High-level coproduction of the bacteriocins nisin A andlactococcin A by Lactococcus lactis

Antonio Fernandez1, Nikki Horn2, Michael J Gasson2, Helen M Dodd2 and Juan M Rodrıguez1*

1 Departamento de Nutricion y Bromatologıa III, Facultad de Veterinaria, Universidad Complutense de Madrid, 28040 Madrid, Spain2 Food Safety Science Division, BBSRC Institute of Food Research, Norwich Research Park, Colney, Norwich NR4 7UA, UK

Received 19 May 2003 and accepted for publication 20 July 2003

In this study, a two-plasmid system for enhanced and consistent biosynthesis of the modellactococcal bacteriocin lactococcin A in non-producing Lactococcus lactis hosts was developed.The system comprised a plasmid carrying the genes lcnA and lciA under the control of the nisin-inducible nisA promoter, and a second plasmid harbouring the lcnC and lcnD genes. Theintroduction of both plasmids into two strains containing the nisRK genes required for nisin-controlled expression, Lc. lactis FI5876 (a nisin A-producer strain) and FI7847, resulted inproduction of extracellular lactococcin A at a higher level than that for the parental strain, Lc.lactis WM4. In addition, transformation of the nisin-producing host with both plasmids led to ahigh-level production of both lactococcal bacteriocins, which may provide a means to exploittheir complementary properties in cheese ripening.

Keywords: Bacteriocin, lactococcin A, nisin, Lactococcus lactis.

Lactococci and other lactic acid bacteria (LAB) play a keyrole in the production of a wide variety of fermented foods.Since concerns on the safety of cheeses and other dairyproducts have increased as a result of outbreaks caused byListeria monocytogenes and other emerging food-bornepathogens, the bacteriocins produced by dairy lactic acidbacteria have been the subject of considerable researchand industrial interest in recent years due to their potentialas food preservatives (Jack et al. 1995). The term ‘bacterio-cin’ comprises a large and diverse group of ribosomallysynthesized antimicrobial peptides or proteins, some ofwhich undergo posttranslational modifications (Nes &Eijsink, 1999).

Occasionally, industrial application of bacteriocin-producing LAB may face practical limitations, such asnarrow antimicrobial spectrum, low-level or inconsistentproduction, and inability to grow in foods in which thebacteriocin(s) produced would be of particular interest(Abee et al. 1995). However, in recent years, improvedtechniques and tools for the genetic manipulation of theseindustrially important organisms have allowed bacteriocinoverexpression, engineering of bacteriocin variants withimproved properties (Kuipers et al. 1996) and transfer ofbacteriocin biosynthesis genes to other strains or species(Rodrıguez et al. 2002). In particular, Lactococcus lactis is

proving to be very versatile and advances in the stabilizationof gene maintenance and in the expression control haveincreased its potential usefulness (Venema et al. 1999).

Lactococcin A is a bacteriocin produced by some Lc.lactis strains and its biosynthesis requires the expression offour genes arranged in two independent operons (Stoddardet al. 1992). The first operon contains the structural (lcnA)and immunity (lciA) genes, while the second comprisesthe lcnC and lcnD genes that encode the lactococcin Asecretory machinery. In this study, a two-plasmid systemfor enhanced and consistent biosynthesis of lactococcin Awas successfully applied to heterologous Lc. lactis strains.The system comprised a plasmid carrying the lcnA andlciA genes under the control of the nisin-inducible nisApromoter (PnisA), and a second plasmid harbouring thelcnC and lcnD genes. In addition, transformation of anisin-producing strain with both plasmids led to a high-level production of both lactococcal bacteriocins, whichmay provide a means to exploit their complementaryproperties.

