Cloning Vectors Based on Cryptic Plasmids Isolated from Lactic Acid Bacteria:Their Characteristics...

Critical Reviews in Biotechnology, 24(4):155–208 (2004)

Cloning Vectors Based on CrypticPlasmids Isolated from Lactic Acid Bacteria:Their Characteristics and Potential Applicationsin Biotechnology

Julie Shareck,1,2 Young Choi,2 Byong Lee,1,3 and Carlos B. Miguez2

1Department of Food Science and Agricultural Chemistry, McGill University, Ste-Anne-de-Bellevue,Quebec, Canada; 2Microbial & Enzymatic Technology Group, Bioprocess Platform, Biotech-nology Research Institute, National Research Council of Canada, Montreal, Quebec, Canada;3The Food Research and Development Center, Agri-Food and Agriculture Canada, Ste-Hyacinthe,Canada

Address correspondence to Carlos B. Miguez, Microbial & Enzymatic Technology Group, BioprocessPlatform, Biotechnology Research Institute, National Research Council of Canada, 6100 Royalmount Ave.,Montreal, Quebec HYP 2R2, Canada. E-mail: [email protected]

ABSTRACT: Lactic acid bacteria (LAB) are Gram positive bacteria, widely distributed in nature, andindustrially important as they are used in a variety of industrial food fermentations. The use of geneticengineering techniques is an effective means of enhancing the industrial applicability of LAB. However,when using genetic engineering technology, safety becomes an essential factor for the application ofimproved LAB to the food industry. Cloning and expression systems should be derived preferablyfrom LAB cryptic plasmids that generally encode genes for which functions can be proposed, but nophenotypes can be observed. However, some plasmid-encoded functions have been discovered in crypticplasmids originating from Lactobacillus, Streptococcus thermophilus, and Pediococcus spp. and can beused as selective marker systems in vector construction. This article presents information concerningLAB cryptic plasmids, and their structures, functions, and applications. A total of 134 cryptic plasmidscollated are discussed.

KEY WORDS: lactic acid bacteria, genetic engineering, cryptic plasmid, cloning vector, food-gradevector construction, heterologous gene expression.

1. INTRODUCTION

Lactic acid bacteria (LAB) constitutea group of Gram-positive bacteria, includ-ing Lactococcus, Lactobacillus, Leuconostoc,Pediococcus, Streptococcus, and Bifidobac-terium species that share the ability to fer-ment sugars primarily into lactic acid. Severalspecies of LAB have a long history of use inthe traditional production of fermented foods,

beverages, and animal feed and are there-fore generally recognized as safe (GRAS)(Davidson et al., 1996; Stiles and Holzapfel,1997; Axelsson, 1998). LAB are alsoused as production organisms for variousfood ingredients such as preservatives, fla-vor compounds, gums and thickeners, andsome cheese flavoring enzymes. Metabolitesare secreted directly into the fermentationmedium, which is quite advantageous from a

0738-8551/03/$.50Copyright C© Taylor & Francis Inc.DOI: 10.1080/07388550490904288

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technological standpoint. To maximize pro-duction, commercial strains can be geneti-cally modified to improve inherent properties,introduce desirable characteristics and novelphenotypes, or remove unwanted traits.

While genetic engineering of LAB couldhave a great positive impact on the food andpharmaceutical industries, its progress couldbe impeded by legal issues related to thecontroversy surrounding this technology. Thesafe use of genetically modified LAB requiresthe development of food-grade cloning sys-tems composed solely of DNA from the ho-mologous host or GRAS organisms and thatdo not rely on antibiotic markers.

The rationale for the development ofcloning vectors derived from LAB crypticplasmids is the need for new food-gradegenetic engineering tools. Cryptic plasmidsare extrachromosomal DNA elements thatencode no recognizable phenotype besidestheir replication functions (von Wright andSibakov, 1998). A strategy to construct vec-tors is to use the replicons of small crypticplasmids and incorporate selectable markers.

Many cryptic plasmids originating fromLAB species have been isolated and character-ized (Wang and Lee, 1997). Vectors based onthese plasmids have been developed and usedin the cloning and expression of several het-erologous genes (Mercenier, 1990; Pouwelsand Leer, 1993; de Vos and Simons, 1994;Klaenhammer, 1995; Kullen and Klaenham-mer, 1999). In this article, cryptic plasmidsisolated from various LAB including Bifi-dobacterium will be compiled and vectors de-rived from these plasmids will be comparedon the basis of size, mode of replication, se-lection markers, segregational and structuralstability, host-range, and overall capacity oftransformation of other bacteria.

2. LACTIC ACID BACTERIA

Originally, the term lactic acid bacterium,dating back to the late nineteenth century, was

a synonym of “milk-souring organisms.” Thefirst pure culture of LAB obtained in 1873 byJ. Lister was Bacterium lactis (i.e. Lactococ-cus lactis) (Axelsson, 1998). LAB are Gram-positive, catalase negative, nonsporing, fas-tidious, and acid tolerant cocci or rods. Theyferment carbohydrates and yield mainly lac-tic acid as an end product. While this generalrule applies to homofermentative LAB, het-erofermentative LAB produce lactic acid andother compounds such as acetic acid, CO2,and ethanol. Some LAB are also responsi-ble for producing flavor compounds charac-teristic of fermented products, e.g. diacetyl incultured butter. Moreover, organic acids andadditional metabolites produced during fer-mentation play important roles in the preser-vation of foods; lower pH and bacteriocinsinhibit growth of spoilage and pathogenic or-ganisms, extending the shelf-life of fermentedfoods.

LAB have a long history of use in the fer-mentation of traditional foods, namely dairyproducts (yogurt, butter, cheese, kefir, ku-miss), meat (salami, sausages), vegetables(sauerkraut, pickles, olives), wine, and silage(Geis, 2003). There are references to the leav-ening of (sourdough) bread in the Bible (e.g.Matthew, 13, 33). Fermented dairy products(cheese, yogurt, butter) are mentioned in an-cient texts from Uruk/Warka (Iraq), datedaround 3,200 B.C. (Nissen et al., 1991).

LAB strains used in food fermentationsare generally associated with habitats richin nutrients such as various food products.Other LAB are members of the normal floraof the mouth, intestine, and vagina of mam-mals, namely bifidobacteria. The genus Bifi-dobacterium shares some of the phenotypictraits of genuine LAB, but bifidobacteria arephylogenetically unrelated, belonging to theActinomyces branch of bacteria (Schleifer andLudwig, 1995) and have a unique modeof sugar fermentation. Traditional LAB arefurther differentiated from Bifidobacteriumspecies based on mol% G+C content in theDNA. All LAB have less than 55 mol% G+C

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content in their DNA (low G+C content),while bifidobacteria have more than 55 mol%G+C (high G+C content).

Bifidobacteria are gaining much deservedattention as growing scientific evidence showsthat some strains of Lactobacillus and Bi-fidobacterium have probiotic properties, i.e.have the capacity to modulate and maintaina healthy intestinal microflora by compet-ing with potentially harmful bacteria and bystimulating the growth of preferred microor-ganisms (Salminen et al., 1998). As the mar-ket for probiotic products expands, Bifidobac-terium species are being used for the produc-tion of fermented milks, yogurts, and cheeses(Sanders and Huis in’t Veld, 1999). Interest inimproving characteristics of Bifidobacteriumstrains by genetic manipulation is driving thedevelopment of cloning vectors, which is whybifidobacteria are to be included in this articlewith the more traditional LAB.

3. CRYPTIC PLASMIDS IN LACTICACID BACTERIA

Present in prokaryotes and in some lowereukaryotes, plasmids are extrachromosomalDNA elements, usually double-strandedmolecules that are autonomous and self-replicating. Plasmids are extremely diversein terms of size (1.5 kb to more than 600kb), copy number (1 to several hundredsper cell), and phenotypes conferred to theirhosts (Osborn et al., 2000). Plasmids areinherited independently from the bacterialchromosome, but some do rely on proteinsencoded by the host for their replicationand transcription. While plasmids are notnormally essential for the growth of bacteria,specific phenotypes such as (1) hydrolysis ofproteins, (2) metabolism of carbohydrates,amino acids and citrate, (3) production ofbacteriocins, exopolysaccharides, pigments,and (4) resistance to antibiotics, heavy metals,and phages have been found to be plasmid-encoded (Wang and Lee, 1997). Nevertheless,

many of the plasmids that occur in LAB arecryptic, a term used to describe plasmids thatdo not have any apparent function.

Usually small and abundant, cryptic plas-mids have no known effect on the host’s phe-notype, range in size from 1 to >100 kb, andhave been reported in many groups of LAB(von Wright and Sibakov, 1998). The mini-mum requirement for a plasmid is its abilityto replicate, and hence, the replicon is consid-ered the most important feature of a crypticplasmid. The replicon is the region on a plas-mid that encodes rep genes, essential for plas-mid replication. Rarely exceeding 3 kb, thereplicon consists of (1) an origin of replication(ori); (2) cop/inc gene(s) involved in the con-trol of the initiation of replication, and (3) repgene(s) encoding Rep proteins required forreplication (Espinosa et al., 2000). A repliconmainly allows plasmid replication, its mainte-nance in a host cell, and transfer between cells(Becker and Meyer, 2003; Grohmann et al.,2003). The degree of plasmid autonomy, re-flected in plasmid promiscuity, depends on thefunctionality of a replicon in different hosts.Narrow host-range plasmids only replicate ina few closely related hosts, while broad host-range plasmids have a replicon that is func-tional in a wide range of hosts.

3.1. Plasmid Replication Mechanisms

The mode of replication of plasmids hasan important impact on some characteris-tics of plasmid-derived vectors, namely host-range, stability, and copy number. In LAB, themost common replication mechanisms are thesigma and theta modes of replication.

3.1.1. Sigma-Replicating Plasmids(Rolling-Circle Mechanism)

The sigma mode of replication, alsoknown as rolling-circle replication (RCR),seems to be restricted to relatively small

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cryptic plasmids (Khan, 1997). RCR plas-mids constitute a group of small, multicopyreplicons that are widely spread among bac-teria. Genetic elements that are involved inRC replication are the rep gene that encodesthe replication initiation protein (Rep) con-trolled by a repressor and its target site, theplus origin of replication or double-strandedorigin (dso). Additionally, most RCR plas-mids have a minus origin of replication orsingle strand origin (sso), a specific sequencethat enables the conversion of ssDNA inter-mediates into double-stranded DNA (dsDNA)molecules (Gruss and Ehrlich, 1989).

The Rep protein is a site-specific nucle-ase, which produces a single-stranded nick atthe plus origin (dso), initiating positive strandreplication and terminating it when a lead-ing strand (ssDNA) is synthesized (Gruss andEhrlich, 1989). The leading strand replicationgenerates: (1) a dsDNA molecule constitutedby the parental [−] strand and the newly syn-thesized [+] strand and (2) a ssDNA inter-mediate that corresponds to the parental [+]strand. Generation of ssDNA is the trademarkcharacteristic of RC replication. Finally, lag-ging strand synthesis occurs and ssDNA inter-mediates are converted to dsDNA at the minusorigin (sso). The last step involves supercoil-ing of the replicated DNAs by the host DNAgyrase (Espinosa et al., 2000).

Cloning vectors based on RC repliconsusually have low segregational stability due toaccumulation of ssDNA intermediates (Posnoet al., 1991a; Vujcic and Topisirovic, 1993)and insertion of foreign DNA may further re-duce their stability (Gruss and Ehrlich, 1989).Accumulation of ssDNA is generally due tothe absence of a sso, consequently hinderingthe conversion of ssDNA to dsDNA molecules(del Solar et al., 1987).

Based on similarities in the structure ofRep proteins and dso, RC replicons fromGram-positive bacteria have been groupedinto five families: pMV158/pE194, pC194,pT181, pSN2, and pTX14-3 (Khan, 1997).Lactococcal RC replicons belong to the

pMV158/pE194 family, except for pWC1,which was included in the pC194 family(Pillidge et al., 1996). Lactobacillus RCRplasmids are divided between the pE194and pC194 families while S. thermophilusreplicons mostly belong to the pC194 group.Leuconostoc RCR plasmids seem to beassociated with several different groups:pCI411 belongs to the pE194 family (Coffeyet al., 1994); pLo13 is included in the pC194group (Fremaux et al., 1993a) while pRS1,pOg32, and pFR18 are grouped with thepT181 family (Alegre et al., 1999; Britoet al., 1996; Biet et al., 1999). RC repliconshave yet to be detected in Pediococcus. Asfor bifidobacterial plasmids, most follow theRC mechanism of replication, except pMB1(Matteuzzi et al., 1990).

3.1.2. Theta-Replicating Plasmids

Although it was assumed that rolling-circle plasmids were the most widespreadin Gram-positive bacteria, a large numberof theta-replicating plasmids recently havebeen isolated and characterized, many origi-nating from LAB. Theta-replicating plasmidstend to be medium- and large-size plasmidsthat encode important metabolic functions,enzymes such as lactase (pSK11L, Hornget al., 1991), lactase-protease (pUCL22, Frereet al., 1993), and citrate permease (pSL2,Jahns et al., 1991), bacteriophage resistance(pCI528, Lucey et al., 1993), exopolysaccha-ride production (pNZ4000, van Kranenburgand de Vos, 1998), and pediocin production(pSMB74, Motlagh et al., 1994; pMD136,Kantor et al., 1997).

Some cryptic plasmids have also beenshown to follow theta replication: pVS40(von Wright and Raty, 1993), pCI305 (Hayeset al., 1991), and pWV02 (Kiewiet et al.,1993). Moreover, theta replication is no longerexclusively reserved to large plasmids, assome very small plasmids replicate via thetheta mode, namely p4028 (4.4 kb, Zuniga

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et al., 1996), pTXL1 (2.7 kb, Biet et al., 2002),and pMB1 (1.9 kb, Matteuzzi et al., 1990;Corneau et al., 2004).

The main difference between sigma andtheta replication is that theta-replicating plas-mids do not produce ssDNA intermediates.This results in greater structural and segrega-tional stability, making theta-replicating plas-mids better candidates for vector constructionas they can stably maintain large heterologousDNA inserts (up to 16.8 kb) (Kiewiet et al.,1993; de Vos and Simons, 1994).

Theta replication has three key compo-nents: (1) an initiator protein (Rep) necessaryfor strand opening, (2) an origin of replication(ori) with specific DNA structural organiza-tion for strand opening and initiator-proteinbinding and, (3) a host-encoded polymeraseI for nascent strand DNA synthesis (delSolar et al., 1998; Alpert et al., 2003). Inthe theta mode, sites for priming of leading-and lagging-strand synthesis are locatedclose to one another within the replicationorigin. During replication, both DNA strandsremain covalently closed, except during theresolution of daughter molecules (de Vos andSimons, 1994).

3.2. Lactococcus Cryptic Plasmids

Members of the Lactococcus (L .) genusare mesophilic LAB used extensively in milkfermentation to produce a number of differ-ent products. The genus includes L. garviae,L. plantarum, L. piscium, and L. raffinolactis,but L. lactis remains the best characterizedLactococcus species with regards to physiol-ogy and molecular genetics. Plasmids in L.lactis are a common component: they vary innumber from 2 to 11 per cell, but most strainsusually possess 4 to 7 plasmids, ranging insize from 3 kb to more than 130 kb (David-son et al., 1996). L. lactis subsp. cremorisstrain Wg2 harbors 5 plasmids: pWV01 (2.2kb), pWV02 (3.8 kb), pWV03 (7 kb), pWV04(19 kb), and pWV05 (27kb) (Kok et al., 1984;

Kiewiet et al., 1993; Seegers et al., 1994)that follow a theta mode of replication, exceptfor RCR plasmid pWV01. This supports thetheory that many theta-replicating plasmidscan be maintained in the same cell, but onlyone RCR plasmid can stably co-exist (Seegerset al., 1994).

Lactococcal plasmids are of great impor-tance as some industrially relevant charac-teristics are plasmid-encoded, such as sugarmetabolism (Gasson, 1990), proteolysis (Kok,1990), phage resistance, conjugal transfer(McKay and Baldwin, 1990; de Vos andDavies, 1984), and bacteriocin production(Klaenhammer, 1993). L. lactis also harborscryptic plasmids that do not encode anyknown metabolic functions. These plasmidshave replicons that are homologous to RCRreplicon pWV01 or to theta-type repliconspWV02/pCI305.

