Plasmids from Food Lactic Acid Bacteria: Diversity, Similarity, and New Developments

Abstract

Plasmids are widely distributed in different sources of lactic acid bacteria (LAB) as self-replicating extrachromosomal genetic materials, and have received considerable attention due to their close relationship with many important functions as well as some industrially relevant characteristics of the LAB species. They are interesting with regard to the development of food-grade cloning vectors. This review summarizes new developments in the area of lactic acid bacteria plasmids and aims to provide up to date information that can be used in related future research.

1. Introduction

Lactic acid bacteria (LAB) are not a collective noun in classification, but are a heterogeneous group of Gram-positive, microaerophilic, non-sporulating and low G + C microorganisms which can ferment a range of carbohydrates to produce lactic acid [1,2]. LAB are commonly found in a variety of natural habitats, and are important industrial microbes that are used to produce a variety of industrial fermented food (dairy products, meat, wine, and silage etc.), macromolecules, enzymes, and metabolites. Some of them attract more attention from researchers as probiotics to maintain and regulate the human intestinal microflora [1,2].

Plasmid is a self-replication DNA molecule, which is non-attached to the cell chromosome and nuclear area DNA. The plasmids are not necessary genetic material for the survival of bacteria, but they often carry some special genes. They allow host strains to survive in a harsh environment and give the host strains greater competitiveness than other microorganisms, which are in the same environments [3]. A large number of plasmids were isolated and characterized from different sources of LAB as self-replicating extrachromosomal genetic materials [4]. Although most plasmids remain cryptic, some plasmids have been found that are associated with many important functions of LAB species, including (1) hydrolysis of proteins; (2) amino acid, citrate, and carbohydrate metabolism (e.g., lactose/galactose utilization, and oligopeptide transport); (3) production of bacteriocin, exopolysaccharide, and pigments; (4) resistance to antibiotic, bacteriophage, heavy metal, and other stress responses; and (5) DNA restriction-modification systems [4,5,6,7,8]. A variety of industrially relevant characteristics are encoded on the LAB plasmids, including the degradation of casein, acidification by lactic acid, and production of flavor compounds, which contribute to the desired flavor and texture of the fermentation product and to optimal growth of strains in milk [6,7,8,9,10]. These plasmids confer adaptive advantages improving the growth and behavior of their host cells. The latter are more suitable to be used in the food industry as starter cultures. Meanwhile, LAB plasmids are widely used for construction of expression systems of LAB, which is an effective means of enhancing the industrial applicability of LAB and minimizing their negative effects [7]. Identification, classification, construction, and application of LAB plasmids have attracted considerable scientific and technological attention. At present, there are over 400 LAB plasmids which have been isolated and studied [3,4,5,6,7,8,9,10]. This review presents information concerning the diversity of LAB plasmids, their replication mechanisms, structures, functions, and applications. The article will focus on the food LAB plasmids.

2. Diversity and Similarity of Plasmids from LAB

Plasmids are most commonly present in different LAB species, involved in 11 genera, including Bifidobacterium, Brevibacterium, Enterococcus, Lactobacillus, Lactococcus, Leuconostoc, Oenococcus, Pediococcus, Streptococcus, Tetragenococcus, and Weissella. LAB plasmids are extremely diverse in terms of size (0.87 kb to more than 250 kb), copy number (from 1 to more than 100 plasmids per cell), and phenotypes conferred to their hosts [5,6,8,9,10,11,12].

2.1. Plasmids of Genus Lactobacillus

Lactobacillus is the largest genus of the LAB group, with over 100 species in total [1,13]. They are widely distributed in variety of natural habitats including the oral, vaginal, and intestinal regions of many animals, fermented food, wine and other alcoholic beverages [1,2]. To date, 22 species of Lactobacillus have been identified that contain plasmids. Lactobacillus plasmids vary widely in size (from 1.81–242.96 kb), number (from 1–10 different plasmids in a single strain), and gene content [6,14,15].

Up to date, Lactobacillus plantarum contains the largest plasmids in the genus Lactobacillus. It is commonly isolated from plant material, and the gastrointestinal tract of animals [16,17]. This organism is used in the production of fermented foods, namely sauerkraut, kimchi and sourdough bread etc. [16]. It is also of interest as a probiotic to maintain and regulate the human intestinal microflora [18,19]. L. plantarum often harbors one or more natural plasmids of various sizes. L. plantarum strain 16 harbors the largest plasmid complement reported for this species to date, 10 plasmids (pLp16A–pLp16L), which range in size from 6.46–74.08 kb [20]. The strain 16 possesses broad-spectrum antifungal activities and has the potential to be used as a biopreservative agent to improve the shelf life of foods.

At present at least 56 plasmids from L. plantarum have been sequenced (Table S1, http://www.ncbi.nlm.nih.gov). Although most L. plantarum plasmids are cryptic, several plasmids encode some important properties, including antibiotic resistance [21,22,23,24], exopolysarccharide biosynthesis [20], chloride or potassium transport [20,25], bacteriophage resistance [26], as well as bacteriocin production [27].