Materials and Methods

Microbiological techniques, strains and plasmids

Bacterial strains and plasmids used in this study are listedin Table 1. Lactococcal strains were routinely grown inM17 medium (Terzaghi & Sandine, 1975) supplemented*For correspondence; e-mail : jmrodrig@vet.ucm.es

Journal of Dairy Research (2004) 71 216–221. f Proprietors of Journal of Dairy Research 2004 216DOI: 10.1017/S0022029904000123 Printed in the United Kingdom

with 5 g glucose/l (GM17 medium) at 30 8C without agi-tation. Escherichia coli was grown in L broth (Lennox, 1955)at 37 8C on an orbitary shaker. Agar plates were made byaddition of 1.5 g agar/l to broth media. Antibiotics (Sigma-Aldrich, Dorset, UK) were added as selective agents whenappropriate: chloramphenicol (Cm), 5 mg/ml for lactococciand 15 mg/ml for Esch. coli, ampicillin (Ap), 200 mg/ml,and erythromycin (Ery), 5 mg/ml. When required for in-duction purposes, nisin A (Aplin & Barret Ltd., Beaminster,UK) was added to the media used for the lactococcalgrowth at a concentration of 100 ng/ml. Antimicrobial ac-tivity in cultures was assayed by a plate diffusion bioassayperformed as previously described (Dodd et al. 1992) withLactococcus lactis FI9182 (nisin A sensitive, lactococcinA resistant) and Lc. lactis FI9921 (nisin A resistant, lacto-coccin A sensitive) as the indicator organisms.

Molecular techniques

Plasmid DNA isolation was carried out using the Qiagenplasmid mini kit (Qiagen, Crawley, UK). Restriction en-zymes and other DNA-modifying enzymes from varioussources were used according to the suppliers recommen-dations. Recombinant plasmids were recovered by trans-formation of Esch. coli as described previously by Doddet al. (1992) or by electroporation of Lc. lactis according tothe method of Holo & Nes (1989) with the modificationsof Dodd et al. (1992). Primers were purchased from Sigma-Genosys (Pampisford, UK). Fragments generated for theconstruction of vectors were amplified using BIO-X-ACT

(Bioline, London, UK) and cloned into pCR2.1 (InvitrogenLtd, Paisley PA4 9RF, UK). For routine PCR screeningof recombinant clones Taq DNA polymerase (AmershamBiosciences UK Ltd, Little Chalfont, UK) was used. Confir-mation of nucleotide sequences was carried out on puri-fied plasmid DNA using a DNA sequencer model 373A(Applied Biosystems, Warrington, UK) and the manu-facturer’s ABI Prism BigDye Terminator Cycle Sequencingkit.

Construction of pFI2396, Lc. lactis FI9943 andLc. lactis FI995

The technique of spliced overlap extension was used in theconstruction of a fragment containing the lactococcin Astructural (lcnA) and immunity (lciA) genes under thecontrol of the nisin promoter PnisA. This initially involvedthe amplification of two DNA fragments. Primers AD1(5k-CCTGAATAATATAGAGATAGGTT-3k) and AD3 (5k-AAATTTAATTGATTTTTCATTTTGAGTGCCTCC-3k) wereemployed to amplify a 271 bp fragment (fragment 1) con-taining PnisA, using plasmid pFI1003 (Karakas Sen et al.1999) as the template. The 17 nucleotides forming a tailat the 5k end of primer AD3 (underlined) are complemen-tary to the region of the lcnA gene encoding the amino-terminal sequence of the lactococcin A leader. Primers AD2(5k-GGAGGCACTCAAAATGAAAAATCAATTAAATTT-3k)and LCN2 (Horn et al. 1998) (5k-ACCCCGGGATTGATG-CCAGCTC-3k) were used to amplify a 702 bp fragment(fragment 2), containing lcnA and lciA genes, using

Table 1. Lactococcal strains and plasmids used in this study

Strainno. Host Plasmid

Bacteriocin determinantsReferenceor sourceL-lcnA-lciA* N-lcnA-lciA† lcnCD‡ nis· Dnis¶

FI5876 + Dodd et al. (1990)FI9951 FI5876 +

pFI2396 + This studyFI9953 FI5876 +

pFI2396 + This studypFI2148 +

FI7847 + Dodd et al. (1996)FI9941 FI7847 +

pFI2396 + This studyFI9943 FI7847 +

pFI2396 + This studypFI2148 +

FI9165 MG1614pFI2149 + + Horn et al. (1999)

FI9166 IL1403pFI2149 + + Horn et al. (1999)