Most RCR lactococcal cryptic plasmids,namely pWV01 (Kok et al., 1984; Leenhoutset al., 1991), pSH71 (de Vos et al., 1987),pD125 (Xu et al., 1990), pCL2.1 (Changet al., 1995), and pBM02 (Sanchez and Mayo,2003) belong to the pE194 family of RCRplasmids, the exception being pWC1 (Pillidgeet al., 1996), which is a pC194-type of repli-con (Table 1). Recently, pSRQ700, a theta-replicating plasmid encoding an anti-phageresistance mechanism was found to containa second replication module, a RC replicon(Boucher et al., 2001).

The advantage of RC lactococcal repli-cons is that most are functional in Gram-positive bacteria and in E. coli. Wide host-range vectors have been derived from thesereplicons (i.e. pGK-, pNZ-, pFX-series) andproven to be very useful to transform LABand other hosts (de Vos and Simons, 1994).However, these vectors tend to suffer fromsegregational instability due to accumulationof ssDNA intermediates.

To solve vector stability problems, the fo-cus has been on developing vectors basedon theta-replicating plasmids. Although suchvectors have a narrow host-range, they have

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TABLE 1Characteristics of Lactococcus lactis RCR Cryptic Plasmids

Plasmid Origin Size (kb)Mode of

replicationFamily ofreplicon References

pWV01 L. lactis subsp.cremoris

2.2 RCR pE194 Kok et al., 1984; Leenhoutset al., 1991

pSH71 L. lactis 2.1 RCR pE194 de Vos et al., 1987pD125 L. lactis subsp.

lactis5.1 RCR pE194 Xu et al., 1990

pCL2.1 L. lactis subsp.lactis

2.1 RCR pE194 Chang et al., 1995

pWC1 L. lactis 2.8 RCR pC194 Pillidge et al., 1996pBM02 L. lactis subsp.

cremoris3.9 RCR pE194 Sanchez et al., 2003

the advantages of (1) being compatible withendogenous RCR plasmids (Hayes et al.,1991), (2) having greater segregational stabil-ity (Kiewiet et al., 1993), and (3) having thecapacity to carry large fragments of heterol-ogous DNA. Theta-replicating plasmids arewidespread, the lactococcal prototype beingpCI305 (Hayes et al., 1991). It was suggestedthat pCI305 followed a theta mode of replica-tion as no homology was found between repA,repB, and replication regions of well-knownRCR plasmids. In addition, ssDNA interme-diates could not be detected.

Theta-replicating cryptic lactococcalplasmids are members of a family of highlyrelated, compatible replicons, as first iden-tified for plasmid pCI305. The group iscomprised of several plasmids, includingpWV02 (Kiewiet et al., 1993), a 28-kb plas-mid used to construct nisin-resistant pVS40(von Wright et al., 1990; von Wright andRaty, 1993), pWV04, pWV05, pIL7 (Seegerset al., 1994), pCIS3 (Seegers et al., 2000),pJW563 (Gravesen et al., 1995), pND324(Duan et al., 1999), and L. lactis strain FG2plasmid replicon (Liu et al., 1997a).

Several theta-replicating cryptic plasmidsfound in Lactococcus are interesting for thedevelopment of vectors, as they naturally en-code selectable markers: pVS40 encodes nisinresistance, pND302, cadmium resistance (Liu

et al., 1996, 1997b), and pND324 has a ther-mosensitive replication region (Table 2).

3.3. Lactobacillus Cryptic Plasmids

The genus Lactobacillus (Lb.) is com-prised of many relatively diverse species,which include Lb. plantarum, Lb. pentosus,Lb. fermentum, Lb. reuteri, Lb. acidophilus,Lb. casei, Lb. helveticus, Lb. hilgardii, Lb.curvatus, Lb. delbrueckii bulgaricus, Lb. del-brueckii lactis, and Lb. sakei. Lactobacilli arewidespread in nature and many have beenused in food fermentation processes, includ-ing milk, meat, and plant material. In addition,a few species of Lactobacillus are used as pro-biotic microorganisms in functional foods.

First discovered by Chassy et al. (1976),plasmids are found in most, but not allLactobacillus species (reviewed by Pouwelsand Leer, 1993; Klaenhammer, 1995; Wangand Lee, 1997). Lb. delbrueckii bulgaricusis known to harbor very few plasmids: itwas recently found that out of 48 strains,only one contained a cryptic plasmid pLBB1(Azcarate-Peril and Raya, 2002). It is gener-ally thought that most Lactobacillus strainsharbor one or more plasmids, the size of whichcan vary from 1.2 kb to 150 kb.

To this date, many lactobacilli plas-mids have been found, but most remain

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TABLE 2Characteristics of Lactococcus lactis Theta-Replicating Plasmids

Plasmid Origin Size (kb) Mode of replication References

pCI305 L. lactis subsp.lactis

8.7 Theta Hayes et al., 1990, 1991

pSL2 L. lactis subsp.lactis biovardiacetylactisBu2

7.8 Theta Jahns et al., 1991

pVS40 L. lactis subsp.lactis biovardiacetylactis

7.8 Theta von Wright et al., 1990; vonWright and Raty, 1993

pSK11L L. lactis subsp.cremoris

47.3 Theta Horng et al., 1991


3.8 Theta Kiewiet et al., 1993

pCI528 L. lactis subsp.cremoris

46 Theta Lucey et al., 1993

pUCL22 L. lactis subsp.lactis

40 Theta Frere et al., 1993

pCT1138 L. lactis subsp.lactis biovardiacetylactis

5.5 MDa Theta Pedersen et al., 1994


19 Theta Seegers et al., 1994


27 Theta Seegers et al., 1994

pIL7 L. lactis 31 Theta Seegers et al., 1994pJW563 L. lactis subsp.

cremoris11.5 Theta Gravesen et al., 1995

pND302 L. lactis subsp.lactis

8.8 Theta Liu et al., 1996, 1997b

FG2 plasmid L. lactis subsp.lactis FG2

1.8 Theta Liu et al., 1997a

pNZ4000 L. lactis 40 Theta van Kranenburg and de Vos,1998

pND324 L. lactis subsp.lactis

3.6 Theta Duan et al., 1999

pCIS3 L. lactis subsp.cremoris

6.1 Theta Seegers et al., 2000

pCI2000 L. lactis subsp.lactis

60 Theta Kearney et al., 2000

MDa: Megadaltons.

cryptic (Wang and Lee, 1997). However, somefunctions have been found to be plasmid-encoded that relate to lactose metabolism,antibiotic resistance, bacteriocin productionand immunity, DNA restriction or modifi-

cation (R-M), exopolysaccharide production,N-acetyl glucosamine fermentation, and cer-tain amino acid (cysteine) transport (reviewedby Pouwels and Leer, 1993; Arihara andLuchansky, 1994).

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Cryptic plasmids in lactobacilli are nu-merous and quite diversified with regards totheir size, mode of replication, and family ofreplicons. The larger-sized plasmids tend tofollow a theta mode of replication, while thesmaller plasmids replicate via RC. Althoughthis is generally true, two theta-replicatingplasmids appear to be very small, 3.3 kbpLJ1 from Lb. helveticus (Takiguchi et al.,1989) and 4.4 kb pKC5b from Lb. fermentum(Pavlova et al., 2002), while 9.3 kb pLP9000from Lb. plantarum (Daming et al., 2003)and 9.8 kb pGT633 from Lb. reuteri (Tannocket al., 1994) are RCR plasmids, despite theirfairly large sizes (Table 3).

Based on Rep protein structure anddso homologies, completely or partially se-quenced RCR plasmids either belong to thepE194 or to the pC194 general classes ofRC replicons. pA1, pLB4, a 7 kb plasmid(Lb. plantarum), pLF1311 (Lb. fermentum),pLC2 (Lb. curvatus), and pLA106 (Lb. aci-dophilus) have so far been determined asmembers of the pE194 family of replicons.RCR plasmids of the pC194 type are pC30il,pLP1, p8014-2, pLP2000 (Lb. plantarum),p353-2 (Lb. pentosus), pLEM3 (Lb. fermen-tum), pLAB1000 (Lb. hilgardii), pGT232, andpTC82 (Lb. reuteri). Lb. plantarum plasmidpLP9000 (Daming et al., 2003) also replicatesvia RC, but has not been assigned to any repli-con family, as no homologies could be foundwith any known RC replicons. Some RC plas-mids are also in the same situation (Tannocket al., 1994; Fortina et al., 1993; Pridmoreet al., 1994).

Much interest is focused on lactobacillitheta-replicating plasmids, the intention be-ing to construct narrow host-range cloningvectors that have increased stability. Severaltheta-replicating plasmids have been identi-fied as such because they do not exhibit typ-ical RCR traits (Table 3): pKC5b (Lb. fer-mentum), pLJ1 and pLH1 (Lb. helveticus),pRV500, and pSAK1 (Lb. sakei). Other plas-mids such as pLA103 and pLA105 (Lb. aci-dophilus), pLKS (Lb. plantarum), and pLBB1

(Lb. delbrueckii bulgaricus) are believed tohave a theta mode of replication, but confir-mation is still pending.

3.4. Streptococcus thermophilusCryptic Plasmids

Streptococcus (S.) thermophilus is theonly streptococcal species used in food fer-mentations: its ability to grow at high temper-atures (52◦C) combined with its limited spec-tra of fermentable sugars distinguish it fromother streptococci. Phylogenetically, it is themost closely related species to L. lactis. S.thermophilus is mainly used as a starter cul-ture to manufacture yogurt and some cheesessuch as mozzarella and Swiss, its main func-tion being the production of lactic acid.

Like Lb. delbrueckii bulgaricus, S. ther-mophilus strains carry very few plasmids(Mercenier et al., 1990). While it has been re-ported that less than 20% of S. thermophilusstrains carry one or two small plasmids(Girard et al., 1987; Herman and McKay,1985; Janzen et al., 1992; Somkuti andSteinberg, 1986; Geis et al., 2003), one grouprecently found that 60% of the S. thermophilusstrains screened harbored plasmids (Turgeonet al., 2001).

Several small S. thermophilus plasmidshave been sequenced and analyzed, and mosthave remained cryptic, as no apparent pheno-typic traits seem to be associated to their pres-ence (Table 4). Several plasmids have beenfound to encode small heat shock proteins(hsp): pER341 (Somkuti et al., 1998), pCI65st(O’Sullivan et al., 1999), pND103 (Su et al.,2002), pST04 and pER1-1 (Geis et al., 2003),pt38 (Petrova et al., 2003) in addition to pER7,pER16, pER26, pER35, pER36, and pER41(Somkuti and Steinberg, 1999; Solow andSomkuti, 2000). It was initially thought thatthe presence of hsp influenced plasmid main-tenance during fermentation at elevated tem-peratures (O’Sullivan et al., 1999). Accord-ing to Geis et al. (2003), expression of these

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TABLE 3Characteristics of Lactobacillus spp. Cryptic Plasmids



pcaT Lb. plantarum 8.5 NA NA Jewel and Thompson-Collins, 1989pA1 Lb. plantarum 2.8 RCR pE194 Vujcic and Topisirovic, 1993pLP1 Lb. plantarum 2.1 RCR pC194 Bouia et al., 1989p8014-2 Lb. plantarum 1.9 RCR pC194 Leer et al., 1992pC30il Lb. plantarum 2.1 RCR pC194 Skaugen et al., 1989pLB4 Lb. plantarum 3.5 RCR pE194 Bates and Gilbert, 1989pLP2000 Lb. plantarum 2.1 RCR pC194 Daming et al., 2003pLP9000 Lb. plantarum 9.3 RCR NA Daming et al., 2003a 7 kb Lb. plantarum 7 RCR pE194 Cocconcelli et al., 1991, 1996

plasmidpLKL Lb. plantarum 6.8 NA NA Eguchi et al., 2000pLKS Lb. plantarum 2.0 Theta — Eguchi et al., 2000pMD5057 Lb. plantarum 11.0 NA NA Danielsen, 2002

Danielsen, 2002pLY2 Lb. fermentum 15.6 NA NA Iwata et al., 1986pLY4 Lb. fermentum 57.8 NA NA Iwata et al., 1986pLEM3 Lb. fermentum 5.7 RCR pC194 Fons et al., 1997pLF1311 Lb. fermentum 2.4 RCR pE194 Aleshin et al., 1999pKC5b Lb. fermentum 4.4 Theta — Pavlova et al., 2002p353-1 Lb. pentosus 1.7 NA NA Posno et al., 1991ap353-2 Lb. pentosus 2.3 RCR pC194 Leer et al., 1992pLUL631 Lb. reuteri 10.2 NA NA Axelsson et al., 1988pLAR33 Lb. reuteri 18 NA NA Rinckel and Savage, 1990pGT633 Lb. reuteri 9.8 NA NA Tannock et al., 1994pGT232 Lb. reuteri 5.1 RCR pC194 Heng et al., 1999pTE15 Lb. reuteri 15 NA NA Lin et al., 1999pTE80 Lb. reuteri 7.0 NA NA Lin et al., 1999pTC82 Lb. reuteri 7.0 RCR pC194 Lin et al., 1996a, 2001pLAB1000 Lb. hilgardii 3.3 RCR pC194 Josson et al., 1989, 1990pLAB2000 Lb. hilgardii 9.1 NA NA Josson et al., 1989pLC2 Lb. curvatus 2.6 RCR pE194 Vogel et al., 1991; Klein et al., 1993pWS97 Lb. delbrueckii 60 NA NA Zink et al., 1991pLB10 Lb. delbrueckii

bulgaricus2.7 NA NA Chagnaud et al., 1992

pLBB1 Lb. delbrueckiibulgaricus

6.1 Theta — Azcarate-Peril and Raya, 2002

pJBL2 Lb. delbrueckiilactis

8.7 Theta — Bourniquel et al., 2002

pN42 Lb. delbrueckiilactis

8.1 Theta — Bourniquel et al., 2002

p1 Lb. acidophilus 1.6 NA NA Damiani et al., 1987p3 Lb. acidophilus 4.2 NA NA Damiani et al., 1987pPM4 Lb. acidophilus NA NA NA Luchansky et al., 1988pLA103 Lb. acidophilus 14.0 Theta — Kanatani et al., 1992, 1995bpLA105 Lb. acidophilus 3.2 Theta — Kanatani et al., 1995apLA106 Lb. acidophilus 2.9 RCR pE194 Sano et al., 1997pLJ1 Lb. helveticus 3.3 Theta — Takiguchi et al., 1989pCP53 Lb. helveticus 11.5 NA NA Yamamoto and Takano, 1996

(Continued on next page)

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TABLE 3Characteristics of Lactobacillus spp. Cryptic Plasmids (Continued)


ReplicationFamily ofreplicon References

pLH1 Lb. helveticus 19.4 Theta — Fortina et al., 1993; Thompsonet al., 1999

pLH2 Lb. helveticus 5.7 RCR NA Fortina et al., 1993pLH3 Lb. helveticus 3.4 RCR NA Fortina et al., 1993pLH4 Lb. helveticus 2.6 RCR NA Pridmore et al., 1994pSAK1 Lb. sakei 19 Theta — Unpublished; GenBank accession

no. Z50862pRV500 Lb. sakei 13 Theta pUCL287 Alpert et al., 2003pLZ15 Lb. casei 28.3 NA NA Chassy and Flickinger, 1987p121BS Lactobacillus

spp.4.2 NA NA Whitehead et al., 2001

NA: not available; —: not applicable to theta replicons.