Many small plasmids from L. plantarum replicate via the rolling-circle replication (RCR) mechanism, while some large plasmids, for example pMD5057, pWCFS103, and pST-III, are predicted to replicate via the theta mechanism [15,22,25,28,29,30,31,32,33]. Studies indicated that a rolling-circle plasmid pMRI 5.2 from a potentially probiotic strain L. plantarum BFE 5092, had two different plasmid-encoded replication initiation proteins from different replicon families, i.e., pMV158 and pC194 family [30]. This result suggests that the genes for these replication initiation proteins may have originated from different plasmids [30].

Megaplasmids of sizes ranging from 120–490 kb are found in Lactobacillus salivarius, Lactobacillus acidophilus, Lactobacillus hamster, Lactobacillus intestinalis, Lactobacillus kalixensis, Lactobacillus ingluviei, and Lactobacillus equi, including pMP118 (242.44 kb), pHN3 (242.96 kb), pWW1 (194.77 kb) [14,34,35,36,37,38] etc. Studies indicated that megaplasmid pMP118 from L. salivarius UCC118 was involved in rhamnose, sorbitol, and ribose utilization [35]. At the same time, it was speculated that pMP118 was likely to contribute to host colonization or probiotic properties [34].

A large number of vectors based on native plasmids from Lactobacillus strains have been developed in the past 20 years [6,7,39,40,41,42,43,44,45,46,47,48,49,50]. The most common expression system is the pSIP expression system, which is derived from Lactobacillus sakei, and is based on the regulatory system of antimicrobial peptides sakacin A or sakacin P and the quorum sensing mechanism [39,40]. The pSIP vectors have been used to express high amounts of heterologous proteins in lactobacilli, such as β-glucuronidase, amino peptidase, amylase, and β-galactosidases etc. [39,40,41,42]. However, due to the use of an erythromycin antibiotic resistance gene as selection marker, the potential of the pSIP system for food applications has been limited.

Therefore several new food-grade selection markers and corresponding expression systems have been developed [43,44]. Nguyen et al. developed a food-grade system for inducible gene expression in L. plantarum using an alanine racemase-encoding selection marker, which is a complementation selection marker, and can be used only with alr deletion mutants of L. plantarum [43]. A novel vector pM4aB for LAB was developed using a bile salt hydrolase gene from L. plantarum as a potential food-grade selection marker [44]. The vector pM4aB contains replicon of L. plantarum plasmid pM4, and it has expressed a catalase gene from L. sakei in L. paracasei [31,44]. Recently, a novel expression system for L. plantarum has been developed, which is based on the manganese starvation-inducible promoter from the specific manganese transporter of L. plantarum NC8 [45]. Its advantages are that no addition of an external inducing agent is required, and additionally, no further introduction of regulatory genes is necessary.

A series of Escherichia coli/Lactobacillus shuttle vectors was constructed, which was useful as a gene manipulation tool for LAB [46,47,48,49,50]. The shuttle vector pLES003 contains a replication origin from Lactobacillus brevis plasmid pLB925A03, a ColE1 origin, and the multi-cloning site from pUC19 [46]. pLES003 can replicate in cells of E. coli, L. brevis, L. plantarum, Lactobacillus helveticus and Enterococcus hirae [46]. The shuttle vector pGYC4α was constructed based on the RCR plasmid pYC2 from L. sakei BM5 isolated from kimchi [47]. pGYC4α expressed α-amylase from Bacillus licheniformis in E. coli, Lactococcus lactis MG1363, L. lactis MG1614, Leuconostoc citreum C16, and Leuconostoc mesenteroides C12 [48]. Replicons of theta-type-replicating plasmids pRCEID2.9 and pRCEID13.9 from Lactobacillus casei strain TISTR1341, have been used to develop E. coli/L. casei compatible shuttle vectors, which were stably maintained in different genetic backgrounds [49]. Recently, a novel plasmid pMC11 was isolated from L. casei MCJ, which is a starter culture for a traditional yoghurt product in China [50]. It contains two distinct replicons both of which replicate via a theta replication mechanism. The shuttle vectors pEL5.7 and pEL5.6 were constructed with two replicons of pMC11, respectively [50]. Meanwhile, the corresponding expression vectors pELX1 and pELX2 successfully expressed a green fluorescent protein in different Lactobacillus species [50]. These shuttle vectors provide efficient genetic tools for DNA cloning and heterologous gene expression in LAB.

2.2. Plasmids of Genus Lactococcus

The genus Lactococcus is widely used in food fermentation [51]. The member of Lactococcus genus L. lactis remains the best characterized Lactococcus species with regards to physiology and molecular genetics [52]. It is found in diverse environments, e.g., plant and animal habitats [53]. L. lactis is one of the most extensively used starter cultures in the LAB group, for production of various fermented dairy products, for example cheese, sour cream and fermented milks etc. [53,54].

L. lactis strains carry plenty of plasmids [9,10]. Most L. lactis strains usually contain 4–7 plasmids, which range in size from 0.87 kb to more than 80 kb [11,55]. The plasmid profile analysis of Lactococcus strains has shown that 150 dairy starter cultures (90 Lactococcus lactis subsp. cremoris, 30 Lactococcus lactis subsp. lactis, and 30 Lactococcuslactis subsp. lactis biovar diacetylactis) gave an average of seven plasmids per strain, ranging from 2–14. In the plant strains, the average was less two plasmids per strain, ranging from 0–4. The results showed that industrial dairy strains possessed a higher average plasmid complement than non-dairy strains. Meanwhile, the former strains contained a greater abundance of plasmids smaller than 10 kb [52].