WM4 + + Stoddard et al. (1992)

*L-lcnA-lciA contains the lactococcin A structural (lcnA) and immunity (lciA) genes preceded by their own promoter

†N-lcnA-lciA contains the lactococcin A structural (lcnA) and immunity (lciA) genes preceded by the nisin promoter

‡lcnCD contains the genes encoding the secretory and processing machinery of lactococcin A preceded by their own promoter

· nis contains the nisin gene cluster

¶Dnis, contains the nisin gene cluster with a frameshift mutation in codon 16 of the nisin structural gene impeding nisin biosynthesis ; the sequences of the

downstream nisin cluster genes are unaffected

Nisin and lactococcin A coproduction by Lc. lactis 217

colonies of Lc. lactis WM4 as template. Primer AD2 wasdesigned with a 5k tail containing 16 nucleotides (under-lined) that are complementary to the 3k end of fragment 1.Fragments 1 and 2 were diluted (1/200) in distilled water,and equal quantities were mixed. The mixture (2 ml) wasused as the template to amplify a 938 bp fragment withprimers AD1 and LCN2. The fragment was cloned intopCR2.1 resulting in the plasmid pFI2400, which wasintroduced into Esch. coli MC1022 (Casadaban & Cohen,1980) to give Esch. coli FI9959. Nucleotide sequenceanalysis of the inserted fragment confirmed that it wascomposed of sequences corresponding to the lcnA andlciA genes under the control of PnisA, with the exception ofa single base substitution that changed the codon encod-ing residue 42 of lciA from TCT (serine) to CCT (proline).The cloned genes and upstream promoter region wereisolated from pFI2400 as an EcoRI fragment and subclonedinto pTG262 (Shearman et al. 1989) to generate pFI2396(direct orientation). Transformation of Lc. lactis FI7847 andLc. lactis FI5876 with this recombinant plasmid generatedstrains FI9941 and FI9951, respectively. Subsequently,transformation of Lc. lactis FI9941 and FI9951 with

pFI2148 (containing lcnCD genes) (Horn et al. 1999) gen-erated strains FI9943 and FI9953, respectively.

Results

Production of lactococcin A

The production of lactococcin A in Lc. lactis FI7847 andFI5876 relied on the introduction of two compatibleplasmid vectors, one containing the PnisA-controlled lcnAand lciA genes and the other carrying lcnC and lcnDgenes. First, the PnisA-lcnA-lciA construction was generatedby PCR using the spliced overlap extension technique andcloned into pTG262, to generate pFI2396. The secondplasmid, pFI2148, is a pIL277-derivative and contains thegenes (lcnCD) encoding the secretory machinery of lacto-coccin A. In the case of the FI7847-derivatives, platediffusion bioassays, using the lactococcin A-sensitive in-dicator organism Lc. lactis FI9921, showed that lacto-coccin A was only produced by FI9943, the derivativecarrying both plasmids, provided the PnisA-controlledsystem was induced with nisin (Fig. 1). No activity was

1 2 3

4 5

6 7 8

9 10

Fig. 1. Plate diffusion bioassays for detection of lactococcin A activity against Lc. lactis FI9921. Wells contained supernatantsextracted from Lc. lactis cultures: 1, FI9943 (non-induced) ; 2, WM4; 3, FI5876; 4, FI9166; 5, FI9165; 6, FI9943 (induced) ; 7,FI9953; 8, FI9951; 9, FI9941 (induced); and 10, FI9941 (non-induced).

218 A Fernandez and others

detected in the supernatants of FI9941, a strain that lackspFI2148. The zones of inhibition caused by FI9943 werehigher than those displayed by Lc. lactis WM4 and FI9165,and similar to those displayed by Lc. lactis 9166. In thecourse of this study it was observed that the lactococcinA activity in Lc. lactis WM4 culture supernatants variedconsiderably between bioassays (data not shown) and itcould be rather low (Fig. 1). A significant finding from thiswork was the more consistent production of lactococcinA by the two-plasmid system in both of the productionhosts employed. The elevated levels of production were veryreproducible without the yield fluctuation that character-ized the parent strain.

