-ture that double-glycine type leader peptides tend tobe recognized by non-cognate bacteriocin transportmachineries. Nevertheless, several studies have indi-cated that optimizing the leader peptide^secretionmachinery combination may lead to increased bac-teriocin yields [6,8]. It is thus conceivable that thee¤ciency of the present system for producing pedio-cin PA-1 or piscicolin 61 can be increased by havingsecretion of the bacteriocin directed by the sakacin Aleader peptide.

Acknowledgments

We would like to thank Birgitta Baardsen andIngvild Rosshaug for excellent technical assistance.This work was supported in part by two grantsfrom the Norwegian Research Council (104988/110and 107897/120).

References

[1] Stiles, M.E. (1996) Biopreservation by lactic acid bacteria.Antonie van Leeuwenhoek 70, 331^345.

[2] Klaenhammer, T.R. (1993) Genetics of bacteriocins producedby lactic acid bacteria. FEMS Microbiol. Rev. 12, 39^86.

[3] Nes, I.F., Diep, D.B., Haîvarstein, L.S., Brurberg, M.B., Eij-sink, V. and Holo, H. (1996) Biosynthesis of bacteriocins inlactic acid bacteria. Antonie van Leeuwenhoek 70, 113^128.

[4] Allison, G.E., Worobo, R.W., Stiles, M.E. and Klaenhammer,T.R. (1995) Heterologous expression of the lactacin F peptidesby Carnobacterium piscicola LV17. Appl. Environ. Microbiol.61, 1371^1377.

[5] Chikindas, M.L., Venema, K., Ledeboer, A.M., Venema, G.

FEMSLE 8434 23-10-98

L. Axelsson et al. / FEMS Microbiology Letters 168 (1998) 137^143142

and Kok, J. (1995) Expression of lactococcin A and pediocinPA-1 in heterologous hosts. Lett. Appl. Microbiol. 21, 183^189.

[6] van Belkum, M.J., Worobo, R.W. and Stiles, M.E. (1997)Double-glycine-type leader peptides direct secretion of bacter-iocins by ABC transporters: colicin V secretion in Lactococcuslactis. Mol. Microbiol. 23, 1293^1301.

[7] Brurberg, M.B., Nes, I.F. and Eijsink, V.G.H. (1997) Phero-mone-induced production of antimicrobial peptides in Lacto-bacillus. Mol. Microbiol. 26, 347^360.

[8] Horn, N., Martinez, M.I., Martinez, J.M., Hernandez, P.E.,Gasson, M.J., Rodriguez, J.M. and Dodd, H.M. (1998) Pro-duction of pediocin PA-1 by Lactococcus lactis using the lac-tococcin A secretory apparatus. Appl. Environ. Microbiol. 64,818^823.

[9] Miller, K.W., Schamber, R., Chen, Y.L. and Ray, B. (1998)Production of active chimeric pediocin AcH in Escherichia coliin the absence of processing and secretion genes from thePediococcus pap operon. Appl. Environ. Microbiol. 64, 14^20.

[10] Haîvarstein, L.S., Diep, D.B. and Nes, I.F. (1995) A family ofABC transporters carry out proteolytic processing of theirsubstrates concomitant with export. Mol. Microbiol. 16,229^240.

[11] Axelsson, L. and Holck, A. (1995) The genes involved inproduction of and immunity to sakacin A, a bacteriocinfrom Lactobacillus sake Lb706. J. Bacteriol. 177, 2125^2137.

[12] Tichaczek, P.S., Nissen-Meyer, J., Nes, I.F., Vogel, R.F. andHammes, W.P. (1992) Characterization of the bacteriocinscurvacin A from Lactobacillus curvatus LTH1174 and sakacinP from L. sake LTH673. Syst. Appl. Microbiol. 15, 460^468.

[13] Marugg, J.D., Gonzalez, C.F., Kunka, B.S., Ledeboer, A.M.,Pucci, M.J., Toonen, M.Y., Walker, S.A., Zoetmulder,L.C.M. and Vandenbergh, P.A. (1992) Cloning, expression,and nucleotide sequence of genes involved in production ofpediocin PA-1, a bacteriocin from Pediococcus acidilacticiPAC1.0. Appl. Environ. Microbiol. 58, 2360^2367.

[14] Holck, A.L., Axelsson, L. and Schillinger, U. (1994) Puri¢ca-tion and cloning of piscicolin 61, a bacteriocin from Carno-bacterium piscicola LV61. Curr. Microbiol. 29, 63^68.

[15] De Bruyn, I.N., Holzapfel, W.H., Visser, L. and Louw, A.I.(1988) Glucose metabolism by Lactobacillus divergens. J. Gen.Microbiol. 134, 2103^2109.

[16] Huëhne, K., Axelsson, L., Holck, A. and Kroëckel, L. (1996)Analysis of the sakacin P gene cluster from Lactobacillus sakeLb674 and its expression in sakacin-negative Lb. sake strains.Microbiology 142, 1437^1448.

[17] Diep, D.B., Haîvarstein, L.S. and Nes, I.F. (1996) Character-ization of the locus responsible for the bacteriocin productionin Lactobacillus plantarum C11. J. Bacteriol. 178, 4472^4483.

[18] Horton, R.M. and Pease, L.R. (1991) Recombination andmutagenesis of DNA sequences using PCR. In: Directed Mu-tagenesis : A Practical Approach (McPherson, M.J., Ed.),pp. 217^247. IRL Press, Oxford, UK.

[19] Holck, A., Axelsson, L., Birkeland, S.-E., Aukrust, T. andBlom, H. (1992) Puri¢cation and amino acid sequence of sa-kacin A, a bacteriocin from Lactobacillus sake Lb706. J. Gen.Microbiol. 138, 2715^2720.

[20] Holck, A.L., Axelsson, L., Huëhne, K. and Kroëckel, L. (1994)Puri¢cation and cloning of sakacin 674, a bacteriocin fromLactobacillus sake Lb674. FEMS Microbiol. Lett. 115, 143^150.

[21] Anderssen, E.L., Diep, D.B., Nes, I.F., Eijsink, V.G.H. andNissen-Meyer, J. (1998) Antagonistic activity of Lactobacillusplantarum C11: two new two-peptide bacteriocins, plantari-cins EF and JK, and the induction factor plantaricin A.Appl. Environ. Microbiol. 64, 2269^2272.

[22] Blom, H., Katla, T., Hagen, B.F. and Axelsson, L. (1997) Amodel assay to demonstrate how intrinsic factors a¡ect di¡u-sion of bacteriocins. Int. J. Food Microbiol. 38, 103^109.

[23] Worobo, R.W., Henkel, T., Sailer, M., Roy, K.L., Vederas, J.and Stiles, M.E. (1994) Characteristics and genetic determi-nant of a hydrophobic peptide bacteriocin, carnobacteriocinA, produced by Carnobacterium piscicola LV17A. Microbiol-ogy 140, 517^526.

[24] van der Vossen, J.M., van der Lelie, D. and Venema, G.(1987) Isolation and characterization of Streptococcus cremo-ris Wg2-speci¢c promoters. Appl. Environ. Microbiol. 53,2452^2457.

[25] von Wright, A., Tynkkynen, S. and Suominen, M. (1987)Cloning of a Streptococcus lactis subsp. lactis chromosomalfragment associated with the ability to grow in milk. Appl.Environ. Microbiol. 53, 1584^1588.

FEMSLE 8434 23-10-98

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