Plasmids of L. lactis are involved in essential functions, e.g., bacteriocin production [56,57,58,59], cadmium resistance [60,61], antibiotic resistance [62], as well as in industrially relevant and significant characteristics, namely citrate utilization [63], casein utilization [55,64], lactose utilization [55,63,64,65], oligopeptide transport [63,65,66], orotate transport [67], cation transport [62,65,68], stress response and adaption [62,63,68,69], exopolysaccharide production [70], anti-phage restriction/modification systems [61,62,63,71,72,73,74], folate biosynthesis [65], proteolysis [65,75], and conjugal transfer [76].

Some plasmid-encoding characters can be used as selectable markers for the development of vectors, which are needed for cloning and to stably maintain the plasmid during bacterial growth, such as nisin resistance, cadmium resistance, zinc resistance, and thermo stability etc. [60,66,77,78]. Most of the lactococcal vectors rely on antibiotic resistance markers, for example erythromycin and chloramphenicol [7]. However, the use of antibiotics limits the application of vectors with antibiotic resistance markers, especially in the food industry and practical vaccines production [79]. Therefore some food-grade selectable marker systems are being developed, which are based on the complementation of auxotrophs and mutations in metabolic genes [80,81,82,83,84,85]. The use of bacteriocins as selecting agents and their cognate immunity genes as selectable marker genes have also been explored as food-grade cloning strategies [83,84,85]. Some bacteriocins have been used, e.g., nisin, lacticin 481 and lacticin 3147 [83,84,85]. Recently, bacteriocin lactococcin 972 (Lcn972) gene cluster has been used as a food-grade post-segregational killing system to stabilize recombinant plasmids in L. lactis in the absence of antibiotics [59]. Once the Lcn972 recombinant plasmid is inside the cells, it is maintained without any obvious deleterious consequences for the cells and antibiotic pressure is no longer needed, providing a useful tool for the development of safer and more sustainable biotechnological applications of L. lactis [59].

Studies on lactococcal plasmids have attracted considerable interest. At present, approximately 86 lactococcal plasmids have been completely sequenced, and 14 plasmids have been partially sequenced (Table S2, http://www.ncbi.nlm.nih.gov).

For a comparative analysis of replication proteins encoded by lactococcal plasmids, a phylogenetic analysis of 96 Rep proteins from lactococcal plasmids and 3 Rep proteins from pUB110, pA1, pCW7 was performed, and it categorized lactococcal plasmids into two groups based on their Rep proteins (Figure 1).

Most Rep proteins (87/99) belong to group I. Subgroup I-1: includes 73 Rep proteins from lactococcal plasmids. This is the most commonly detected replicon in lactococcal plasmids. Members of this subgroup exhibit a conserved Rep_3 domain (Pfam database, PF01051) and L. lactis RepB_C domain (Pfam database, PF06430).The latter domain is found in the C-terminal region of RepB proteins from L. lactis. Some members of this subgroup, pCI305 [86], pWVO2 [87], pW563 [88], and pCD4 [89], are shown experimentally to replicate via the theta replication, suggesting that these plasmids in subgroup I-1 may replicate via theta replication mechanism.

Subgroup I-2 encompasses two Rep proteins from pSK11-1 and pAF07, which contain the Rep_3 domain (Pfam database, PF01051). Comparing with members of subgroup I-1, this subgroup has not L. lactis RepB_C domain.

Subgroup I-3 contains Rep proteins from pCI2000 and pNP40. This subgroup shows a conserved RepA_N domain (Pfam database, PF06970) at the N-terminal region, which is noted to contain a helix-turn-helix motif. This is a remarkable feature of pLS32-type theta replication proteins [90]. Meanwhile, a 40-bp sequence directly repeated two and three quarter times is present on Rep proteins of pCI2000 and pNP40 [73,78,91]. Such repeats, also termed iterons, are common elements of the origin of replication (ori) for many theta replicating plasmids [92].

Subgroup I-4 contains 3 Rep proteins with a special DUF536 domain (Pfam database, PF04394) at C-terminal, which is found in several bacterial proteins of unknown function that may be involved in a theta-type replication mechanism. One of three plasmids, pUCL22, was shown to replicate via theta replication mechanism, suggesting that the replication mode of other plasmids in subgroup I-4 (pSK11-3 and pCIS4) may follow theta replication [93,94].

Subgroup I-5 consists of Rep proteins of pVF50, pQA554, pCIS7, pKF147A, pGdh442, pNCDO2118, and pLP712, containing a conserved Rep_3 domain (Pfam database, PF01051) and a conserved Phg_2220_c domain (Pfam database, PF09524). The latter domain is found exclusively in bacteriophage and in the bacterial prophage region, but the functions of this domain are unknown. It was confirmed that the region upstream of repA of pGdh442 contained motifs characteristic of the replication origins of lactococcal theta-type replicons [66]. Therefore this result suggests that members of subgroup I-5 replicate via theta replication. Interestingly, the plasmids pVF50, pQA554, pCIS7, and pLP712, are isolated from dairy niche, however, pGdh442, pKF147A, and pNCDO2118 originated from plant niche [55,64,66,68,69,95,96], suggesting there is extensive horizontal gene transfers among these plasmids.