Dual production of lactococcin A and nisin A

With respect to FI5876-derivatives, bioassays using thelactococcin A-sensitive indicator organism Lc. lactisFI5876, also revealed that both plasmids were requiredfor lactococcin A biosynthesis since only FI9953 was ableto produce extracellular lactococcin A (Fig. 1). The singlebase substitution involving residue 42 of LciA (the proteinconferring immunity to lactococcin A) did not affect this

function, since the lactococcin A-producing FI5876 deriva-tive was immune to this bacteriocin. The halos of inhi-bition observed around the supernatants of FI9953 weresimilar to those produced by FI9943 (Fig. 1). Lc. lactisFI5876 is a nisin A-producing strain and, therefore, itwas expected that its derivatives retained this property. AsFig. 2 shows, Lc. lactis FI9953 simultaneously producedlactococcin A and nisin A, being the nisin production levelhigher than that of the parental strain and FI9951.

Discussion

In this work, transformation of Lc. lactis FI5876 (Dodd et al.1990) (a nisin A-producer strain) and FI7847 (Dodd et al.1996) (a non-nisin producer FI5876-derivative with a de-letion in the nisA gene) with two vectors, one containingthe PnisA-lcnA-lciA construction and the second encodinglcnCD genes, resulted in enhanced and consistent pro-duction of lactococcin A compared with that of the par-ental strain, Lc. lactis WM4. Additionally, Lc. lactis FI9953was able to coproduce lactococcin A and nisin A. The factthat the activity of Lc. lactis FI9953 on the indicator

1 2 3

4 5

6 7 8

9 10

Fig. 2. Plate diffusion bioassays for detection of nisin A activity against Lc. lactis FI9182. Wells contained supernatants extracted fromLc. lactis cultures: 1, non-inoculated GM17 broth; 2, FI7847 (non-induced); 3, FI7847 (induced); 4, FI9953; 5, FI5876; 6, FI9943(induced); 7, FI9951; 8, FI9941 (induced) ; 9, FI9943 (non-induced); and 10, FI9941 (non-induced).

Nisin and lactococcin A coproduction by Lc. lactis 219

FI9182 (nisin-sensitive and lactococcin A-resistant) washigher than that of the parental strain and FI9951 (Fig. 2)may indicate that nisin activity undermines immunity tolactococcin A.

Production of lactococcin A in naturally non-producinglactococcal and non-lactococcal hosts has been previouslyattempted. Chikindas et al. (1995) described expressionand secretion of the lactococcin A in Pediococcus acid-ilactici PAC1.0, a pediocin PA-1-producing strain. In theirwork, introduction of pKV4, a plasmid containing the fourgenes required for lactococcin A biosynthesis, togetherwith their own gene regulatory elements, into such hostresulted in coproduction of both active pediocin PA-1 andlactococcin A. However, production of lactococcin A wasnot compared with that of a naturally-producing strain(Chikindas et al. 1995). Secretion and processing of mostLAB bacteriocins requires a dedicated transport andprocessing system integrated by an ABC (ATP-bindingcassette) export protein and an accessory protein (Franke,1998). Exploitation of the significant amino acid homo-logies among leader peptides and dedicated transporters ofa large number of bacteriocins, including lactococcin A(Havarstein et al. 1994, 1995), constitutes an attractivepossibility for heterologous production of these anti-microbial peptides. In this context, van Belkum & Stiles(1995) showed that the secretory apparatus of leucocin Awas able to direct, to a limited extent, secretion of lacto-coccin A in Leuconostoc gelidum UAL187-22 cells trans-formed with pMB553 (encoding lcnA and lciA). Lc. lactisIL1403 has been often selected as a host for lactococcin Aproduction since in this strain the translocation function(lcnC and lcnD) necessary for processing and secretion oflactococcins are provided by chromosomal gene analogs(Venema et al. 1996) and, therefore, only the lcnA andlciA genes are required. Horn et al. (1998) cloned thesegenes of Lc. lactis WM4 into the shuttle vector pTG262, togenerate pFI2058, which was introduced into Lc. lactisIL1403. Although Lc. lactis IL1403 harbouring pFI2058(strain FI8817) produced lactococcin A, this host has limi-tations such as the low copy number of the chromosomallcnC and lcnD gene analogs and the fact that these genesare not identical to the equivalent lactococcin A trans-locatory machinery (Venema et al. 1996), resulting in a lessefficient secretion or processing of the bacteriocin. A re-duced yield of lactococcin A expressed in IL1403 deriva-tives has been previously reported (van Belkum et al.1991; Holo et al. 1991).