TABLE 4Characteristics of Streptococcus thermophilus Cryptic Plasmids



pA2 S. thermophilus 2 RCR NA Mercenier et al., 1990pA33 S. thermophilus 6.9 RCR NA Mercenier et al., 1990pST1 S. thermophilus 2.1 RCR pC194 Janzen et al., 1992pST1 no. 29 S. thermophilus 2.8 RCR pC194 Hashiba et al., 1993pER8 S. thermophilus 2.2 RCR pC194 Somkuti and Steinberg, 1986pER371 S. thermophilus 2.7 RCR pC194 Solaiman and Somkuti, 1998pER341 S. thermophilus 2.8 RCR pC194 Somkuti et al., 1998pER36 S. thermophilus 3.7 NA NA Somkuti and Steinberg, 1986pER13 S. thermophilus 4.2 NA NA Somkuti and Steinberg, 1986pER16 S. thermophilus 4.5 NA NA Somkuti and Steinberg, 1986pER342 S. thermophilus 9.5 NA NA Somkuti and Steinberg, 1986pER35 S. thermophilus 11.0 NA NA Somkuti and Steinberg, 1986pER372 S. thermophilus 14.8 NA NA Somkuti and Steinberg, 1986pER7 S. thermophilus 4.5 NA NA Somkuti and Steinberg, 1999pER26 S. thermophilus 4.5 NA NA Somkuti and Steinberg, 1999pER41 S. thermophilus 3.6 NA NA Somkuti and Steinberg, 1999pCI65st S. thermophilus 6.5 RCR pC194 O’Sullivan et al., 1999pSMQ172 S. thermophilus 4.2 RCR pMV158/pE194 Turgeon et al., 2001pND103 S. thermophilus 3.5 RCR pC194 Su et al., 2002pSt04 S. thermophilus 3.1 RCR pC194 Geis et al., 2003pER1-1 S. thermophilus 3.4 RCR pC194 Geis et al., 2003pJ34 S. thermophilus 3.4 RCR pC194 Geis et al., 2003pSt08 S. thermophilus 7.5 RCR pC194 Geis et al., 2003pER1-2 S. thermophilus 4.4 NA NA Geis et al., 2003pSt06 S. thermophilus 5.3 NA NA Geis et al., 2003pSt0 S. thermophilus 8.1 NA NA Geis et al., 2003pSt22-2 S. thermophilus 4.2 NA NA Geis et al., 2003pt38 S. thermophilus 2.9 RCR pC194 Petrova et al., 2003

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proteins is induced by elevated temperaturesand low pH, increasing thermo- and acid resis-tance of the strains that carry hsp. Moreover,the promoter of hsp16.4 of pER341 is underinvestigation for potential use in temperature-controlled expression of heterologous genesin LAB (Somkuti et al., 1998). Genes forrestriction-modification (R/M) systems havealso been identified on plasmids, as reportedfor pER35 (Solow and Somkuti, 2001), pSt08and pSt0 (Geis et al., 2003), and pCI65st(O’Sullivan et al., 1999).

S. thermophilus plasmids can be groupedaccording to their sizes (Janzen et al., 1992):group I consists of small plasmids rangingin size from 2.1 to 3.5 kb; group II, 4.2kb; group III, 5.2 kb; group IV, 6.8 kb; andgroup V, 7.4 kb. Based on mechanism ofreplication, S. thermophilus plasmids isolatedso far replicate via RC and belong to thepC194 family of replicons (Janzen et al.,1992; Hashiba et al., 1993; O’Sullivan et al.,1999; Solaiman and Somkuti, 1998; Somkutiand Steinberg, 1986; Somkuti et al., 1998;Su et al., 2002; Geis et al., 2003; Petrovaet al., 2003), except pSMQ172 (Turgeon andMoineau, 2001), which has been assigned tothe pMV158/pE194 family of replicons. Al-though the presence of ssDNA intermediateshas never been demonstrated in any of theplasmids besides in pSMQ172 and pt38, itis still assumed that they replicate via RC,as suggested by homologies to known RCRplasmids.

TABLE 5Characteristics of Leuconostoc and Oenococcus oeni Cryptic Plasmids

Plasmid Origin Size (kb) Mode of replication Family of replicon References

pLo13 Ln. oenos 3.9 RCR pC194 Fremaux et al.,1993a

pOg32 Ln. oenos 2.5 RCR pT181 Brito et al., 1996p4028 Ln. oenos 4.4 Theta — Zuniga et al., 1996pRS1 O. oeni 2.5 RCR pT181 Alegre et al., 1999pFR18 Ln. mesenteroides 1.8 RCR pT181 Biet et al., 1999pTXL1 Ln. mesenteroides 2.7 Theta — Biet et al., 2002pCI411 Ln. lactis 2.9 RCR pE194 Coffey et al., 1994

3.5. Leuconostoc and Oenococcusoeni Cryptic Plasmids

Strains of Leuconostoc (Ln.) can be foundin many natural and man-made habitats, suchas grass, herbage, and silage. The genus Leu-conostoc is comprised of heterofermentativeorganisms that play an important role in thefermentation of vegetables (cabbage, cucum-bers) by initiating spontaneous lactic acid fer-mentations (Davidson et al., 1996). The abil-ity of some species to ferment citric acid inmilk to the flavor compound diacetyl, the“butter flavor” in dairy products, has led totheir use as industrial dairy starters. Leuconos-toc species contribute to both flavor develop-ment and preservation of food (Johansen andKibenich, 1992). Although Ln. oenos, primar-ily used in malolactic fermentations, has beenreclassified as Oenococcus oeni (Dicks et al.,1995), it is more convenient to include it in thissection. O. oeni is an acid and alcohol toler-ant LAB that is found naturally in fruit mashesand related habitats. Used in winemaking, itconverts malate to lactate and reduces wineacidity, thereby improving the organolepticproperties and the stability of the final product(Davis et al., 1985).

Few studies report the presence of plas-mids in Leuconostoc spp. (Table 5). Janseet al. (1987) reported that after screening42 strains of Leuconostoc, only 11 plasmidscould be recovered from 8 strains of Ln. oenos.These plasmids were small, between 2.47 kb

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and 4.61 kb and had a low copy number,factors supporting the statement that in thisgenus, little genetic information is plasmid-encoded. However, some Leuconostoc plas-mids have been shown to encode metabolicfunctions such as lactose utilization and cit-rate permease activity (David et al., 1992) aswell as bacteriocin production and immunity(Hastings et al., 1991).

Nonetheless, most Leuconostoc plasmidscharacterized to date remain cryptic (Cof-fey et al., 1994), replicate via RCR and be-long to various replicon families, includingpT181, pE194, and pC194. Among LAB RCRplasmids, the only plasmids assigned to thepT181 class of RC replicons have been foundin Leuconostoc species (pOg32, pRS1, andpFR18).

Two plasmids, p4028 from Ln. oenos andpTXL1 from Ln. mesenteroides, reportedlyfollow theta replication, a surprising discov-ery considering the size of the plasmids, 4.4 kband 2.7 kb, respectively (Zuniga et al., 1996;Biet et al., 2002).

3.6. Pediococcus andTetragenococcus halophilaCryptic Plasmids

Pediococci are a group of homofermen-tative LAB that are ecologically, morpho-logically, and physiologically similar to thelactococci (Gonzalez and Kunka, 1983).Comparison of 16S rRNA sequences con-firmed the heterogeneous phylogeny of thePediococcus (P .) genus and P. halophiluswas reclassified as Tetragenococcus (T.)halophila (Benachour et al., 1997). Pedio-cocci are naturally found on plant material, infermented vegetables and in beer. They forma group of economically important microor-ganisms as they are used as starter culturesfor sausage-making, fermentation of vegeta-bles and soy milk, and for silage inocula-tion (Davidson et al., 1996). Moreover, T.halophila is salt tolerant and is used in the

brewing of soy sauce (Uchida, 1982) and inthe curing of salted anchovies (Villar et al.,1985).

Pediococci, mainly P. pentosaceus and P.acidilactici, harbor many different plasmidsthat encode a variety of traits, namely uti-lization of raffinose, melibiose, and sucrose(Gonzalez and Kunka, 1986) as well as bacte-riocin production (Klaenhammer, 1993). Pe-diocin, an anti-listerial bacteriocin, is pro-duced by several pediococcal strains (Marugget al., 1992; Motlagh et al., 1994). A semi-pure form of pediocin is produced commer-cially (AltaTM2341) and is used as a preser-vative in ready-to-eat meats (Rodriguez et al.,2002), but pediocin has yet to be authorizedas a fully licensed food additive.

Studies have shown that genes forpediocin production are plasmid-encoded:pSRQ11 (9.4 kb) and pSMB74 (8.9 kb)from P. acidilactici and pMD136 (19.5 kb)from P. pentosaceus are involved in pediocinproduction (Gonzalez and Kunka, 1983;Motlagh et al., 1994; Kantor et al., 1997)(Table 6). Since pediocin can be used as aselection marker in cloning vectors, plasmidsthat carry the genes for its production are in-teresting with regards to the development offood-grade cloning vectors.

Unlike other pediococcal species, T.halophila harbors plasmids that are gener-ally thought to be cryptic (Kayahara et al.,1989). pUCL287 from T. halophila, the firstfully sequenced pediococcal plasmid, wasshown to be a theta-replicating plasmid andrepA287 encoded a protein involved in plas-mid replication (Benachour et al., 1995).No homology could be detected betweenRepA287 and replication proteins of well-characterized theta-type replicons, such aslactococcal Rep22 (Frere et al., 1993; Seegerset al., 1994) and the enterococcal pAMβ1-pIP501 group (Le Chatelier et al., 1993). Itwas concluded that pUCL287 represents anew theta-type replicon family and RepA287belongs to a new family of replicationproteins.

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TABLE 6Characteristics of Pediococcus spp. Cryptic Plasmids


replication References

pSRQ1 P. pentosaceus 30 MDa NA Gonzalez and Kunka, 1983pSRQ7 P. pentosaceus 12 MDa NA Gonzalez and Kunka, 1983pSRQ8 P. pentosaceus 17 MDa NA Gonzalez and Kunka, 1983pSRQ9 P. acidilactici 6.7 MDa NA Gonzalez and Kunka, 1983pSRQ10 P. acidilactici 23 MDa NA Gonzalez and Kunka, 1983pSRQ11 P. acidilactici 9.4 NA Gonzalez and Kunka, 1983;

Marugg et al., 1992pSMB74 P. acidilactici 8.9 Theta Motlagh et al., 1994; Benachour

et al., 1997.pUCL287 T. halophila 8.7 Theta Benachour et al., 1995, 1997pMD136 P. pentosaceus 19.5 Theta Kantor et al., 1997

MDa: Megadaltons; NA: not available.

Plasmids pMD136 (Kantor et al., 1997)and pSMB74 (Benachour et al., 1997) aretwo other pediococcal plasmids that have beenreported to follow theta replication. Whilethe pMD136 replicon showed homology totheta-type replicons of L. lactis plasmids,the putative replication protein encoded bypSMB74 was found to be highly homologousto RepA287, suggesting that pSMB74 mightbe a member of the pUCL287 replicon family.

3.7. Bifidobacterium CrypticPlasmids

Bifidobacteria share phenotypic and mor-phological attributes with many lactobacilli,which led to the belief that they belongedto the genus Lactobacillus. Classification bymolecular methodologies has provided evi-dence that this is indeed a distinct genus that ismore closely related to the Actinomycetaceafamily (Stiles and Holzapfel, 1997).

Bifidobacteria are natural inhabitants ofthe gastrointestinal tract, where they playan important role in the health of thehost. They compete with intestinal putrefac-tive bacteria such as enterobacteria (Ibrahimand Bezkorovainy, 1993) and clostridia(Bezirtzoglou and Romond, 1990). They

lower the gut pH by releasing acetate and lac-tate from carbohydrate metabolism and alsoproduce bacteriocins, factors that combine to-gether to inhibit pathogenic bacteria and pre-vent intestinal infections. Some epidemiolog-ical and clinical studies have also providedevidence that fermented milks containing cer-tain Bifidobacterium (B.) species have anti-carcinogenic and immunostimulating prop-erties (Van’t Veer et al., 1989; Reddy andRivenson, 1993; Lee et al., 1993). Besides oc-curring naturally in the GI tract, B. animalisand B. lactis have been found in a variety ofcommercial dairy products. B. longum, B. bi-fidum and B. infantis are now being added tofoods and therapeutic preparations, i.e. probi-otic products, for their health benefits (Stilesand Holzapfel, 1997).

First reported in 1982 by Sgorbati et al.,plasmids in the genus Bifidobacterium werethought to be present only in a few species.Approximately 20% of bacterial isolates thatwere screened contained detectable plasmids,and only several species were represented,namely B. longum, the predominant speciesin the human intestine, B. globosum, a speciescommon to animals, and B. indicum and B.asteroides, exclusively present in the West-ern and Asiatic honeybees (Sgorbati et al.,1982). Iwata and Morishita (1989) later

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demonstrated that B. breve also harbored plas-mids, as many as four. More recently, a plas-mid was discovered in a strain of B. pseu-docatenulatum (Smeianov et al., 2002). Suchdiscoveries refute the initial belief that plas-mids could only be found in a few bifidobac-terial species.

To this date, several bifidobacterial plas-mids have been fully sequenced (Table 7): tenfrom B. longum, and one from each species,including B. breve, B. asteroides, and B. pseu-docatenulatum. Another B. breve plasmid(pNBb1) was partially sequenced (Bourgetet al., 1993). Based on the absence of ssDNAintermediates, it was determined that pNBb1did not replicate via RC (Bourget et al., 1993).However, recent findings have shown thatpNBb1 contains the conserved Pfam domainRep (GenBank accession no. PF01446) andcould thus replicate via RC (Corneau et al.,2004).

Plasmids pKJ36, pKJ50, pMG1 (Parket al., 1999, 2000, 2003), and pNAC1

TABLE 7Characteristics of Bifidobacterium spp. Cryptic Plasmids

Plasmid Origin Size (kb) Mode of replication References

pMB1 B. longum 1.9 Theta Matteuzzi et al., 1990pVS809 B. globosum 2.8 NA Mattarelli et al., 1994pTB6 B. longum NA NA Matsumura et al., 1997pKJ50 B. longum 5.0 RCR Park et al., 1997pKJ36 B. longum 3.6 RCR Park et al., 1997pMG1 B. longum 3.9 RCR Park et al., 2003pNBb1 B. breve 5.6 RCR Bourget et al., 1993pCIBb1 B. breve 5.8 RCR O’Riordan and Fitzgerald, 1999pAP1 B. asteroides 2.1 Unknown Kaufmann et al., 1997; GenBank

accession no. Y11549p4M B. pseudocatenulatum 4.5 RCR Smeianov et al., 2002; GenBank

accession no. AF359574pB44 B. longum 3.6 RCR Smeianov et al., 2002; GenBank

accession no. AY066026pDOJH10L B. longum 10 RCR Lee, J. H. et al., 2002; GenBank

accession no. AF538868pBLO1 B. longum 3.6 RCR Schell et al., 2003; GenBank

accession no. AF540971pNAC1 B. longum 3.5 RCR Corneau et al., 2004pNAC2 B. longum 3.7 RCR Corneau et al., 2004pNAC3 B. longum 10.2 Unknown Corneau et al., 2004

NA: not available; unknown: family of replicon is unknown.

(Corneau et al., 2001) have been shownto follow RCR because ssDNA intermedi-ates have been found. Curiously, RepA ofpKJ50, and RepB of pKJ36 showed sig-nificant amino acid homology with vari-ous replication proteins from theta-replicatingplasmids (respectively, 43% and 37% withLb. acidophilus pLA103 and 54% and 43%with T. halophilus pUCL287). Moreover, thethree B. longum plasmids (pKJ36, pKJ50,and pMG1) contained iteron-like sequences,much like B. longum pNAC1, pNAC2, andpNAC3 (Corneau et al., 2004). Although quitecommon, iterons are not restricted to theta-replicating plasmids, as these repeating se-quences have been reported in plasmids thatuse the strand-displacement mechanism or theRC mechanism (del Solar et al., 1998).

Bifidobacterial plasmids have been di-vided into five groups based on Rep aminoacid sequence homology (Corneau et al.,2004). Most plasmids were grouped withRCR plasmids, except pMB1 (Matteuzzi

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et al., 1990), which showed similarity toColE2, a theta-replicating plasmid from E.coli. B. longum pNAC3 and B. asteroidespAP1 seemed to be more closely related to areplicase coded by pDOJH10L and the threeplasmids formed a group of plasmids of un-known replication type (Corneau et al., 2004).Bifidobacterium is the only genus where allthe isolated and characterized plasmids havebeen deemed cryptic. To this date, no phe-notypic trait has been found to be plasmid-encoded.