Group II contains 9 Rep proteins from lactococcal plasmids, Rep protein of pA1 (pE194/pMV158 family of RCR plasmid), and Rep protein of pUB110 (pC194-type of RCR plasmid). All plasmids of this group replicate by means of rolling circle, which involves the synthesis of ssDNA intermediates. Most RCR lactococcal plasmids belong to the pE194/pMV158 family, except to pWC1 and pMN5, which are pC194-type of replicons [58,62,97,98,99,100,101,102,103].

RCR lactococcal plasmids exhibit a broad host range, being able to replicate a range of Gram-positive and Gram-negative bacteria, e.g., E. coli, which is the most widely studied prokaryotic model organism and used widely for genetic operation in the fields of biotechnology and microbiology [99,101,104]. A series of wide host range vectors (i.e., pCK-, pNZ-, pFIAV-series) were constructed from lactococcal RCR plasmids, and used for transformation LAB and other hosts [7,105].

However, RCR plasmids are incompatible with other RCR plasmids, so a single lactococcal strain never contains more than one RCR plasmid [99]. At the same time, RCR plasmids have been shown to be structurally and segregationally unstable due to their single-stranded mode of replication, which can produce accumulation of ssDNA intermediates [106]. RCR plasmids have a limited replicon size (10 kb), therefore cloning vectors based on the replicons of RCR plasmids can only be used to clone relatively small DNA fragments due to their structural and segregational instability with large inserts [106]. The above-mentioned factors limit the application of the derived vectors of RCR plasmids.

To solve vector stability problems, the focus has been on developing vectors based on theta-replicating plasmids [7]. In contrast to RCR plasmids, theta-type plasmids replicate by means of a double-stranded rather than a single-stranded replication intermediate, which results in better structural stability, allowing for the insertion of large heterologous DNA fragments. This property is useful for the construction of cloning vectors. Meanwhile, lactococcal theta derived vectors are compatible with endogenous RCR plasmids. Most theta-replicating lactococcal plasmids are members of a family of highly related, compatible replicons, so they can coexist in the same L. lactis strain [107]. It would be a great advantage to have a series of compatible vectors available.

The pathogen Lactococcus garvieae 21881 harbors five circular plasmids, namely pGL1 (4.54 kb), pGL2 (4.57 kb), pGL3 (12.95 kb), pGL4 (14.01 kb), and pGL5 (68.8 kb). The plasmid pGL2 replicates via the rolling circle mechanism, however, the other four plasmids are theta-replicating [108]. The plasmids pGL1, pGL2, and pGL5 encode putative proteins related with the synthesis, secretion, and immunity of bacteriocin. The plasmid pGL5 harbors genes (txn, orf5 and orf25) encoding proteins that could be considered putative virulence factors [108].

Figure 1. A Neighbor-joining tree was constructed with the ClustalW using Rep proteins from Lactococcus lactis plasmids. Bootstrap values for a total of 100 replicates are shown at the nodes of the tree. The plasmid pCW7 (a pT181 type RCR plasmid) was used as an out-group. Bar 1% sequence divergence.

Figure 1. A Neighbor-joining tree was constructed with the ClustalW using Rep proteins from Lactococcus lactis plasmids. Bootstrap values for a total of 100 replicates are shown at the nodes of the tree. The plasmid pCW7 (a pT181 type RCR plasmid) was used as an out-group. Bar 1% sequence divergence.

2.3. Plasmids of Genus Pediococcus

The genus Pediococcus possesses a homofermentative metabolism, and is commonly found in a variety of natural habitats, including the surface of plants and fruits, and fermented food (e.g., cured meat, raw sausages, and marinated fish etc.). It is applied industrially in food fermentations, and used for biotechnological processing and preservation of foods [109].

Pediococci species, mainly Pediococcus pentosaceus and Pediococcus acidilactici, harbor many different plasmids, ranging in size from 1.82–190 kb. Some plasmids encode a variety of traits, such as utilization of raffinose and sucrose [110,111], antibiotic resistance [112,113], as well as bacteriocin production and immunity [114,115,116,117,118,119].

Currently, there are a number of pediococcal plasmids which have been completely sequenced. pEOC01, a plasmid (11.661 kb) from P. acidilactici NCIMB 6990, encodes multidrug resistance (i.e., clindamycin, erythromycin, and streptomycin). The plasmid contains a streptomycin resistance gene aadE gene which holds 100% identity to an aadE gene found in Gram-negative bacterium Campylobacter jejuni plasmid. This observation is significant in that it provides evidence for recent horizontal transfer of streptomycin resistance from a lactic acid bacterium to a Gram-negative intestinal pathogen and as such infers a role for such plasmids for dissemination of antibiotic resistance genes possibly in the human gut [120].

P. pentosaceus can be isolated from a variety of plant materials and bacteria-ripened cheeses. This organism is used as an acid producing starter culture in the fermentation of some sausages, cucumbers, green beans, soy milk, and silage. It is also a typical component of the adventitious microflora of most cheese varieties during ripening. Some strains have been reported to contain several resident plasmids that render the bacterium capable of fermenting some sugars (raffinose, melibiose, and sucrose), as well as producing bacteriocins [114,117,118,121,122].