Horn et al. (1999) improved lactococcin A productionby two approaches. First, they cloned the genes lcnC andlcnD into the vector pIL277, generating pFI2148. Thisplasmid is compatible with the pTG262-based plasmidpFI2058, carrying the lcnA and lciA genes, allowing thetwo plasmids to be stably maintained in the hosts Lc. lactisMG1614 and IL1403. In the second approach, the geneslcnC and lcnD were cloned into pFI2058 to generatepFI2149, which harbours all four genes of the lactococcinA cluster arranged in two operons. It is noteworthy that the

yields were influenced by the particular lactococcal strainemployed as a production host, since IL1403 consistentlyachieved higher activity levels. This observed yield differ-ence may reflect metabolic differences between Lc. lactissubsp. lactis (IL1403) and Lc. lactis subsp. cremoris(MG1614). In this work, the zones of inhibition caused byFI9943 and FI9953 (both MG1614-derived strains) weresimilar to those displayed by Lc. lactis FI9166, a IL1403derivative. Therefore, a significant increase in lactococcinA production was observed by placing lcnA and lciA genesunder the control of the nisin promoter. The auto-regulatoryprocess involved in nisin biosynthesis (Kuipers et al. 1995),based on signal transduction by the two-component regu-latory system encoded in the nisRK genes of the nisin genecluster (Engelke et al. 1994), has been successfully appliedfor development of the so-called nisin-controlled ex-pression (NICE) systems (de Ruyter, 1998). Currently, suchsystems are considered among the best inducible systemsfor LAB because of their many advantages (Kuipers et al.1997).

The particular properties of nisin A and lactococcinA, and the beneficial effects of their coproduction, arefeatures that can be exploited to extend their potentialapplication in the food industry. Nisin is a broad spectrumbacteriocin which inhibits the growth of Gram-positivespoilage and pathogenic bacteria, such as clostridia, staphy-lococci and listeria, and even that of Gram-negativebacteria when combined with other bacterial hurdles.At present, nisin is the only bacteriocin licensed for useas food additive (E234) in 45 countries. In contrast, one ofthe unique features of lactococcin A is its very narrowantimicrobial spectrum, since this bacteriocin only killsother lactococci (Holo et al. 1991). Unlike the broad-spectrum bacteriocins, which can be applied in the foodindustry as biopreservatives, applications for narrow-spectrum bacteriocins are not immediately obvious (Morganet al. 1995). However, lactococcin A has a lytic effect onsensitive cells (Holo et al. 1991; Morgan et al. 1995),probably caused by the resulting cell damage, which pro-motes uncontrolled degradation of the cell wall by theautolysin AcmA (Martınez-Cuesta et al. 2000). Therefore,this bacteriocin could have a technological role in theripening of cheeses with a maturation time long enoughto allow gradual lysis of starter bacteria (Morgan et al. 1995).The lysis of starter culture cells, and the subsequent releaseof intracellular enzymes into the cheese matrix, is a slowprocess that plays an important role in the development offlavour and other organoleptic properties (Collins et al.2003). Therefore, the addition of lactococcal strain with alytic effect on the starter culture would accelerate cheeseripening reactions. Although using pure bacteriocins asfood additives may be controversial, the potential benefitsof these peptide compounds can be exploited by employ-ing ‘ food-grade’ organisms as production strains (Holzapfelet al. 1995). Work is in progress in order to constructfood-grade lactococci with the properties acquired in thisstudy.

220 A Fernandez and others

This work was partially supported by grant AGL2002-04609-C02-02 from the Comision Interministerial de Ciencia yTecnologıa (CICYT), Madrid, Spain. A. F. holds a fellowship fromthe Universidad Complutense de Madrid, Spain.

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