4. VECTORS DERIVED FROM LACTICACID BACTERIA CRYPTIC PLASMIDS

4.1. General Properties of CloningVectors

An essential prerequisite for effective ge-netic manipulation of organisms is the avail-ability of suitable vectors that ensure replica-tion and maintenance of both vector DNA andinserted foreign DNA. Useful cloning vectorsmust have (1) an origin of replication, ori, (2)selectable markers to readily identify trans-formed cells, (3) one or more unique restric-tion endonuclease sites, (4) a low molecularweight, smaller vectors being more stable andhaving a higher copy number, and (5) highsegregational and structural stability.

A vector used in industrial fermentationshas to remain structurally intact and be main-tained in the host cell in the absence ofselective pressure during the fermentationprocess. Structural instability, the deletion ofspecific plasmid sequences, is very difficult toovercome because it is not fully understood.Segregational instability, the loss of entireplasmid molecules, arises from a failure todistribute plasmid to both daughter cells uponcell division and is affected mainly by modeof replication.

Generally, RCR plasmids are more proneto segregational instability than theta-typeplasmids. First, RCR plasmids appear to lack

a partitioning function, and plasmids are prob-ably randomly distributed over daughter cells(Novick, 1987). Second, the accumulation ofssDNA intermediates also results in reducedsegregational stability, as it disturbs copynumber control (Pouwels and Leer, 1993).Segregational instability of RCR plasmid vec-tors also increases with the size of DNA in-serts (Leer et al., 1992).

Segregational instability can be overcomeby (1) using theta-replicating plasmids that donot produce ssDNA intermediates and stablymaintain DNA inserts (Janniere et al., 1990),(2) using high copy number plasmid vectors,since there is always a probability that at leastone plasmid molecule will be transmitted to adaughter cell at each cell division, (3) apply-ing constant selective pressure in the growthmedium, and (4) integrating foreign DNA intothe host chromosome.

Before a cloning trial, compatibility of avector with other vectors or with endogenousplasmids should be considered. Plasmid in-compatibility can be caused by competitionof different plasmids with a similar replica-tion mechanism for host cell proteins essen-tial in DNA replication (i.e. Rep proteins).Generally, only one type of plasmid will re-main in the host, especially if favored by se-lective pressure. However, it has been shownthat while only one RCR plasmid can be main-tained in a host, four to five theta-replicatingplasmids can exist stably in the same host(Kiewiet et al., 1993). Hence, vectors derivedfrom theta-type plasmids tend to suffer lessfrom incompatibility problems.

The past two decades have seen the de-velopment of a panoply of cloning vec-tors for the engineering of various bacte-rial species, some of which are still usedtoday. Currently used vectors for LAB orother Gram-positive bacteria can be di-vided into two major classes. Class 1 vec-tors, represented by streptococcal pIP501and enterococcal pAMβ1 and their deriva-tives, are large conjugative plasmids that areresistant to macrolides, lincosamides, and

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spectogramin B (MLS resistance) and otherantibiotics (Simon and Chopin, 1988; Daoand Ferretti, 1985). pIP501 and pAMβ1 aretheta-replicating plasmids and their deriva-tives have exhibited segregational and struc-tural stability, even upon insertion of large for-eign DNA fragments (Kiewiet et al., 1993).These broad host-range plasmids can repli-cate in many Gram-positive bacteria, includ-ing various LAB such as Lactococcus spp.,Lactobacillus spp., and Pediococcus spp. (deVos and Simons, 1994). However, as strep-tococci and enterococci are not GRAS or-ganisms, pIP501, pAMβ1, their derivativescannot be considered food-grade, and are notsuitable for food applications.

Class 2 vectors are derived from smallcryptic plasmids from several lactococcalspecies. Once they are tagged with a se-lectable marker, usually an antibiotic resis-tance gene, they can be used as cloning vec-tors. Prototype vectors, based on lactococcalreplicons pSH71 and pWV01, contain oneor more genes coding for resistance to an-tibiotics. These vectors tend to suffer fromstructural and segregational instability due totheir mechanism of replication (RCR) and canrarely maintain large DNA fragments; they areonly useful for the cloning of smaller genes.Nonetheless, pSH71 and pWV01 repliconsand their derivatives are still useful as theyhave a broad host-range.

In addition to this class of vectors, crypticplasmids originating from other LAB specieshave also been marked with antibiotic resis-tance genes to serve as broad or narrow host-range cloning vectors. Research is now beingfocused on the development of stable cloningvectors derived from plasmids with GRASorigins.

4.2. Vectors Derived fromLactococcus Cryptic Plasmids

The most widely used plasmid vectorswere constructed twenty years ago from L.

lactis replicons pSH71 and pWV01 (de Vos,1987; Kok et al., 1984). These two repli-cons share some common features: they areboth small, lactococcal cryptic plasmids, withnearly identical sequences and can replicatein E. coli and many Gram-positive bacteria.They differ only in copy number in differentstrains of L. lactis, low for pWV01 and highfor pSH71. The drawback of pWV01- andpSH71-based vectors is the often encounteredstructural and segregational instability, espe-cially upon insertion of large foreign DNAfragments (Gruss and Ehrlich, 1989; Kiewietet al., 1993).

Many pWV01-based derivatives havebeen constructed, mainly by varying vectorsize, marker genes, and inserting multiplecloning sites (MCS). Plasmid pGK12, the pro-totype vector of the pGK-series, is based onpWV01, and carries chloramphenicol and ery-thromycin resistance genes for selection inL. lactis, B. subtilis, and E. coli (Kok et al.,1984). Several species in the genus Lacto-bacillus have also served as hosts for pGK12-type vectors (Table 8). Kok et al. (1984) foundthat pGK12 replicated in E. coli at a high copynumber, while it showed low copy numberin L. lactis and B. subtilis. pGK-vectors havebeen used to clone and express various het-erologous genes in several hosts.

pMG-vectors, also based on pWV01,were developed for the cloning and expres-sion of bacteriocin and lysozyme genes (vanBelkum et al., 1989; van de Guchte et al.,1989). More recently, pIAV-vectors have beendeveloped (Perez-Arellano et al., 2001). Plas-mid copy numbers of pIAV7 and pIAV9 weresignificantly higher than those reported forother pWV01 derivatives in L. lactis (Koket al., 1984; Kiewiet et al., 1993). When cul-tured in antibiotic-free medium, both vectorswere also maintained stably in host cells formore than 40 generations; after 120 genera-tions, 35% of pIAV7 transformants still exhib-ited antibiotic resistance and 28% for pIAV9.

In addition to cloning vectors, integra-tion vectors were also derived from pWV01.

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TABLE 8Vectors Derived from Lactococcus lactis Cryptic Plasmids and Their Characteristics

Vector Replicon Origin Size (kb)Geneticmarker

Shuttlevector

Transfermethod Host range References

pGK1 pWV01 L. lactis 5.0 Cmr No ppf B. subtilis Kok et al., 1984pGK12 pWV01 L. lactis 4.4 Cmr Emr Yes ppf L. lactis Kok et al., 1984

B. subtilisE. coli

elp Lb. acidophilus Posno et al., 1991aelp Lb. breviselp Lb. caseielp Lb. delbrueckii Zink et al., 1991elp Lb. fermentum Posno et al., 1991aelp Lb. helveticus de los Reyes-Gavilan

et al., 1990elp Lb. pentosus Posno et al., 1991aelp Lb. plantarum Badii et al., 1989;

Posno et al., 1991aelp Lb. reuteri Luchansky et al.,

1989pGKV1 pWV01 L. lactis 4.6 Cmr Emr Yes elp Lb. acidophilus Luchansky et al.,

1988pGKV10 pWV01 L. lactis 4.6 Cmr Yes ppf B. subtilis van der Vossen et al.,

1985pGKV110 pWV01 L. lactis 4.6 Cmr Emr Yes ppf L. lactis van der Vossen et al.,

1985B. subtilis

pGKV11 pWV01 L. lactis 4.6 Emr Cmr Yes ppf L. lactis van der Vossen et al.,1985

B. subtilispGKV2 pWV01 L. lactis 4.7 Cmr Emr No ppf L. lactis van der Vossen et al.,

1985B. subtilisE. coli

Yes elp Lb. plantarum Josson et al., 1989pGKV21 pWV01 L. lactis 4.8 Cmr Emr Yes elp Lb. acidophilus van der Vossen et al.,

1994elp Lb. plantarum Pouwels and Leer,

1993elp Lb. casei Gaier et al., 1992

pGKV210 pWV01 L. lactis 4.4 Cmr Emr Yes ppf L. lactis van der Vossen et al.,1987

B. subtiliselp Lb. casei Gaier et al., 1992elp Lb. johnsonii Fremaux et al., 1993belp Lb. reuteri Djordjevic et al.,

1994pGKV13 pWV01 L. lactis 5.1 Cmr Emr Yes elp Lb. plantarum Badii et al., 1989pGKV500 pWV01 L. lactis 7.4 MDa Emr No ppf L. lactis Kok et al., 1985

B. subtilispMG24 pWV01 L. lactis 4.4 Kmr No elp L. lactis van Belkum et al.,

1989(Continued on next page)

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TABLE 8Vectors Derived from Lactococcus lactis Cryptic Plasmids and Their Characteristics (Continued)


Shuttlevector


pMG36 pWV01 L. lactis 3.7 Kmr No elp L. lactis van de Guchte et al.,1989

pMG36e pWV01 L. lactis 3.7 Emr No elp L. lactis van de Guchte et al.,1989

No elp Lb. gasseri Roy et al., 1993pTRK170 pWV01 L. lactis 6.6 Cmr Yes tdc Lb. acidophilus Raya and

Klaenhammer,1992

tdc Lb. gasseritdc Lb. helveticus

pGIP212 pWV01 L. lactis 8.2 Cmr Kmr Spr Yes elp Lb. plantarum Hols et al., 1994pIAV1 pWV01 L. lactis 6.1 Emr Cmr lacZ Yes elp Lb casei Perez-Arellano

et al., 2001pIAV6 pWV01 L. lactis 3.5 Cmr Yes elp Lb. caseipIAV7 pWV01 L. lactis 5.2 Emr Yes elp Lb. caseipIAV9 pWV01 L. lactis 5.2 Cmr Yes elp Lb. caseipNZ11 pSH71 L. lactis 4.9 Cmr Kmr No ppf B. subtilis de Vos et al., 1986

L. lactisE. coli

pNZ12 pSH71 L. lactis 4.1 Cmr Kmr No ppf B. subtilis de Vos et al., 1986L. lactisE. coli

No elp Lb. casei de Vos et al., 1987;Olukoya et al.,1993

No elp Lb. curvatus Vogel et al., 1992;Gaier et al., 1990

No elp Lb. plantarum Bringel and Hubert,1990

No elp Lb. sakei Gaier et al., 1990pNZ121 pSH71 L. lactis 4.1 Cmr Kmr NA NA B. subtilis de Vos et al., 1987

Sp. aureusE. coliL. lactis

pNZ122 pSH71 L. lactis 3.5 Cmr Kmr No elp L. lactis de Vos et al., 1986pNZ123 pSH71 L. lactis 2.8 Cmr NA NA L. lactis de Vos et al., 1986

NA elp Lb. acidophilus Kim et al., 1994NA elp Lb. casei Platteeuw et al.,

1994Lb. plantarum

pNZ124 pSH71 L. lactis 2.8 Cmr Yes elp Lb. casei Platteeuw et al.,1994

Lb. plantarumpNZ17 pSH71 L. lactis NA NA NA NA NA de Vos, 1987pNZ18 pSH71 L. lactis NA Cmr Kmr Yes elp Lb. casei Solaiman et al.,

1992pNZ19 pSH71 L. lactis NA Cmr Kmr Yes elp Lb. casei Solaiman et al.,

1992 ; Somkutiet al., 1992

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Shuttlevector


pNZ220 pSH71 L. lactis NA Kmr No elp L. lactis de Vos et al., 1989B. subtilisE. coli

pNZ272 pSH71 L. lactis 4.7 Cmr Yes elp Lb. casei Platteeuw et al.,1994

Lb. plantarumpCK1 pSH71 L. lactis 5.5 Cmr Kmr Yes ppf B. subtilis Gasson and

Anderson, 1985L. lactisE. coli

pCK17 pSH71 L. lactis 5.9 Cmr Kmr Yes ppf B. subtilis Gasson andAnderson, 1985

L. lactisE. coli

pCK21 pSH71 L. lactis 5.9 Cmr Kmr Yes ppf B. subtilis Gasson andAnderson, 1985

L. lactisE. coli

pMIG1 pSH71 L. lactis 4.8 Cmr Kmr Yes elp L. lactis Wells et al., 1993pMIG2 pSH71 L. lactis 4.0 Cmr Yes elp L. lactispMIG2H pSH71 L. lactis 4.2 Cmr Yes elp L. lactispMIG3 pSH71 L. lactis 5.5 Cmr Yes elp L. lactispVS2 pSH71 L. lactis 5.1 Cmr Emr No ppf L. lactis von Wright et al.,

1987No elp Lb. plantarum Aukrust and Blom,

1992No elp Lb. reuteri Ahrne et al., 1992No elp Lb. sakei Aukrust and Blom,

1992; Axelssonet al., 1993

pVSB1 pSH71 L. lactis 4.0 Cmr lac Yes elp Lb. sakei Axelsson and Holck,1995

pRW1 pSH71 L. lactis 9.7 Emr Tcr Apr Yes ppf L. lactis von Wright andRaty, 1993

pBN183 pSH71 L. lactis 10.1 Apr Cmr Kmr Yes elp Lb. casei Solaiman et al.,1992

pBN183A pSH71 L. lactis 8.7 Apr Cmr Kmr Yes elp Lb. casei Solaiman et al.,1992

pDBN183 pSH71 L. lactis 8.8 Apr Cmr Kmr Yes elp Lb. casei Solaiman et al.,1992

pGIP331 pSH71 L. lactis NA Cmr Kmr Spr Yes elp Lb. plantarum Hols et al., 1994pFX1 pD125 L. lactis 5.5 Cmr Yes elp L. lactis Xu et al., 1990, 1991pFX3 pD125 L. lactis 4.5 Cmr Yes elp L. lactis Xu et al., 1990, 1991pCP12 pWC1 L. lactis 3.9 Cmr No elp L. lactis Pillidge et al., 1996

L. garviaeS. thermophilus

(Continued on next page)

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Shuttlevector


En. faecalisSp. aureus

p21-22 pBM02 L. lactis NA AprEmr Yes elp L. lactis Sanchez and Mayo,2003

Lb. caseiLb. plantarumB. subtilis





pCI3340 pCI305 L. lactis 5.7 Cmr Yes elp L. lactis Hayes et al., 1990pCI374 pCI305 L. lactis 4.9 Cmr Yes elp L. lactis Hayes et al., 1990pVS40 pVS40 L. lactis 7.8 Nisr No ppf L. lactis von Wright et al.,

1990pKMP1 pSK11L L. lactis 20.6 Cmr Emr No elp L. lactis Horng et al., 1991pCI534 pCI528 L. lactis 12.5 Cmr No elp L. lactis Lucey et al., 1993pLR300 pWV02 L. lactis 5.6 Emr No NA L. lactis Kiewiet et al., 1993

NA P. acidilacticipND302 pND302 L. lactis 8.8 Cdr No elp L. lactis Liu et al., 1996pND304 pND302 L. lactis 13.2 Tcr Apr Yes elp L. lactispND624 pND302 L. lactis 13.4 Apr Nisr Cdr No elp L. lactispND625 pND302 L. lactis 10.4 Nisr Cdr No elp L. lactispND421 pND324 L. lactis 15.1 Apr Emr Nisr No elp L. lactis Duan et al., 1999

L: Lactococcus; Lb: Lactobacillus; S: Streptococcus; Ln: Leuconostoc; P: Pediococcus; Sp: Staphylococcus; E: Escherichia;En: Enterococcus; B: Bacillus; Apr: ampicillin resistance; Cmr: chloramphenicol resistance; Emr: erythromycin resistance;Spr: spectinomycin resistance; Tcr: tetracycline resistance; Nisr: nisin resistance; Cdr: cadmium resistance; Kmr: kanamycinresistance elp: electroporation; tdc: transduction; ppf: protoplast fusion.

pVE6002, a thermosensitive plasmid derivedfrom pWV01, was isolated from pGK12 aftermutagenesis: it was non-replicative in L. lac-tis at temperatures above 35◦C (Maguin et al.,1992). Ori+ vectors, which contained onlythe replication origin of pWV01, were con-structed by physically separating ori from itsreplicon, repA. Helper strains carrying repAsupport replication of pOri+ when RepA isprovided in trans. When RepA is absent, Ori+

vectors integrate into the host chromosome(Leenhouts et al., 1996).