Pediocin PA-1/AcH, an anti-listerial class IIa bacteriocin, is produced primarily by several pediococcal strains, including P. acidilactici strains PAC1.0, H, E, F, M, K10, HA-6111-2, HA-5692-3, MM33; Pediococcus parvulus ATO34, ATO77 and P. pentosaceus FBB61 [119]. The genetic determinants of the biosynthesis of pediocin PA-1/AcH are located within a plasmid-borne operon cassette in all producing LAB strains examined to date, including pSRQ11 (9.4 kb), pSMB74 (8.9 kb), pATO77 (3.509 kb), pS34 (3.509 kb), pWHE92 (3.510 kb), and pMD136 (19.5 kb) etc. [116,117,118,119,123,124,125,126,127].

Pediocin PA-1/AcH is also synthesized by other LAB, except pediococcal strains, including L. plantarum WHE92 [128], L. plantarum DDEN 11007 [129], L. plantarum Acr2 and E. faecium Acr4 with plasmid-coding [130,131]. The plantaricin 423 from L. plantarum 423, is encoded in plasmid pPLA4. The operon structure of plantaricin 423 is similar to pediocin PA-1/AcH from P. acidilactici. The plaC and plaD genes are virtually identical to pedC and pedD of the pediocin PA-1 operon, as well as coaC and coaD of the coagulin operon [27]. The antilisterial bacteriocin coagulin, produced by Bacillus coagulans I₄, has an operon which shows high similarity with the pediocin operon [132]. The results show horizontal gene transfer among these plasmids from different species. It has been shown that the plasmids responsible for production in P. acidilactici H can be transferred intragenerically by conjugation [133]. Recently the flanking regions of the pediocin PA-1/AcH (pediocin PA-1) operon were characterized in order to evaluate mobile genetic elements in intergeneric and interspecific pediocin producing LAB [130]. Studies showed that ISLpl1, tyrosine recombinase and mobilization regions were found, which were known to be associated with transfer of genes linked to bacteriocin production, antibiotic resistance, and sugar utilization [130].

In several strains, the sizes and organization of the various pediocin-encoding plasmids are similar, including pATO77 from P. parvulus ATO77, pS34 from P. pentosaceus S34, and pWHE92 from L. plantarum WHE92 [119,123,125,126,127,128,134,135] (Figure 2). Structure, immunity and secretion system genes are linked together in the operons, and the promoter sequences are the same. Pediocin can be used as a selection marker in cloning vectors, therefore, plasmids that carry the genes for its production, can be used for development of food-grade cloning vectors [122].

Figure 2. Organization of the gene clusters of pediocin and related bacteriocins from different plasmids. (a) pS34 from P. pentosaceus (Pediococcus pentosaceus) S34 (NG_035883.1); (b) pATO77 from P. parvulus (Pediococcus parvulus) ATO77 (NG_035882.1); (c) pSMB74 from P. acidilactici (Pediococcus acidilactici) H (NC_004832.1); (d) pWHE92 from L. plantarum WHE92 (NG_035884.1); (e) pPLA4 from L. plantarum 423 (AF304384.2); (f) pI4 from B. coagulans I₄ (NG_035346.1); and (g) pEnt4 from E. faecium Acr4 (NG_041274.1). Open reading frames (ORFs) encoding the related proteins are marked with a different color. The number of amino acid residues within each encoded protein is shown above the corresponding ORF.

Figure 2. Organization of the gene clusters of pediocin and related bacteriocins from different plasmids. (a) pS34 from P. pentosaceus (Pediococcus pentosaceus) S34 (NG_035883.1); (b) pATO77 from P. parvulus (Pediococcus parvulus) ATO77 (NG_035882.1); (c) pSMB74 from P. acidilactici (Pediococcus acidilactici) H (NC_004832.1); (d) pWHE92 from L. plantarum WHE92 (NG_035884.1); (e) pPLA4 from L. plantarum 423 (AF304384.2); (f) pI4 from B. coagulans I₄ (NG_035346.1); and (g) pEnt4 from E. faecium Acr4 (NG_041274.1). Open reading frames (ORFs) encoding the related proteins are marked with a different color. The number of amino acid residues within each encoded protein is shown above the corresponding ORF.

Pediococcus claussenii is a common beer spoilage organism. P. claussenii ATCC BAA-344T contained eight plasmids (pPECL-1 to pPECL-8) that encode a variety of traits, including drug resistance, conjugation protein, the toxin-antitoxin (TA) system, and bacteriocin etc. [136]. From the point of view of beer spoilage, several genes are interesting, including the already-known beer spoilage-associated gene horA found on pPECL-8. The plasmids pPECL-6 and pPECL-7 are not circularized due to repetitive transposon regions found in both plasmids, making PCR-based gap closing very difficult. The overall G + C content of the genome is 36.8%, whereas that of the plasmids ranges from 34.9%–42.5%. The results show that there is a horizontal gene transfer in this bacterium.

2.4. Plasmids of Genus Enterococcus

Enterococci are Gram-positive cocci that can survive harsh conditions in nature. They can be found in soil, water, and plants. Some strains are used in the manufacture of foods, and improved the typical taste and flavor of many foods (such as cheeses and sausages) by their proteolytic and lipolytic activities [137,138]. However, some enterococci are opportunistic pathogens causing serious human and animal infections because of their virulence genes and resistance to antibiotics [138,139].

Enterococci harbor abundant plasmids, some of which are characterized encoding antibiotic resistance (e.g., erythromycin, tetracycline, gentamicin, teicoplanin, and macrolide-lincosamide-streptogramin B antibiotics) [12,140,141,142,143,144], bacteriocins (e.g., enterocin 1071A, enterocin 1071B, enterocin I, enterocin J, enterocin Q, bacteriocin 51, and lactococcin) [145,146,147,148,149] as well as virulence factors, namely aggregation substance protein [150,151], surface exclusion protein [152], extracellular surface protein [144,152], cell wall surface anchor family protein [149], cytolysin [144], toxin [153], and sex pheromone [154]. The abilities of enterococci to produce bacteriocins and to adapt to different environmental conditions are important characteristics for the food industry [138,155].

The plasmid pAR6 from E. faecium, contains a newly characterized heat shock promoter (P_hsp), which was found to regulate the expression of α-crystallin heat shock protein (hsp) in the plasmid [156]. P_hsp was used for construction of a novel Lactobacillus vector pAR1801, which can replicate in L. plantarum and L. lactis [156].

2.5. Plasmids of Streptococcus thermophilus

Streptococcus thermophilus is the only streptococcal species used in food fermentations, and has been widely used as a yogurt manufacturing starter culture to produce yogurt together with Lactobacillus delbrueckii subsp. bulgaricus for thousands of years throughout the world.

Like L. delbrueckii subsp. bulgaricus, S. thermophiles strains carry very few plasmids [7,157]. Most plasmids of S. thermophiles are cryptic, as no apparent phenotypic traits seem to be associated with their presence. Some plasmids have been found to encode small heat shock proteins, including pER341 [158], pCI65st [159], pND103 [160], pST04 and pER1-1 [161], pt38 [162], pER7, pER16, pER26, pER35, pER36, and pER41 [163,164], pK1002C2, and pK2007C6 [165].

Some researches indicated that these small heat shock proteins were induced by elevated temperatures and low pH, and expression of these proteins increases thermo- and acid resistance of the strains that carry heat shock proteins [161,164]. Therefore the promoter of heat shock protein gene hsp16.4 of pER341 is under investigation for potential use in temperature controlled expression of heterologous genesin LAB [158]. Genes for restriction-modification (R/M) systems have also been identified on plasmids pCI65st [159], pSt08, pSt0 [161], and pER35 [164].

Most S. thermophilus plasmids replicate via the RCR mechanism. Most RCR plasmids belong to the pC194 family, however pSMQ172 has been assigned to the pE194/pMV158 family [166]. The plasmids pSMQ-316 and pSMQ-312b replicate via the theta-replicating mode [167].

2.6. Plasmids of Genus Bifidobacterium

The genus Bifidobacterium shares some of the phenotypic traits of genuine LAB, but bifidobacteria are phylogenetically unrelated to other LAB, belonging to the phylum Actinomyces branch of bacteria, and have a unique fructose-6-phosphate phosphoketolase pathway of sugar fermentation [1,168,169]. Some Bifidobacterium strains naturally colonize the human gastrointestinal tract (GIT) and vagina, are beneficial to health by means of the production of short chain fatty acids and the exclusion of intestinal pathogens, as well as modulation of the immune function [170]. Some Bifidobacterium strains are used in the food industry because of their probiotic activities. Therefore researchers show considerable scientific and technological attention to this genus.

Plasmids have been detected in nine of the 31 species of Bifidobacterium, including Bifidobacterium asteroids [171,172], Bifidobacterium breve [173,174], Bifidobacterium bifidum [174], Bifidobacterium catenulatum [175,176], Bifidobacterium indicum [171,172], Bifidobacterium longum [177,178,179,180,181,182,183,184,185], Brevibacterium linens [186], Bifidobacterium pseudolongum subsp. globosum [187], and Bifidobacterium pseudocatenulatum [188]. Most plasmids are rolling circular plasmids, and their size varies from 1.847–10.22 kb. A few plasmids showed characters of theta replication plasmids [175,176,179,181]. The ori region of pSP02 was used to construct a series of first generation cloning vectors able to replicate in many bifidobacterial species [185].

2.7. Plasmids of Genus Oenococcus

The genus Oenococcus only contained two species Oenococcus oeni (formerly called Leuconostoc oenos), and Oenococcus kitaharae. As its name implies, O. oeni plays a pivotal role in the field of oenology. During the fermentation of wine, O. oeni is responsible for performing malolactic conversion, an important secondary fermentationin the production of wine [189,190,191].