Despite the efforts to derive vectors frompWV01, the fact remained that most construc-tions showed low copy number in L. lactis,which sparked the development of high copynumber plasmid vectors for use in lactococci.The pNZ-series of vectors, such as pNZ12and pNZ121 (both 4.1 kb), were shown toreplicate in B. subtilis, S. aureus, and E. coli(de Vos, 1986) with high and low copy num-ber, respectively. Plasmids pNZ18 and pNZ19were constructed by inserting a useful MCSinto pNZ12 and pNZ121, respectively. Many

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vectors were further derived, welcomed ad-ditions to the growing pNZ-family, and usedto clone heterologous genes in various Gram-positive and -negative bacteria (de Vos andSimons, 1994). High copy number pCK1 (5.5kb) based on pSH71 was also constructed,but lacked cloning sites in addition to beingquite unstable (Gasson and Anderson, 1985).To remedy this situation, pMIG vectors basedon pCK1 were developed (Wells et al., 1993).They contained a MCS and could transformGram-positive bacteria and E. coli at both highand low copy number, respectively.

Other lactococcal replicons have also beenused for vector development. pD125 (5.5 kb)was genetically marked with the cat-194gene of pC194 resulting in the constructionof pFX1 and its MCS-containing derivative,pFX3 (4.5 kb). pFX3 was used to clone thetagatose 1,5-biphosphate aldolase gene in L.lactis and E. coli, although the expressionof the gene was not verified in either host(Xu et al., 1991). The latest lactococcal RCRplasmid to be discovered (pBM02, 3.85 kb)showed a wide host-range and could replicatein Gram-positive and Gram-negative bacteria,including Lb. plantarum, Lb. casei, B. subtilis,and E. coli (Sanchez and Mayo, 2003). Un-like the previously described vectors, pCP12based on the cryptic plasmid pWC1 couldtransform several Gram-positive bacteria, butnot E. coli, mainly because it belongs tothe pC194 family of replicons, rather thanthe pE194 (Pillidge et al., 1996). Generally,pE194-type replicons have a wide host-rangeand are functional in E. coli (del Solar et al.,1998).

Since vectors derived from RCR plasmidssuffer from structural and segregational in-stability, vectors based on lactococcal theta-replicating plasmids have been developed.Narrow host-range vectors pCI374 andpCI3340 based on theta-replicating plasmidpCI305 were constructed; both had a low copynumber and could only transform L. lactis(Hayes et al., 1990). pLR300, derived frompWV02, could transform lactococci and P.

acidilactici and was stably maintained for atleast 100 generations without selective pres-sure at a copy number of 5–10 plasmidsper chromosome equivalent (Kiewiet et al.,1993).

A few other lactococcal vectors are worthmentioning. The food-grade vector pVS40(7.9 kb) was derived from a 28-kb theta-replicating cryptic plasmid and uses nisin re-sistance as a selective marker (von Wrightet al., 1990). An integration vector pND421was derived from a lactococcal thermostableplasmid (pND324). With nisin and ery-thromycin resistance genes, pND421 wasshown to integrate into host chromosomalDNA. The integrated plasmid was quite sta-ble for up to 100 generations without selec-tion at 30◦C, which is desired for commercialapplications (Duan et al., 1999). Food-gradepND625 uses cadmium and nisin resistanceas markers (Liu et al., 1996). However, theuse of cadmium in foods as a means of se-lective pressure remains questionable, unlessit is only used for initial transformant selec-tion and the construct has demonstrated highstability.

4.3. Vectors Derived fromLactobacillus Cryptic Plasmids

To date, a plethora of cloning vectorshas been constructed from Lactobacilluscryptic plasmids. Virtually every newly iso-lated lactobacilli plasmid has been taggedwith antibiotic resistance markers, sometimeseven with a replication origin of Gram-negative bacteria, and used to clone andexpress heterologous genes in various Lac-tobacillus species, in Gram-positive hostssuch as Bacillus spp., Carnobacterium di-vergens, Carnobacterium piscicola, Entero-coccus faecalis, Lactococcus lactis, Staphy-lococcus aureus, Streptococcus sanguis,Streptococcus gordonii, Streptococcus mu-tans, as well as in E. coli (Pouwels and Leer,1993; Klaenhammer, 1995; Wang and Lee,

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1997). Most derived vectors have broad host-ranges that seem rather limited when com-pared to the host-ranges of lactococcal repli-cons.

Nevertheless, some lactobacilli repli-cons have proven to be functional in E.coli, namely pA1, pPSC20/pPSC22, pGT633,pLC2, pLA106, and pLF1311 (Table 9)(Vujcic and Topisirovic, 1993; Cocconcelliet al., 1991; Tannock et al., 1994; Klein et al.,1993; Sano et al., 1997; Aleshin et al., 2000).They are mostly members of the pE194-family of RC plasmids, a group of repliconsknown to have a wide host-range (del Solaret al., 1998). Derivatives of these plasmidscould be propagated in Lactobacillus spp.,Bacillus spp., and E. coli.

Some vectors derived from Lb. fermen-tum (Iwata et al., 1986; Fons et al., 1997),Lb. reuteri (Ahrne et al., 1992; Heng et al.,1999; Lin and Chung, 1999; Lin et al., 2001),Lb. helveticus (Hashiba et al., 1990, 1992),Lb. acidophilus (Damiani et al., 1987), Lbcrispatus (Pouwels and Leer, 1993), and Lb.delbrueckii bulgaricus (Zink et al., 1991;Chagnaud et al., 1992) exhibit a narrow host-range called host-specific replication, as theycannot replicate in other bacteria. Such vec-tors are interesting with regards to safety as-pects associated with the use of viable re-combinant organisms or genetically modifiedmicroorganisms in food products or their useas vaccine carriers. Narrow host-range vec-tors are deemed intrinsically safer, as theyare less likely to be horizontally transferredto other bacteria species than vectors basedon broad host-range replicons (Pouwels andLeer, 1993).

Concerning stability, RC-based vectorslike pULP8 and pULP9 were lost from trans-formed cells after 20 generations (Bringelet al., 1989); after 13 generations, only 30%of transformants still harbored pLAB1000-derived vectors (Josson et al., 1989); Lb. del-brueckii bulgaricus pLE16 transformants lostresistance to chloramphenicol after 28 gener-ations (Chagnaud et al., 1992). It can be ar-

gued that industrial strains of LAB used in theproduction of fermented foods only undergo afew generations, thus implying that even vec-tors that suffer from segregational instabilitycould still be suitable for cloning.

Nonetheless, vectors based on theta-replicating plasmids showed remarkable sta-bility: 100% of pULA105E transformants stillexhibited erythromycin resistance after 100generations (Kanatani et al., 1992); pKC5b-derived shuttle vector pSP1 was maintained inLactobacillus spp. for 100 generations with-out selective pressure (Pavlova et al., 2002);pTC82 and its derivative pTC82-RO bothshowed exceptional segregational stability, as100% of transformants still harbored recom-binant plasmids after 216 generations (Lin andChung, 1999).

However, not all vectors derived fromtheta-replicating plasmids are exceptionallystable: pRV566 (Lb. sakei) was maintainedin transformed cells for only 20 generations.Such a loss rate was deemed suitable consider-ing Lb. sakei strains used as starters in sausagemaking grow for about 12 generations duringthe fermentation process (Alpert et al., 2003).Some vectors also tend to show excellent seg-regational stability in vitro, but do quite poorlyin vivo. A recombinant plasmid, pNCHK104derived from Lb. reuteri pGT232, was foundto be structurally and segregationally stableunder laboratory growth conditions (>97%erythromycin-resistant cells after being cul-tured 7 days without selective pressure ), butwas poorly maintained in vivo (<33% resis-tant cells after being associated with mice for14 days) (Heng et al., 1999).

Lactobacillus strains are generally resis-tant to ampicillin, cloxacillin, gentamycin,kanamycin, neomycin, penicillin, strepto-mycin, tetracycline, amongst others (Okluoyaet al., 1993; Vescovo et al., 1982), but doshow sensitivity to chloramphenicol and ery-thromycin. Vectors used to transform lacto-bacilli carry the erythromycin resistance genefrom pE194 or pAMβ1 or the chlorampheni-col resistance gene from pC194 or pBR322

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TAB

LE

9V

ecto

rsD

eriv

edfr

om

Lac

tob

acill

us

spp

.Cry

pti

cP

lasm

ids

and

Th

eir

Ch

arac

teri

stic

s

Vec

tor

Rep

licon

Ori

gin

Size

(kb)

Gen

etic

mar

ker

Shut

tleve

ctor

Tra

nsfe

rm

etho

dH

ost

rang

eR

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ence

s

pcaT

pcaT

Lb.

plan

taru

m8.

5C

mr

NA

elp

C.d

iver

gens

Ahn

etal

.,19

92C

.pis

cico

laL

b.ca

sei

Lb.

plan

taru

mm

obC

.pis

cico

lapA

1pA

1L

b.pl

anta

rum

4.0

Cm

rN

oN

AE

.col

iV

ujci

can

dTo

pisi

rovi

c,19

93L

b.de

lbru

ecki

iL

b.pl

anta

rum

pUL

P8pL

P1L

b.pl

anta

rum

6.6

Apr

Em

rY

esel

pB

.sub

tili

sB

ring

elet

al.,

1989

Lb.

plan

taru

mpU

LP9

pLP1

Lb.

plan

taru

m6.

8A

prE

mr

Yes

elp

B.s

ubti

lis

Bri

ngel

etal

.,19

89L

b.pl

anta

rum

pLP8

25p8

014-

2L

b.pl

anta

rum

7.6

Apr

Cm

rY

esel

pL

b.ac

idop

hilu

sPo

sno

etal

.,19

91a

elp

Lb.

brev

isPo

sno

etal

.,19

91a

elp

Lb.

case

iL

eer

etal

.,19

92;

Posn

oet

al.,

1991

ael

pL

b.fe

rmen

tum

Posn

oet

al.,

1991

ael

pL

b.he

lvet

icus

delo

sR

eyes

-Gav

ilan

etal

.,19

90el

pL

b.pe

ntos

usPo

sno

etal

.,19

91a;

Lee

ret

al.,

1992

elp

Lb.

plan

taru

mB

adii

etal

.,19

89;

Lee

ret

al.,

1992

;Po

sno

etal

.,19

91a;

Duc

kwor

thet

al.,

1993

pLP8

2Hp8

014-

2L

b.pl

anta

rum

5.9

Apr

Cm

rY

esel

pL

b.ca

sei

Lee

ret

al.,

1992

elp

Lb.

pent

osus

Duc

kwor

thet

al.,

1993

Lb.

plan

taru

m(C

onti

nued

onne

xtpa

ge)

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onl

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TAB

LE

9V

ecto

rsD

eriv

edfr

om

Lac

tob

acill

us

spp

.Cry

pti

cP

lasm

ids

and

Th

eir

Ch

arac

teri

stic

s(C

on

tin

ued

)

Vec

tor

Rep

licon

Ori

gin

Size

(kb)

Gen

etic

mar

ker

Shut

tleve

ctor

Tra

nsfe

rm

etho

dH

ost

rang

eR

efer

ence

s

pLPC

37p8

014-

2L

b.pl

anta

rum

3.3

Cm

rN

oel

pL

b.ca

sei

Lee

ret

al.,

1992

Lb.

pent

osus

Lb.

plan

taru

mpP

SC1

a7

kbpl

asm

idL

b.pl

anta

rum

4.1

Cm

rY

esel

pB

.sub

tili

sC

occo

ncel

liet

al.,

1991

Lb.

acid

ophi

lus

Lb.

ferm

entu

mL

b.he

lvet

icus

Lb.

plan

taru

mL

b.re

uter

iL

.lac

tis

pPSC

10a

7kb

plas

mid

Lb.

plan

taru

m3.

0E

mr

Yes

elp

B.s

ubti

lis

Coc

conc

elli

etal

.,19

91L

b.ac

idop

hilu

sL

b.fe

rmen

tum

Lb.

helv

etic

usL

b.re

uter

iL

.lac

tis

pPSC

11a

7kb

plas

mid

Lb.

plan

taru

m2.

9C

mr

Yes

elp

B.s

ubti

lis

Coc

conc

elli

etal

.,19

91L

b.ac

idop

hilu

sL

b.fe

rmen

tum

Lb.

helv

etic

usL

b.re

uter

iL

.lac

tis

pPSC

20a

7kb

plas

mid

Lb.

plan

taru

m5.

5C

mr

Em

rY

esel

pB

.sub

tili

sC

occo

ncel

liet

al.,

1991

;Ves

covo

etal

.,19

91L

b.ac

idop

hilu

sL

b.fe

rmen

tum

Lb.

helv

etic

us

178

Cri

tical

Rev

iew

s in

Bio

tech

nolo

gy D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y Se

rial

s U

nit -

Lib

rary

on

10/2

1/13

For

pers

onal

use

onl

y.

Lb.

reut

eri

L.l

acti

spP

SC22

a7

kbpl

asm

idL

b.pl

anta

rum

4.3

Cm

rE

mr

Yes

elp

B.s

ubti

lis

Coc

conc

elli

etal

.,19

91L

b.ac

idop

hilu

sL

b.fe

rmen

tum

Lb.

helv

etic

usL

b.re

uter

iL

.lac

tis

NA

cryp

ticpl

asm

idL

b.pl

anta

rum

NA

Apr

Yes

NA

E.c

oli

May

oet

al.,

1989

NA

Lb.

plan

taru

mpL

PV10

6p2

56L

b.pl

anta

rum

NA

Apr

Em

rY

esel

pL

b.pl

anta

rum

Hol

cket

al.,

1992

Lb.

sake

pLPV

III

p256

Lb.

plan

taru

m4.

2E

mr

lac

Yes

elp

Lb.

sake

Axe

lsso

nan

dH

olck

,19

95pL

Y2

pLY

2L

b.fe

rmen

tum

15.6

Tcr

NA

ppf

Lb.

ferm

entu

mIw

ata

etal

.,19

86pL

Y4

pLY

4L

b.fe

rmen

tum

57.8

Em

rN

App

fL

b.fe

rmen

tum

Iwat

aet

al.,

1986

pLE

M5

pLE

M3

Lb.

ferm

entu

m3.

4E

mr

No

elp

Lb.

ferm

entu

mFo

nset

al.,

1997

pLE

M7

pLE

M3

Lb.

ferm

entu

m3.

5E

mr

No

elp

Lb.

ferm

entu

mFo

nset

al.,

1997

pLFV

M2

pLF1

311

Lb.

ferm

entu

m5.

0C

mr

Yes

conj

mob

Lb.

brev

isA

lesh

inet

al.,

1999

Lb.

buch

neri

L.l

acti

sE

n.fa

ecal

isE

n.fa

eciu

mB

.sub

tili

sB

.thu

ring

iens

issu

bsp.

gall

eria

eB

.thu

ring

iens

issu

bsp.

kurs

taki

B.t

huri

ngie

nsis

subs

p.fin

itim

usB

.am

ylol

ique

faci

ens

B.fl

avum

E.c

oli

(Con

tinu

edon

next

page

)

179

Cri

tical

Rev

iew

s in

Bio

tech

nolo

gy D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y Se

rial

s U

nit -

Lib

rary

on

10/2

1/13

For

pers

onal

use

onl

y.

TAB

LE

9V

ecto

rsD

eriv

edfr

om

Lac

tob

acill

us

spp

.Cry

pti

cP

lasm

ids

and

Th

eir

Ch

arac

teri

stic

s(C

on

tin

ued

)

Vec

tor

Rep

licon

Ori

gin

Size

(kb)

Gen

etic

mar

ker

Shut

tleve

ctor

Tra

nsfe

rm

etho

dH

ost

rang

eR

efer

ence

s

NA

pLF1

311

Lb.

ferm

entu

mN

AN

AY

esN

AG

ram

-pos

itive

Ale

shin

etal

.,20

00E

.col

ipS

P1pK

C5b

Lb.

ferm

entu

m9.