Several small cryptic plasmids of O. oeni have been sequenced and described to date, i.e., pOM1, pLo13, p4028, pOg32, pRS1, pRS2, pRS3, pUBLO1, pUBLO5, and pUBLO6 [192,193,194,195,196,197]. The plasmids pOENI-1 (18.3 kb) and pOENI-1v2 (21.9 kb) are detected in three industrial starters (C9, C10, and C6) and a new isolate S11 [198]. Sequence analyses of plasmids indicate that they carry two genes possibly involved in wine adaptation encoding a predicted sulphite exporter (tau E) and a NADH: flavin oxidoreductase [198]. Due to the important role of O. oeni in the process of wine-making, various attempts have been made to develop cloning vectors and transformation protocols for O. oeni based on these plasmids [192,193,194,195,196,197,198,199,200]. At the beginning, small RCR plasmids pRS1, pRS2 and pRS3 from O. oeni were used for development of vectors [196,200]. However, RCR plasmids are less stable than theta-type-replicating plasmids [7]. Therefore, researchers begin to pay attention to the large theta-replicating plasmids from O. oeni, for example, pOENI-1 (18.3 kb), pOENI-1v2 (21.9 kb), and pRS7 (~20 kb) [198,201,202]. The plasmid pRS7 from O. oeni CT86, is a different plasmid to pOENI-1 and pOENI-1v2 but possesses a repA gene homologous to those of the plasmids of the “pOENI-1 family” [202]. Recently, a shuttle vector, pRS7Rep (6.1 kb), was constructed using the replication region of pRS7. The vector pRS7Rep can replicate in E. coli and some LAB, including P. acidilactici, L. plantarum, L. casei, L.citreum, and Enterococcus faecalis. Meanwhile, it contains single restriction sites useful for cloning purposes, and can improve characteristics of some LAB starter strains by means of expression of different exogenous proteins.

O. kitaharae was isolated from a composting distilled Shochu residue [203]. Comparing with O. oeni, the genome of O. kitaharae contains more genes which are involved in cellular defense, such as bacteriocins, antimicrobials, restriction-modification systems and a CRISPR locus [204]. One plasmid was found in O. kitaharae DSM 17330 [204].

3. Plasmid Replication Mechanisms

In LAB, the most common replication mechanisms are the Sigma and Theta modes of replication. The mode of replication of plasmids has an important impact on some characteristics of plasmid-derived vectors, namely host range, stability, and copy number [7].

3.1. Rolling-Circle Replicating Plasmids

The sigma mode of replication, is also named as RCR. RCR plasmids are usually small in size (less than 10 kb), have multiple copies, and are tightly organized. RCR plasmids are ubiquitous in Gram-positive bacteria, although they have been reported in many Gram-negative bacteria and archaea [205].

The common components of RCR plasmids are replication initiator (Rep) protein, the double strand origin (dso), and the single strand origin (sso). RCR generally involves two main stages, i.e., leading strand replication and lagging strand replication [92,205]. RCR type plasmids replication initiates with specific binding of the plasmid-encoded Rep protein to the cognate dso, which contains a specific protein binding sequence, and introduces a cut within the nick site of the dso. The leading strand is synthesized at the free 3′OH end at the nick, and produces single-stranded DNA replication intermediates [205,206]. The displaced single strand DNA (ssDNA) is then converted into double strand DNA (dsDNA) through the synthesis of the lagging strand by host proteins initiating at the sso [92,205].

Based on the sequence similarities in initiator proteins and dso, the RCR plasmids can be classified into several families, e.g., pT181, pE194/pMV158, pC194, and pSN2 family [205,206]. In general, plasmids of the RCR-type from Gram-positive bacteria often have a broad host range.

3.2. Theta-Type-Replicating Plasmids

At present six classes of theta replicons have been recognized [90,207,208,209]. Class A includes plasmids which encode a replication protein (Rep) and have a characteristic replication origin, designated oriA, and are independent of DNA polymerase I (PolI). The ori region generally consists of an AT-rich region which usually is followed by three and a half 22 bp direct repeats (called iterons) and two small inverted repeats which overlap the ribosome binding site of the rep gene and the -35 site of the rep promoter [9,87]. A number of plasmids from Gram-negative hosts are grouped in class A. Most LAB theta-type-replicating plasmids belong to class A, for example, pCI305 [86], pWVO2 [87], pW563 [88], pCD4 [89], and pUCL22 [93] etc.

Class B replicons do not encode a Rep protein and lack oriA. Their replications are initiated by processing of a transcript synthesized by the host RNA polymerase. Class C replicons, only two of which are known (the closely related plasmids pColE2 and pColE3), encode a replication protein but do not carry an oriA-like structure and require PolI for replication. Class D, this replication is dependent on a plasmid-encoded replication protein (Rep) but not on a DNA structure typical for origins of most Rep-dependent plasmids, and is initiated by DNA polymerase I (PolI). pAMβl from E. faecalis is recognized as a prototype for class D. Some LAB theta-type-replicating plasmids belong to class D, including the pIP501, pSM19035, and enterococcal plasmid pEF1 [146,210].

The plasmid pLS20 from Bacillus subtilis is representative of class E. The plasmid pLS20 shows no similarity with other known plasmid replicons.The structural organization of the pLS20 minimal replicon is entirely different from that of typical rolling circle plasmids from Gram-positive bacteria. The pLS20 minireplicons replicate in polA5 and recA4 B. subtilis strains. At same time, pLS20 is different from Bacillus natto pLS32 in that the former does not encode a Rep protein [90,209]. Taken together, these results strongly suggest that pLS20 belongs to a new class of theta replicons [209].