4E

mr

Yes

elp

Lb.

ferm

entu

mPa

vlov

aet

al.,

2002

Lb.

jens

enii

Lb.

spp.

Lb.

gass

eri

Lb.

cris

patu

sL

b.jo

hnso

nii

Lb.

sali

vari

usS.

mut

ans

S.go

rdon

iiS.

sang

uis

pLPE

23M

p353

-2L

b.pe

ntos

us3.

7E

mr

No

elp

Lb.

plan

taru

mPo

uwel

san

dL

eer,

1993

pLPE

24M

p353

-2L

b.pe

ntos

us3.

7E

mr

No

elp

Lb.

plan

taru

mpL

PE31

7p3

53-1

Lb.

pent

osus

2.9

Em

rN

oel

pL

b.ca

sei

Posn

oet

al.,

1991

aL

b.pe

ntos

usL

b.pl

anta

rum

pLPE

323

p353

-2L

b.pe

ntos

us3.

6E

mr

No

elp

Lb.

case

iPo

sno

etal

.,19

91a

Lb.

pent

osus

Lb.

plan

taru

mpL

PE35

0p3

53-2

Lb.

pent

osus

3.6

Cm

rN

oel

pL

b.ca

sei

Lee

ret

al.,

1992

Lb.

pent

osus

Lb.

plan

taru

mpL

P353

7p3

53-2

Lb.

pent

osus

6.3

Apr

Em

rY

esel

pL

bac

idop

hilu

sPo

sno

etal

.,19

91a

Lb.

case

iL

b.pe

ntos

usL

b.pl

anta

rum

180

Cri

tical

Rev

iew

s in

Bio

tech

nolo

gy D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y Se

rial

s U

nit -

Lib

rary

on

10/2

1/13

For

pers

onal

use

onl

y.

pLP3

537-

xyl

p353

-2L

b.pe

ntos

us12

.2A

prE

mr xy

lY

esel

pL

b.ca

sei

Posn

oet

al.,

1991

bL

b.pe

ntos

usL

b.pl

anta

rum

pGT

633

pGT

633

Lb.

reut

eri

9.8

Em

rN

Ael

pB

.sub

tili

sTa

nnoc

ket

al.,

1994

En.

faec

alis

Lb.

delb

ruec

kii

Lb.

ferm

entu

mL

b.re

uter

iL

b.ga

sser

iL

b.sa

liva

rius

Sp.a

ureu

sS.

sang

uis

NA

pLU

L63

1+

pVS1

Lb.

reut

eri

NA

Em

rY

espp

fL

.lac

tis

Axe

lsso

net

al.,

1988

NA

E.c

oli

pLU

L63

1pL

UL

631

Lb.

reut

eri

10.2

Em

rN

Ael

pL

b.re

uter

iA

hrne

etal

.,19

92pL

UL

200

pLU

L63

1L

b.re

uter

i6.

0C

mr

Yes

elp

Lb.

reut

eri

pLU

L20

1pL

UL

631

Lb.

reut

eri

7.4

Cm

r Em

rY

esel

pL

b.re

uter

ipL

UL

202

pLU

L63

1L

b.re

uter

i4.

3C

mr

No

elp

Lb.

reut

eri

pLU

L63

4pL

UL

631

Lb.

reut

eri

5.1

Em

rN

oel

pL

b.re

uter

ipN

CK

H10

4pG

T23

2L

b.re

uter

i5.

7E

mr

Yes

elp

Lb.

reut

eri

Hen

get

al.,

1999

pNC

HK

103

pGT

232

Lb.

reut

eri

6.7

Em

rY

esel

pL

b.re

uter

ipT

E15

-RO

pTE

15L

b.re

uter

i6.

7E

mr

Apr

Yes

elp

Lb.

reut

eri

Lin

etal

.,19

99L

b.fe

rmen

tum

pTE

80-R

OpT

E80

Lb.

reut

eri

6.9

Em

rA

prY

esel

pL

b.re

uter

ipT

C82

-RO

pTC

82L

b.re

uter

i7

Em

rN

oel

pL

b.re

uter

iL

inet

al.,

2001

pER

M3.

2pL

AB

1000

Lb.

hilg

ardi

i7.

6E

mr

Apr

Yes

NA

Lb.

plan

taru

mSc

heir

linck

etal

.,19

89pL

AB

1102

pLA

B10

00L

b.hi

lgar

dii

7.5

Cm

rA

prY

esel

pB

.sub

tili

sJo

sson

etal

.,19

89E

n.fa

ecal

isL

b.ca

sei

Lb.

plan

taru

mpL

AB

1301

pLA

B10

00L

b.hi

lgar

dii

5.3

Em

rA

prY

esel

pB

.sub

tili

sJo

sson

etal

.,19

89E

n.fa

ecal

isL

b.ca

sei

Lb.

plan

taru

m(C

onti

nued

onne

xtpa

ge)

181

Cri

tical

Rev

iew

s in

Bio

tech

nolo

gy D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y Se

rial

s U

nit -

Lib

rary

on

10/2

1/13

For

pers

onal

use

onl

y.

TAB

LE

9V

ecto

rsD

eriv

edfr

om

Lac

tob

acill

us

spp

.Cry

pti

cP

lasm

ids

and

Th

eir

Ch

arac

teri

stic

s(C

on

tin

ued

)

Vec

tor

Rep

licon

Ori

gin

Size

(kb)

Gen

etic

mar

ker

Shut

tleve

ctor

Tra

nsfe

rm

etho

dH

ost

rang

eR

efer

ence

s

pLA

B13

04pL

AB

1000

Lb.

hilg

ardi

i5.

2E

mr

Apr

Yes

elp

B.s

ubti

lis

Joss

onet

al.,

1990

En.

faec

alis

Lb.

plan

taru

mpL

AB

1321

pLA

B10

00L

b.hi

lgar

dii

6.9

Em

rA

prY

esel

pB

.sub

tili

sJo

sson

etal

.,19

90E

n.fa

ecal

isL

b.pl

anta

rum

pJK

352

pLC

2L

b.cu

rvat

us5.

9C

mr

Apr

Yes

elp

B.s

ubti

lis

Kle

inet

al.,

1993

Lb.

case

iL

.lac

tis

pJK

352d

pLC

2L

b.cu

rvat

us3.

2C

mr

No

elp

B.s

ubti

lis

Kle

inet

al.,

1993

Lb.

case

iL

.lac

tis

pJK

353

pLC

2L

b.cu

rvat

us5.

8C

mr

Apr

Yes

elp

B.s

ubti

lis

Kle

inet

al.,

1993

Lb.

case

iL

.lac

tis

pJK

354

pLC

2L

b.cu

rvat

us5.

8C

mr

Apr

Yes

elp

B.s

ubti

lis

Kle

inet

al.,

1993

Lb.

case

iL

.lac

tis

pJK

355

pLC

2L

b.cu

rvat

us3.

2C

mr

No

elp

B.s

ubti

lis

Kle

inet

al.,

1993

Lb.

case

iL

.lac

tis

pJK

356

pLC

2L

b.cu

rvat

us3.

2C

mr

No

elp

B.s

ubti

lis

Kle

inet

al.,

1993

Lb.

case

iL

.lac

tis

pJK

300

pWS9

7L

b.de

lbru

ecki

i6.

8C

mr

Apr

Yes

elp

Lb.

delb

ruec

kii

Zin

ket

al.,

1991

pLE

16pL

B10

Lb.

delb

.bu

lgar

icus

7.6

Cm

rT

crY

esel

pL

acto

baci

llus

spp.

89C

hagn

aud

etal

.,19

92

pSS1

pLB

B1

Lb.

delb

.ssp

.bu

lgar

icus

7C

mr

Em

rT

crN

oel

pL

.lac

tis

Azc

arat

e-Pe

rila

ndR

aya,

2002

Lb.

john

soni

ipN

42+

pJD

C9

pN42

Lb.

delb

.lact

isN

AE

mr

Yes

NA

L.l

acti

sB

ourn

ique

leta

l.,20

02(C

onti

nued

onne

xtpa

ge)

182

Cri

tical

Rev

iew

s in

Bio

tech

nolo

gy D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y Se

rial

s U

nit -

Lib

rary

on

10/2

1/13

For

pers

onal

use

onl

y.

pLH

RpL

J1L

b.he

lvet

icus

8.5

Apr

Em

rY

esel

pL

b.he

lvet

icus

Has

hiba

etal

.,19

90pB

G10

pLJ1

Lb.

helv

etic

us6.

0β

-gal

Yes

elp

Lb.

helv

etic

usH

ashi

baet

al.,

1992

pCP5

3dpC

P53

Lb.

helv

etic

us4.

7T

crN

oel

pL

b.he

lvet

icus

Yam

amot

oan

dTa

kano

,199

6L

b.ca

sei

pPV

751

p1L

b.ac

idop

hilu

sN

AT

crY

esel

pL

b.ac

idop

hilu

sD

amia

niet

al.,

1987

pPV

754

p3L

b.ac

idop

hilu

sN

AT

crY

esel

pL

b.ac

idop

hilu

spT

RK

13pP

M4

Lb.

acid

ophi

lus

12.5

Cm

rN

AN

AL

b.ac

idop

hilu

sL

ucha

nsky

etal

.,19

88pT

RK

159

pPM

4L

b.ac

idop

hilu

s10

.3C

mr

Em

rT

crY

esN

AL

b.ac

idop

hilu

sM

uria

naan

dK

laen

ham

mer

,199

1

pUL

A10

5EpL

A10

5L

b.ac

idop

hilu

s7.

8A

prE

mr

Yes

elp

Lb.

acid

ophi

lus

Kan

atan

ieta

l.,19

92L

b.ca

sei

pLA

106P

Vem

pLA

106

Lb.

acid

ophi

lus

3.6

Em

rN

oel

pL

b.ac

idop

hilu

s.Sa

noet

al.,

1997

Lb.

case

iE

.col

ipR

V56

6pR

V50

0L

b.sa

kei

7.3

Em

rA

prY

esel

pL

b.sa

kei

Alp

erte

tal.,

2003

Lb.

plan

taru

mL

b.cu

rvat

usL

b.ca

sei

pLZ

15pL

Z15

Lb.

case

i27

lac

NA

elp

Lb.

case

iC

hass

yan

dFl

icki

nger

,198

7L

.lac

tis

Hem

eet

al.,

1994

pAZ

20pN

CD

O15

1L

b.ca

sei

8.3

Apr

Cm

rY

esel

pL

b.de

lbru

ecki

iZ

ink

etal

.,19

91

C:C

arno

bact

eriu

m;c

onjm

ob:c

onju

gativ

em

obili

zatio

n;m

ob:m

obili

zatio

n;pp

f:pr

otop

last

fusi

on.

183

Cri

tical

Rev

iew

s in

Bio

tech

nolo

gy D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y Se

rial

s U

nit -

Lib

rary

on

10/2

1/13

For

pers

onal

use

onl

y.

as selection markers; ampicillin resistance isused in shuttle vectors for selection in E. coli.

Food-grade selection markers have alsobeen developed for LAB used in food appli-cations. Xylose metabolism, not widespreadin Lactobacillus species, has been used as aselective phenotype in pLP3537-xyl (Posnoet al., 1991b). pBG10 carries β-galactosidasefrom Lb. bulgaricus, a marker that is usefulin bacteria that do not naturally metabolizelactose (Hashiba et al., 1992).

4.4. Vectors Derived fromStreptococcus thermophilusCryptic Plasmids

The lack of plasmids in S. thermophilushas impeded the development of genetic toolsbased on endogenous replicons. The first vec-tors constructed were shuttle vectors derivedfrom S. thermophilus RC replicons pA2 (2kb) and pA33 (6.9 kb) cloned in pVA891and suffered from segregational instability(Mercenier et al., 1989).

More efficient shuttle vectors have beendeveloped since, based on endogenous RCreplicons and exhibiting a host-range limitedto S. thermophilus and E. coli. The pMEUseries of shuttle vectors contain the pER8replicon, the ori of E. coli vectors pUC18/19and antibiotic resistance genes (Emr, Cmr

and Apr) (Solaiman and Somkuti, 1993) (Ta-ble 10). They transformed efficiently intoS. thermophilus and E. coli and exhibitedhigh segregational and structural stability.However, as they are larger, pMEU9 andpMEU10 were not as stable as their coun-terparts pMEU5-pMEU6. Nonetheless, thepMEU-vectors were deemed as effective asthe pUC- and pNZ-series of cloning vectors(Macrina et al., 1980; de Vos, 1987).

pER8-derived vectors were also usedto clone and express several heterologousgenes in S. thermophilus and in E. coli,namely cho and melC operons (Solaiman andSomkuti, 1995a, 1995b), cholesterol oxidase

and indole oxygenase genes (Somkuti et al.,1995; Solaiman and Somkuti, 1996), andmore recently, the pediocin operon (Coderreand Somkuti, 1999; Somkuti and Steinberg,2003). pMEU5a, combined with a fragmentof pER341 including the promoter sequenceof hsp16.4 and the promotorless green flu-orescent protein (gfp) gene from jelly fish,was used to construct pG341Pa and pG341Pb.Both vectors were able to transform S. ther-mophilus, L. lactis, and E. coli, and permit-ted the expression of gfp in all three hosts(Somkuti and Steinberg, 1999). The E. coli-S. thermophilus shuttle vector pPC418 de-rived from pER8 expressed and secreted ac-tive pediocin in S. thermophilus, L. lactis,and En. faecalis (Coderre and Somkuti, 1999;Somkuti and Steinberg, 2003).

Another S. thermophilus replicon(pND103) was used to construct threecloning vectors, of which pND913 (6.4 kb)was shown to be the most efficient. It couldreplicate in S. thermophilus and in L. lactis.The pND103-derived vectors were alsoshown to be segregationally and structurallystable in E. coli: after 30 generations, 95% ofclones still harbored the recombinant plasmid(Su et al., 2002). pSMQ172, another crypticendogenous plasmid, also demonstrated thecapacity to transform L. lactis, but with amuch lower copy number than when trans-forming S. thermophilus or E. coli (Turgeonand Moineau, 2001).

The latest vector reported (pHRM1) is de-rived from S. thermophilus pSt08 and carriesa gene for a small heat shock protein (shsp)from pSt04 (El Demerdash et al., 2003). Vec-tor pHRM1 (6.4 kb) uses shsp as a selectionmarker and can transform S. thermophilus.Transformants exhibited increased resistanceto incubation at 60◦C or pH 3.5 and were ableto grow at 52◦C. In addition, shsp was shownto be as efficient as an erythromycin selectivemarker. Its small size (0.65 kb) favors easycloning and shsp could serve as a food-grademarker. However, it still has to be determinedif it can function in other LAB.

184

Cri

tical

Rev

iew

s in

Bio

tech

nolo

gy D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y Se

rial

s U

nit -

Lib

rary

on

10/2

1/13

For

pers

onal

use

onl

y.

TAB

LE

10V

ecto

rsD

eriv

edfr

om

S.t

her

mo

ph

ilus

Cry

pti

cP

lasm

ids

and

Th

eir

Ch

arac

teri

stic

s

Vec

tor

Rep

licon

Ori

gin

Size

(kb)

Gen

etic

mar

ker

Shut

tleve

ctor

Tra

nsfe

rm

etho

dH

ost-

rang

eR

efer

ence

s

pTG

219

pA2

S.th

erm

ophi

lus

NA

Em

rN

AN

AN

AM

erce

nier

etal

.,19

89pM

EU

5apE

R8

S.th

erm

ophi

lus

5.7

Em

rA

prY

esel

pS.

ther

mop

hilu

sSo

laim

anan

dSo

mku

ti,19

93pM

EU

5bpE

R8

S.th

erm

ophi

lus

5.7

Em

rA

prY

esel

pS.

ther

mop

hilu

spM

EU

6apE

R8

S.th

erm

ophi

lus

5.7

Em

rA

prY

esel

pS.

ther

mop

hilu

spM

EU

6bpE

R8

S.th

erm

ophi

lus

5.7

Em

rA

prY

esel

pS.

ther

mop

hilu

spM

EU

9pE

R8

S.th

erm

ophi

lus

6.9

Em

rC

mr

Apr

Yes

elp

S.th

erm

ophi

lus

pME

U10

pER

8S.

ther

mop

hilu

s6.

9E

mr

Cm

rA

prY

esel

pS.

ther

mop

hilu

spE

U5x

ML

2201

a/b

pER

8S.

ther

mop

hilu

s8.

3E

mr

Apr

Yes

elp

S.th

erm

ophi

lus

Sola

iman

and

Som

kuti,

1995

bpE

U5a

ML

2201

apE

R8

S.th

erm

ophi

lus

8.3

Em

rA

prY

esel

pS.

ther

mop

hilu

spE

U5a

CH

2201

a/b

pER

8S.

ther

mop

hilu

s12

.0E

mr

Apr

Yes

elp

S.th

erm

ophi

lus

pER

82pE

R8

S.th

erm

ophi

lus

4.7

Em

rC

mr

No

elp

S.th

erm

ophi

lus

Som

kuti

etal

.,19

95pE

R82

PbpE

R8

S.th

erm

ophi

lus

4.8

Em

rC

mr

No

elp

S.th

erm

ophi

lus

pER

82Pb

IDpE

R8

S.th

erm

ophi

lus

8.8

Em

rC

mr

Apr

No

elp

S.th

erm

ophi

lus

Sola

iman

and

Som

kuti,

1996

pEU

5aID

2201

pER

8S.

ther

mop

hilu

s8.

4E

mr

Apr

No

elp

S.th

erm

ophi

lus

pG34

1Pa

pER

8S.

ther

mop

hilu

s6.

4E

mr

Apr

Yes

elp

S.th

erm

ophi

lus

Som

kuti

and

Stei

nber

g,19

99pG

341P

bpE

R8

S.th

erm

ophi

lus

6.4

Em

rA

prY

esel

pS.

ther

mop

hilu

spP

C41

8pE

R8

S.th

erm

ophi

lus

9.1

Em

rA

prY

esel

pE

.col

iC

oder

rean

dSo

mku

ti,19

99S.

ther

mop

hilu

sL

.lac

tis

En.

faec

alis

pSM

Q17

2ca

tpS

MQ

172

S.th

erm

ophi

lus

5.7

Cm

rN

oel

pS.

ther

mop

hilu

sT

urge

onan

dM

oine

au,2

001

S.sa

liva

rius

L.l

acti

sE

.col

ipN

D91

3pN

D10

3S.

ther

mop

hilu

s6.

4E

mr

Apr

Yes

elp

S.th

erm

ophi

lus

Suet

al.,

2002

L.l

acti

spN

D91

4pN

D10

3S.

ther

mop

hilu

s14

.3E

mr

Apr

Yes

elp

S.th

erm

ophi

lus

pND

915

pND

103

S.th

erm

ophi

lus

14.3

Em

rA

prY

esel

pS.

ther

mop

hilu

spH

RM

1pS

t08

S.th

erm

ophi

lus

6.4

shsp

No

elp

S.th

erm

ophi

lus

ElD

emer

dash

etal

.,20

03

185

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tical

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iew

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tech

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oade

d fr

om in

form

ahea

lthca

re.c

om b

y Se

rial

s U

nit -

Lib

rary

on

10/2

1/13

For

pers

onal

use

onl

y.

4.5. Vectors Derived fromLeuconostoc, Oenococcus,and Pediococcus Cryptic Plasmids

The situation of recombinant DNA tech-nology in Leuconostoc and Pediococcus iscomparable in both genera, which is why theycan be grouped together. Compared to otherLAB, few plasmids have been isolated andcharacterized in Leuconostoc and Pediococ-cus species; even fewer cloning vectors havebeen constructed from endogenous plasmids,resulting in a shortage of genetic engineeringtools (Table 11).

While several O. oeni (Ln. oenos) crypticplasmids, namely pOg32 (Brito et al., 1996),p4028 (Zuniga et al., 1996), and pRS1 (Alegreet al., 1999), have been studied and deemedsuitable for the development of narrow host-range cloning vectors, vectors based on thesereplicons have yet to be constructed. So far,only three Leuconostoc replicons have beentagged with antibiotic selection markers andused to derive cloning vectors such as pFR18(Biet et al., 1999), pTXL1 (Biet et al., 2002),and pCI411 (Coffey et al., 1994). Derived vec-tors cannot replicate in E. coli, except for shut-tle vectors pFBYC018E and pFBYC050E.However, pFBYC018E suffers from segrega-tional instability due to the large fragment ofE. coli DNA it carries: after cultivation for100 generations under non-selective condi-tions, only 3% of clones carried the recom-binant plasmid.

While most vectors are maintained in Leu-conostoc species, pFBYC50E was also main-tained in P. acidilactici and Lb. sakei. Leu-conostoc vector pCI431 based on pCI411showed a wide range of Gram-positive hoststrains including L. lactis, S. thermophilus, B.subtilis, and Lb. casei.

There are even less vectors derived fromendogenous pediococcal plasmids. Someplasmids originating from P. acidilactici andP. pentosaceus encode pediocin production,a bacteriocin that could serve as a food-grade selection marker for the construction

of cloning vectors (Motlagh et al., 1994).While the idea has been suggested, it hasnever been carried out: there are presentlyno vectors derived from pediococcal repliconsencoding pediocin production. This situationmay change, as pediocin becomes more easilyavailable for selection.

To our knowledge, RepA287-based vec-tors are the only cloning vectors derivedfrom a pediococcal cryptic plasmid pUCL287(Table 12). Two derivatives, pUCB813 andpUCB825, were able to transform and weremaintained in P. acidilactici, Lb. plantarum,Ln. mesenteroides subsp. mesenteroides, andEn. faecalis, but not Lactococcus spp.(Benachour et al., 1995). RepA287 can thusbe a good candidate for the further devel-opment of vectors for LAB except Lacto-coccus species. Since pUCL287 is a theta-replicating plasmid, one would expect it tobe segregationally stable. However, whenpUCB825 was grown under non-selectiveconditions, only 1.2% of clones harboredthe recombinant plasmid after 25 generationsand 0.1% after 56 generations, suggestinglow segregational stability (Benachour et al.,1997).

Many broad host-range vectors can beused to modify Leuconostoc and Pediococ-cus species, but the development of cloningvectors based on endogenous plasmids wouldstill be advantageous with regards to food-grade modification and self-cloning of indus-trial strains used in food applications.

4.6. Vectors Derived fromBifidobacterium Cryptic Plasmids

Ever since plasmids were first reported inthe genus, vectors of the general utility-typehave been constructed and transformed in var-ious Bifidobacterium spp. Most vectors areshuttle vectors, as they carry two origins ofreplication and two selective marker systemsfor Bifidobacterium species and E. coli (Table13).

186

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lthca

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om b

y Se

rial

s U

nit -

Lib

rary

on

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1/13

For

pers

onal

use

onl

y.

TAB

LE

11V

ecto

rsD

eriv

edfr

om

Leu

con

ost

oc

spp

.Cry

pti

cP

lasm

ids

and

Th

eir

Ch

arac

teri

stic

s

Vec

tor

Rep

licon

Ori

gin

Size

(kb)

Gen

etic

mar

ker

Shut

tleve

ctor

Tra

nsfe

rm

etho

dH

ost-

rang

eR

efer

ence

s

pFB

YC

018E

pFR

18L

n.m

esen

tero

ides

5.9

Apr

Em

rY

esel

pL

n.cr

emor

isB

iete

tal.,

1999

Ln.

mes

ente

roid

essu

bsp.

mes

ente

roid

esL

n.m

esen

tero

ides

subs

p.de

xtra

nicu

mL

b.sa

kei

pFB

YC

18E

pFR

18L

n.m

esen

tero

ides

3.5

Em

rN

oel

pL

n.cr

emor

isL

n.m

esen

tero

ides

subs

p.m

esen

tero

ides

Ln.

mes

ente

roid

essu

bsp.

dext

rani

cum

Lb.

sake

ipF

BY

C05

0EpT

XL

1L

n.m

esen

tero

ides

subs

p.m

esen

tero

ides

7.7

Apr

Em

rY

esel

pL

n.cr

emor

isB

iete

tal.,

2002

Ln.

mes

ente

roid

essu

bsp.

mes

ente

roid

esL

n.m

esen

tero

ides

subs

p.de

xtra

nicu

mP.

acid

ilac

tici

Lb.

sake

ipF

BY

C50

EpT

XL

1L

n.m

esen

tero

ides

subs

p.m

esen

tero

ides

5.2

Em

rN

oel

pL

n.cr

emor

is

Ln.

mes

ente

roid

essu

bsp.

mes

ente

roid

esL

n.m

esen

tero

ides

subs

p.de

xtra

nicu

mP.

acid

ilac

tici

Lb.

sake

ipC

I431

pCI4

11L

n.la

ctis

5.8

Cm

rN

oel

pL

n.m

esen

tero

ides

subs

p.m

esen

tero

ides

Cof

fey

etal

.,19

94

Ln.

para

mes

ente

roid

esL

.lac

tis

subs

p.la

ctis

S.th

erm

ophi

lus

B.s

ubti

lis

Lb.

case

i

187

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tical

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iew

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Bio

tech

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gy D

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oade

d fr

om in

form

ahea

lthca

re.c

om b

y Se

rial

s U

nit -

Lib

rary

on

10/2

1/13

For

pers

onal

use

onl

y.

TABLE 12Vectors Derived from Pediococcus (Tetragenococcus) Cryptic Plasmids and Their Characteristics

Size Genetic Shuttle TransferVector Replicon Origin (kb) marker vector method Host-range References

pUCB813 pUCL287 T. halophila 8.3 Emr No elp P. acidilactici Benachour et al.,1995Ln. mesenteroides

subsp. mesenteroidesLb. plantarumEn. faecalis

pUCB825 pUCL287 T. halophila 6.9 Emr No elp P. acidilactici Benachour et al.,1995Ln. mesenteroides

subsp. mesenteroidesLb. plantarumEn. faecalis

B. longum replicon pMB1 has beenused extensively to develop a family of E.coli-bifidobacteria shuttle vectors (Matteuzziet al., 1990; Missich et al., 1994; Rossi et al.,1996, 1998). Plasmid pRM2, an E. coli-B.longum shuttle vector, contains the pMB1 ori,the ColEl ori from E. coli, and a MCS in ad-dition to ampicillin resistance and spectino-mycin resistance that is originated from En.faecalis for selection in E. coli and Bifidobac-terium spp., respectively. Plasmid pRM2 canonly transform B. longum with very low effi-ciency, and was lost after 50 generations with-out selective pressure.

A new set of pMB1-derived shuttlevectors has been developed featuring ery-thromycin, spectinomycin, and chloram-phenicol resistance genes as selective markersfor Bifidobacterium spp. and ampicillin for se-lection in E. coli (Rossi et al., 1996, 1998).These vectors can transform various Bifi-dobacterium spp. pDG7 (a pMB1 fragmentcloned into a pBR322 derivative) was used toclone heterologous genes such as lipase fromPseudomonas fluorescens, α-amylase fromBacillus licheniformis, and cholesterol oxi-dase from Streptomyces spp. in five differentBifidobacterium species (Rossi et al., 1998),but none of the cloned genes were expressed inBifidobacterium spp. under the control of theirown promoters. The smallest pMB1 deriva-tive, pTRE3 (2.8 kb), consists only of the

pMB1 replicon in addition to a MCS and achloramphenicol resistance gene. In additionto transforming Bifidobacterium spp., pTRE3has excellent segregational stability (>95%cells still harbor the plasmid following culti-vation for 100 generations without selectivepressure). pTRE3 was unable to transform E.coli, supporting the argument that the pMB1replication functions do not work in this host.Only pMB1 derivatives containing a func-tional E. coli origin could transform E. coli(Rossi et al., 1996, 1998).

Recently, the segregational and structuralstabilities of pMB1 vectors were studied incontinuous fermentation of B. animalis with-out selective pressure (Gonzalez Vara et al.,2003). It was reported that vector maintenancein a host cell was heavily influenced by overallplasmid molecular weight, not only by the sizeof the inserted DNA fragment, as previouslysuggested (Corchero and Villaverde, 1997).

Besides pMB1, other B. longum crypticplasmids have been used to construct shut-tle vectors. pBLES100, derived from pTB6,was shown to transform two strains of B.longum, and was maintained fairly stably(Matsumura et al., 1997). B. animalis wastransformed by shuttle vectors pBKJ50F andpBKJ50R, derived from B. longum plasmidpKJ50 cloned into pBR322 (Park et al., 1999).Similar results were obtained with pBKJ36and pBES2, shuttle vectors obtained from the

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nit -

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rary

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For

pers

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use

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TABLE 13Vectors Derived from Bifdobacterium longum Cryptic Plasmids and Their Characteristics


Shuttlevector

Transfermethod Host-range References

pMR3 pMB1 B. longum 7.3 Cmr Apr Yes elp E. coli Matteuzzi et al.,1990

pDG7 pMB1 B. longum 7.3 Cmr Apr Yes elp E. coliB. animalis Argnani et al.,

1996pRM2 pMB1 B. longum 6.0 Spr Apr Yes elp B. longum Missich et al.,

1994E. coli

pDH7 pMB1 B. longum 6.6 Cmr Apr Yes elp E. coli Rossi et al., 1996pKG7 pMB1 B. longum 7.0 Cmr Apr Yes elp B. animalis

E. colipNC7 pMB1 B. longum 4.9 Cmr Apr No elp B. animalispDGE7 pMB1 B. longum 9.5 Cmr Apr Emr Yes elp B. animalis

E. colipLF5 pMB1 B. longum 5.7 Cmr Apr Yes elp B. animalis Rossi et al., 1998

B. bifidumB. infantisB. longumB. magnum

pCLJ15 pMB1 B. longum 5.9 Apr Emr Yes elp B. animalisB. bifidumB. infantisB. longumB. magnum

pSPEC1 pMB1 B. longum 5.9 Spr Apr Yes elp B. animalisB. bifidumB. infantisB. longumB. magnum

pTRE3 pMB1 B. longum 2.8 Cmr No elp B. animalis Rossi et al., 1998B. bifidumB. infantisB. longumB. magnum

pBLES100 pTB6 B. longum 9.1 Spr Yes elp B. longum Matsumura et al.,1997

pBKJ50F/R pJK50 B. longum 8.1 CmrApr Yes elp B. animalis Park et al., 1999pBKJ36F/R pKJ36 B. longum 6.8 CmrApr Yes elp B. animalis Park et al., 2000

B. infantispBES2 pMG1 B. longum 7.6 CmrApr Yes elp B. longum Park et al., 2003pLAV pMB1 B. longum 4.3 Cmr Yes elp B. animalis Gonzalez Vara

et al., 2003

cloning of B. longum plasmids into pBR322and pUC19, respectively (Park et al., 2000,2003). These vectors exhibit narrow host-range, transforming several species of bifi-

dobacteria, namely B. longum, B. animalis,and B. infantis. pBES2 was structurally andsegregationally stable as 90% of transfor-mants still carried it after 30 generations

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without selective pressure. This is not com-parable to the segregational stability ex-hibited by theta-replicating pTRE3, but isexpected from an RCR plasmid-derivedvector.

5. FOOD-GRADE CLONING VECTORS

Food-grade cloning vectors have been de-veloped to fulfill an industrial demand forGRAS recombinant products. Although anygenetic manipulation of an organism createsa GMO, food-grade genetic modifications, i.e.modifying an organism with its own DNA orwith DNA originating from GRAS organisms,might not be as ill-perceived as a non food-grade genetic modification. Moreover, self-cloning, i.e. the re-introduction of DNA froma host that is modified or from a closely re-lated strain of the same species, is excludedfrom the EU Directive on the contained use ofgenetically modified microorganisms (CEC-219, 1990) (de Vos, 1999). In this light, or-ganisms that are modified by self-cloning arenot considered GMOs and are considered safeand suitable for food applications.

Food-grade vectors (1) are constructedwith DNA of GRAS organisms (i.e., LABcryptic plasmid) and (2) do not contain antibi-otic resistance genes as markers. Food-gradeselective marker systems have been devised:marker genes originate from GRAS organ-isms (i.e., LAB) and are derived from nat-urally occurring phenotypes such as carbo-hydrate metabolism, resistance or immunityto bacteriocins, proteolytic activity, and DNAsynthesis (de Vos, 1999b).