Class F, the plasmid pLS32 from B. natto was identified as a new family of Gram-positive theta replicons [90]. The family replicons do not possess an AT-rich region, and have a replication initiation protein (RepN), which contains a helix-turn-helix motif. Several iterons are present on RepN proteins, and identified as the origin of replication [73,78,91]. This replication region is structurally dissimilar to those of class A, but has been shown to be DNA polymerase I independent [90]. This family, based on structural organization and homology at the nucleotide and amino acid levels, includes a number of other plasmids of diverse origins, such as Lactobacillus plasmids pLJ1 [211], the enterococcal plasmids pAD1 [212,213], pCF10 [214], and pPD1 [215], as well as the lactococcal plasmids pNP40 [73] and pCI2000 [91].

DNA synthesis of theta plasmids can be bidirectional, may initiate from multiple origins; and produces double-stranded DNA replication intermediates, which result in better structural stability [92]. Therefore theta plasmids have a high segregational stability, and can incorporate large DNA fragments as cloning vectors [7].

4. Mobility of LAB Plasmids

Plasmids can be transmitted from one bacterium to another (even of another species) via three main mechanisms, i.e., transformation, transduction, and conjugation [216]. Conjugation is a key mechanism for horizontal gene transfer in bacteria [216]. In contrast to conjugation, transformation is a slower process as it requires the existence of free DNA and the achievement of a competent cellular state [217].

The transmissible plasmids can be classified according to their transfer machinery and mobilization ability, i.e., conjugative plasmids and mobilizable plasmids [216,218]. Conjugative (self-transmissible) plasmids contain a self-sufficient conjugative transfer system, which codify all the functions required for their HGT (Horizontal Gene Transfer) [219]. However, mobilizable plasmids carry only a mobilization region (mob) encoding specific relaxase and its cognate oriT, are incapable of initiating conjugation, and can be transmissible only in the presence of additional conjugative functions provided by either the recipient chromosome or by other auxiliary (alsotermed “helper”) plasmid [220,221]. Therefore conjugative plasmids tend to be large (˃30 kb) with low copy number, while mobilizable plasmids are small (˂15 kb) and have high copy number [222]. The transfer efficiency of mobilizable plasmids is usually lower than that of conjugative plasmids. However, mobilizable plasmids are more frequently found in nature. Therefore mobilizable plasmids have a tremendous impact in horizontal gene transfer in nature [216,221].

Relaxase is an essential ingredient of the MOB machinery, in both conjugative and mobilizable plasmids. It is a key protein that recognizes the origin of transfer (oriT), and then initiates and terminates conjugative DNA processing [216]. Small mobilizable plasmids were classified in four main families or super families according to the similarity of their relaxases and the phylogenetic relationships among them [216]. Later the conjugative plasmids were added to this classification system, transmissible plasmids were grouped into six MOB families, i.e., MOB_F, MOB_H, MOB_Q, MOB_C, MOB_P, and MOB_V [218,222]. The new additional MOB_F and the MOB_H families are present predominantly in large conjugative plasmids [218]. Most MOB_F plasmids are more than 60 kb, and a few of MOB_F plasmids are 20–60 kb. All MOB_H plasmids are more than 60 kb. The MOB_P and MOB_Q plasmids are uniformly distributed in plasmids of all size ranges. MOB_C is present inmid-sized plasmids (5–60 kb).

Over 50% of MOB_V plasmids are less than 5 kb. Most RCR plasmids of Firmicutes belong to the MOB_V family. The Mob relaxase from the streptococcal plasmid pMV158 is the representative of the family [220,222]. A lot of relaxases from LAB plasmids are identified in that they belong to the MOB_V family, including p141, pAMalpha1, pBM02, pCD034-1, pCD034-2, pF8801, pG6301, pGL2, pK214, pLA106, pLAB1000, pLAC1, pLB4, pLB925A02, pLC88, pM4, pMBLR00, pMRI5.2, pPB1, pPLA4, pS86, pSD11, pSMA23, pTXW, pWCZ, and pYSI8 etc. [27,30,31,32,108,220,223,224,225,226]. Most plasmids are small size (˂15 kb), except pPLA4 (8.135 kb), pAMalpha1 (9.759 kb), and pK214 (29.871 kb).

Most plasmids from B. longum are classified into the MOB_Q family, e.g., pDOJH10L and pDOJH10S from B. longum DJO10A [181]; pKJ36 and pKJ50 from B. longum KJ [177]; pBLO1 from B. longum NCC2705 [178]; pNAC2 from B. longum RW041 [179]; p6043A and p6043B from B. longum DPC6043; pMG1 and pTB6 [180]. All of plasmids are less than 5 kb and replicate by means of rolling-circle replication.

5. Conclusions

In the past 20 years, LAB plasmid biology has become an important area of LAB researches since plasmids associated with many important functions and some industrially relevant characteristics of LAB species; and LAB plasmids may be used to construct new vectors which can change the characters of hosts and accelerate the industrial and biotechnological applications of hosts. At present, a large number of new LAB plasmids have been isolated, characterized, and reconstructed. A series of wide host range vectors have been developed, based on LAB plasmids, and used for transformation of LAB and other hosts. A lot of heterologous proteins have been expressed in LAB by means of LAB plasmid derived vectors. LAB are generally regarded as safe (GRAS) microorganisms, therefore they are used as a delivery vector for therapeutic proteins and antigens, and to develop effective vaccines against potential pathogens and their evolving antibiotic resistance trends. All of these advancements will accelerate the developments of LAB plasmids and LAB.