LAB possess a variety of sugar fermenta-tion phenotypes that can be exploited as se-lectable markers on cloning vectors. Genesfor the metabolism of rare sugars, such as xy-lose, inulin, and melibiose, have been used asmarkers in the construction of cloning vectors(Posno et al., 1991b; Hols et al., 1994; Wankeret al., 1995; Boucher et al., 2002). Lac-tose metabolism, although quite widespread

among LAB, can still be used as a selec-tive marker in lactose-deficient strains or mu-tants. A set of cloning and expression vectorsfor self-cloning of L. lactis using lacF as aselection marker, the lactose-inducible lacApromotor and the lactococcal pSH71 repliconwas developed by Platteeuw et al. (1996), astrategy that had been carried out previously(MacCormick et al., 1995). Lactose comple-mentation markers were also developed forLactobacillus spp. by Takala et al. (2002),who constructed a vector containing lacGfrom Lb. casei that could restore the abilityof a lactose-deficient Lb. casei strain to growon lactose.

Further complementation marker systemshave been developed based on auxotrophies,generally mutations in genes essential forgrowth. One system was comprised of a vectorcarrying an ochre suppressor as a selectablemarker that could rescue a mutation in L.lactis’ purine genes for purine biosynthesis(Dickely et al., 1995). Another similar sys-tem was based on an amber suppressor, supD,encoding an altered tRNASer that could com-plement pyrimidine auxotrophs that had a mu-tated pyrF (Sorensen et al., 2000).

Recently, a spin off of complementa-tion marker systems was reported by Emondet al. (2001). The system involves two plas-mids: RepB−plasmid pCOM1 carries an ery-thromycin resistance gene, while RepB+ vec-tor pVEC1 harbors the DNA insert. SincepCOM1 can only replicate in L. lactis iftrans complemented by RepB, both plasmidshave to be present to transform a host celland enable its survival in the presence oferythromycin. The marker plasmid pCOM1can be removed effectively upon curing inantibiotic-free medium.

Selective markers based on genes fornisin resistance (nsr) or nisin immunity (nisI)have also been reported. The plasmid vectorspVS40, pFM011, and pFK012 containing nsrfrom L. lactis were constructed more than adecade ago and were shown to be quite effec-tive in the direct selection of transformants

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(von Wright et al., 1990; Froseth et al., 1991;Hugues and McKay, 1991). nisI was used asa selection marker in pLEB590, which wasshown to transform efficiently nisin-sensitivestrains of L. lactis and Lb. plantarum (Takalaand Saris, 2002). Similarly, it was suggestedthat the immunity gene to lactacin F, a bacteri-ocin produced by Lb. johnsonii, could serve asa suitable marker to develop new food-gradecloning vectors (Allison and Klaenhammer,1996). This marker is small and versatile andwas found to be applicable in various Lacto-bacillus spp.

Selection markers are of prime impor-tance in recombinant DNA technology. Re-search should definitely focus on the furtherdevelopment of food-grade selective markersystems that are suitable for large scale, in-dustrial applications.

6. INTEGRATIVE GENE CLONINGSYSTEMS

Cloning systems based on plasmid vec-tors, although quite versatile, suffer fromsegregational instability, a problem commonto both naturally occurring and recombinantplasmids. While selective pressure does over-come this problem, it is not always feasiblein an industrial setting or in continuous fer-mentations. Moreover, selective pressure issometimes not desirable in food applications.Ultimately, chromosomal integration of for-eign DNA fragments is the solution to pro-vide stability. Integrative gene cloning re-sults in the specific insertion of a geneticsequence into the chromosome of bacterialhosts. In addition to introduction and stablemaintenance of cloned genes without selec-tion markers, integration also permits disrup-tion or deletion of unwanted genes. Thereare different types of integration, namely (1)transposition via IS-elements, (2) site-specificrecombination using attP/integrase systems,and (3) homologous recombination via sui-cide or temperature-sensitive vectors.

6.1. Transposition

Transposition, performed by transposonsor by bacterial insertion sequences (IS), leadsto the random mutation of chromosomalgenes. ISS1 has been used in combination withnon-replicative vectors to perform integration(Romero and Klaenhammer, 1992). Maguinet al. (1996) used temperature-sensitive pG+

plasmids as a delivery system for lactococ-cal insertion sequence ISS1, resulting in themutagenesis of L. lactis by transposition.An IS1223-based integration vector was alsoconstructed by using pSA3: pTRK327 in-serted randomly into the chromosomes oftwo Lb. gasseri strains where no homologywas detected for the IS element (Walker andKlaenhammer, 1994). The use of transposonsshowed limited success and integration or mu-tagenesis vectors carrying such elements havenot been constructed (reviewed by Ariharaand Luchansky, 1994; Klaenhammer, 1995).

6.2. Site-Specific Recombination

Site-specific recombination systems use aphage integrase-mediated site-specific inser-tion in the host chromosome. Insertion at aspecific chromosomal location (attB) is me-diated by a small region of homology (e.g.<20 bp) on the phage (attP) and a phage-encoded integrase (int) (Klaenhammer, 1995).Integrases catalyze recombination betweensites located either in the same or in sepa-rate DNA molecules. Vectors based on thesite-specific integration apparatus of temper-ate bacteriophages of Lactococcus and Lac-tobacillus have been constructed. Lillehauget al. (1997) devised an integration vector sys-tem based on the site-specific integration ap-paratus of the temperate lactococcal bacterio-phage �LC3. The vector, carrying the �LC3integrase gene (int) and the phage attachmentsite (attP), enabled the integration of a DNAinsert in the �LC3 attB site of the L. lactischromosome.

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Similarly, Cruz Martın et al. (2000)proposed an integrase-based site-specificrecombination system, combined with a β-recombinase. β-recombinases catalyze the in-tramolecular deletions and inversions of DNAsequences, enabling the removal of unwantedDNA sequences left by the integration events.LAB modified by this system are food-gradeand suitable for industrial fermentations. Thevector carrying int-attP was transformed intoLb. casei and integrated into the attB site onthe host chromosome; subsequent expressionof the β-recombinase led to the site-specificremoval of non food-grade DNA (Cruz Martınet al., 2000).

Site-specific recombination is no longerexclusively reserved to bacteria possessing anattB site in their chromosomes. It is now possi-ble to clone attB by homologous integration inall the bacterial chromosomes tested, enablingsite-specific and stable insertion of heterolo-gous DNA fragments (Klaenhammer, 1995).

6.3. Homologous Recombination

Integration by homologous recombina-tion has been exploited to construct inte-gration vectors and carry out gene knock-out, amplification, replacement, and inser-tion. All systems for chromosomal integrationare based on non- or conditionally-replicatingplasmids. The first integration vectors forLAB were based on E. coli, B. subtilis, andS. aureus replicons because of their inabil-ity to replicate in LAB (de Vos and Simons,1994). Homologous DNA fragments carriedby such vectors integrated into chromosomaltarget DNA sequence, as vectors could notreplicate in the host cell.

The pG+series of integration vectors,based on a conditionally-replicating lacto-coccal replicon, were developed and becameimportant tools for LAB gene manipulation.The pG+ replicon has a temperature sensi-tive mode of replication: at permissive tem-

perature (i.e., 28◦C), it can be transformedand maintained in Gram-positive and Gram-negative hosts. However, upon elevation ofgrowth temperature (i.e., above 35◦C), RepAis inactivated, hindering replication and forc-ing the plasmid to integrate in the hostchromosome.

Several gene replacement strategies havebeen devised using this system. Henrich et al.(2002) reported the construction of two typesof vectors based on the pG+ replicon that con-tained sections of either the chromosomal leuoperon of L. lactis or the tel operon from thelactococcal sex factor. Genes cloned into theleu or tel sequences of these vectors were de-livered to the homologous regions of the chro-mosome or the sex factor through two singlecrossover events, resulting in the integrationof the recombinant plasmids and the excisionof the vector portions.

A similar approach was undertaken todelete a gene in L. lactis, by using theRepA+ temperature-sensitive helper plasmidpVE6007 (Maguin et al., 1992) and RepA−

vector pORI280 (Leenhouts et al., 1996),which bore a DNA fragment homologous tothe target sequence on the host chromosome.At the permissive temperature, both plasmidswere maintained in the host cell. Upon tem-perature elevation, repA was inactivated andpORI280, unable to replicate due to the ab-sence of RepA, integrated into the chromo-some, leading to the deletion of ltnA1 in L. lac-tis (Cotter et al., 2003). Other conditionally-replicating plasmids can be exploited to de-velop further integration vectors: pND324,a lactococcal thermosensitive plasmid, wasused to derive an integration vector (Duanet al., 1999).

The simplest form of chromosomal inte-gration occurs when a non-replicating plas-mid with a unique region of homology withthe host’s chromosome integrates via homol-ogous recombination. This integration eventis termed single crossover or Campbell-likeintegration. Single crossover recombinationsresult in the integration of the entire plasmid

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at a defined genetic locus. Genes can there-fore be inactivated or inserted at specificlocations.Several systems of integration rely-ing on single crossover events have been re-ported (Leenhouts et al., 1990; Simons et al.,1993; Kok and van den Burg, 2003; Scheir-linck et al., 1989; McIntyre and Harlander,1993). Generally, single crossover recombi-nation events are reversible and thus, unstable;selective pressure can be applied to maintainthe integrity of the cloned gene.

When two regions of homology arepresent between vector and chromosome,double crossover occurs (gene replacement).Depending on the nature of the sequences inbetween the homologous regions, the finalresult may be a chromosomal deletion or agene insertion. An integrative cloning vectorcontaining the Lb. bulgaricus β-galactosidasegene was constructed using a chromosomalfragment of Lb. acidophilus inserted in E.coli plasmid pBluescript II SK+. It permittedintegration of the gene in Lb. acidophilusby double crossover (Lin et al., 1996b). Thetransformant maintained the integrated genestably without selective pressure: it retainedβ-galactosidase activity after 30 transfers inthe absence of lactose. Gosalbes et al. (2000),by using an integrative vector based on thenon-replicative plasmid pRV300 that con-tained the 3′ end of lacG and the com-plete lacF gene, integrated two genes (E. coligusA gene and L. lactis ilvBN) into the chro-mosome of Lb. casei, which were success-fully expressed under the control of the lacpromoter.

7. CONTROLLED EXPRESSIONSYSTEMS

Controlled expression systems are impor-tant tools that allow genes of interest to beexpressed independently of the growth of theproduction host (Kuipers et al., 1997). Severalinducible expression systems used in LABhave been described and reviewed (Table 14)

(Kok, 1996; Kuipers et al., 1997; de Vos et al.,1997; de Vos, 1999a). Controlled gene expres-sion systems can be based on promoters con-trolled by sugars (lac promoter), salt (gadCpromoter), temperature upshift (tec phagepromoter), pH decrease (P170), or phage in-fection (�31 promoter). Not all systems ful-fill food-grade criteria, as inducing factorsshould be acceptable in foods: small inor-ganic molecules (salts), organic molecules(saccharides, fatty acids), or proteinaceouscompounds originating from LAB. Moreover,changes in growth conditions including pH,temperature, aeration, or even phage infec-tion would be an acceptable way to inducegene expression.

The most versatile system is the NICE sys-tem (nisin-controlled expression). Biosynthe-sis of the antimicrobial peptide nisin is con-trolled by a nisin gene cluster. The promoterof nisA is autoregulated by a two-componentregulatory system, consisting of the sensor ki-nase NisK and the response regulator NisR,which respond to extracellular nisin. The ad-dition of nisin induces co-transcription ofgenes of interest with nisA via membrane-associated NisK and transcriptional regula-tor NisR, resulting in the expression of genescloned downstream of nisA (Geis, 2003). deRuyter et al. (1996a) have developed a seriesof vectors and strains specifically suited forregulated gene expression in L. lactis, basedon transcriptional and translational fusions ofthe nisA promoter. The vectors were derivedfrom lactococcal replicon pSH71, carried theE. coli gusA and L. lactis pepN genes ei-ther translationally or transcriptionally fusedto the nisA promoter. Expression of thegenes was induced by subinhibitory levels ofnisin.

Similarly, Henrich et al. (2002) also re-ported the development of a set of plasmidvectors suited for nisin-inducible gene expres-sion in any strain of L. lactis. Food-graderecombinants of L. lactis were constructedthat had the nisRK genes and the nisin-inducible promoter PnisA fused to peptidase

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TABLE 14Characteristics of Inducible Expression Systems for Lactic Acid Bacteria

LABInductiontreatment

Inducibleelement

Inductionfactor References

L. lactis Lactose lacA or lacR promotor <10 Van Rooijen et al., 1992;Eaton et al., 1993;Payne et al., 1996

L. lactis Lactose LacR <10 Gosalbes et al., 2000L. lactis Lactose lacA/T 7 promotor <20 Wells et al., 1995;

Steidler et al., 1995S. thermophilus Lactose lacS-GalR ∼ 10 Mollet et al., 1993Lb. pentosus Xylose xylA promotor 60–80 Lokman et al., 1994,

1997L. lactis High temperature dnaJ promoter <4 Van Asseldonk et al.,

1993L. lactis High temperature tec-Rro12 >500 Nauta et al., 1996, 1997L. lactis Low pH, low temperature PA170 promotor 50–100 Israelsen et al., 1995L. lactis Low pH gadC-GadR >1000 Sanders et al., 1997,

1998a, 1998bL. lactis Aeration sodA promoter 2 Sanders et al., 1995L. lactis Absence of peptides prtP or prtM promotor <8 Marugg et al., 1995,

1996L. lactis Absence of tryptophan trpE promoter 100 Chopin et al., 1993L. lactis Mitomycin C repressor/operator �r1t 70 Nauta et al., 1996L. lactis �31infection �31promotor and ori >1000 O’Sullivan et al., 1996L. lactis Nisin nisA or nisF promotor >1000 Kuipers et al., 1995; de

Ruyter et al., 1996a,1996b; Henrich et al.,2002

Lb. helveticus NisinLb. casei Nisin Kleerebezem et al., 1997Ln. lactis Nisin

genes (pep). Expression of PnisA::pep fusionsafter induction with nisin was successfullydemonstrated.

The nisin system not only functions inlactococci, but also in other LAB, suchas Lactobacillus spp. and Leuconostoc spp.(Kleerebezem et al., 1997; Kuipers et al.,1998). Controlled expression systems thatrely on nisin for induction are of great in-terest for the overproduction of various pro-teins, as nisin is a food-grade additive and isthus safe to use in food applications. More-over, the system is suitable for industrial scaleapplications.

8. CONCLUSION

Many cryptic plasmids have been isolatedfrom LAB and used to construct cloning orintegration vectors, especially in the generaLactococcus and Lactobacillus and in S. ther-mophilus. There is still much work remain-ing for the Leuconostoc, Pediococcus, andBifidobacterium genera. Research prospectsare clear-cut: (1) additional cryptic plasmidsshould be isolated, especially from the generathat suffer from a deficiency in genetic en-gineering tools; (2) plasmids that have beenisolated, but have yet to be studied in depth,

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should be characterized and sequenced, theirminimal replicons determined as well as theirmodes of replication; (3) vectors that havebeen derived should be tested for their capaci-ties in cloning and expression of heterologousgenes in various hosts; (4) cloning vectorswith high segregational stability and high andlow copy number derivatives should also beconstructed to offer stable expression of tar-get genes; (5) narrow host-range vectors areimportant for the development of vaccine de-livery systems in vivo and should not be over-looked; (6) more importantly, food-grade vec-tors, which use food-grade marker systems,need to be developed to enable the legal ac-ceptation of genetically modified foods; andfinally, (7) food-grade modification strategies,such as integration and controlled expressionsystems, should also be considered for the en-gineering of industrial strains of LAB used infood production; these systems should be de-rived preferably from LAB cryptic plasmids.The development of efficient, food-grade, in-dustrially suitable genetic engineering tools tomodify LAB used for the production of fer-mented foods, for the overproduction of var-ious food ingredients and metabolites, or forthe delivery of oral vaccines is of prime impor-tance. These genetic manipulation tools havebeen awaited, as the food-grade modificationof industrially-used LAB is expected to revo-lutionize the market place.

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Cloning Vectors Based on Cryptic Plasmids Isolated from Lactic Acid Bacteria:Their Characteristics...

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