1. INTRODUCTION
Lactobacillus, one of the lactic acid bacteria, is a natural inhabitant in the gastrointestinal tract of man and animals (Conway et al., 1987) and an important bacterium in the food industries of fermented dairy, meat, and vegetable, and also in animal feed (Chassy, 1985; Lee, 1996).
In addition, it is believed to offer some benefits as probiotics in human health, such as detoxifying carcinogens, enhancing immune response, metabolizing cholesterol, etc. (Bronzetti, 1995; Fernandes et al., 1987; Gilliland et al., 1985; O’Sullivan et al., 1992; Perdigon et al., 1986; Sandine, 1979).
Recently, people are becoming more interested in investigating this microorganism. In order to take further advantage of Lactobacillus, it is necessary to study its molecular biology and improve its specific beneficial characteristics. Generally, current recombinant DNA technology is the most efficient method to fulfill these purposes. Lactobacillus studies are relatively new in the field of molecular biology.
Although rudimentary knowledge of Lactobacillus genetics is not abundant, current molecular biology information and methodologies used for the study of other microorganisms facilitated the progress in the investigation of Lactobacillus at the molecular level. Research achievements include native plasmid detection and characterization, vectors construction, transformation system development, gene isolation, and strain modification. The goal of this review is to update the information on Lactobacillus plasmids/vectors that are essential tools in recombinant DNA technology. Readers who have further interest in this topic may refer to other related reviews (Gruss and Ehrlich, 1989; Pouwels and Leer, 1993)
II. PLASMID DISTRIBUTION
As taxonomic changes have been continuously introduced in Lactobacillus (Pot et al., 1994; Wood and Holzapfel, 1996) and are still under investigation, this review is therefore restricted to a rather general overview of the plasmids from 65 typical Lactobacillus species.
However, it must also be taken into account that some plasmids reported in this review may not be from typical lactobacilli. Among 65 valid taxonomic Lactobacillus species (Pot et al., 1994), at least 25 species (approximately 38% of the genus) contain native plasmids. Lactobacillus plasmids were first isolated from Lactobacillus casei (Chassy et al., 1976) and then from other Lactobacillus.
These 25 species of Lactobacillus are Lactobacillus acidophilus (Klaenhammer and Sutherland, 1980), Lactobacillus alimentarius (Lonner et al., 1990), Lactobacillus brevis (Olukoya et al., 1993; Soeding et al., 1993), Lactobacillus buchneri (Vogel et al., 1991), Lactobacillus casei (Chassy et al., 1976), Lactobacillus crispatus (Pouwels and Leer, 1993), Lactobacillus curvatus (Vogel et al., 1991), Lactobacillus delbrueckii (Vescovo et al., 198 l), Lactobacillus farciminis (Vogel et al., 1991), Lactobacillusfermenturn (Ishiwa and Iwata, 1980), Lactobacillusfructivoruns (Vescovo et al., 198 l), Lactobacillus gasseri (Tannock and Savage, 1987), Lactobacillus halotolerans (Vogel et al., 1991), Lactobacillus helveticus (Smiley and Fryder, 1978), Lactobacillus hilgardii (Josson et al., 1989), Lactobacillus johnsonii (Muriana and Klaenhammer, 1987), Luctobacillus mali (Vogel et al., 1991), Lactobacillus murinus (Tannock and Savage, 1987), Lactobacillus paracasei (Djordjevic et al., 1994), Lactobacillus pentosus (Posno et al., 1991a), Lactobacillus plantarum (Klaenhammer, 1984; Nes, 1984), Lactobacillus reuteri (Vescovo et al., 198l), Lactobacillus sake (Shay et al., 1988), Lactobacillus salivarius (Tannock and Savage, 1987), Lactobacillus sanfrancisco (Lonner et a]., 1990). Among them, Lactobacillus plasmids were detected mostly from L. plantarum and then L. acidophilus, L. casei, and L. helveticus. Lactobacillus appears to contain one or more (usually from 1 to 10) different plasmids, but L. plantarum LPC25 had 16 plasmids (Ruiz-Barba et al., 1991).
The known plasmids are between 1.2 kb and 169 kb from L. plantarum LL3 1 and LL2, respectively (Mayo et al., 1989). Although most Lactobacillus plasmids are less than 10 kb, some plasmids are larger than 100 kb, such as pPM68 (1 10 kb) from L. acidophilus (Muriana and Klaenhammer, 1987), a plasmid (150 kb) fromL. gasseri CNRZ222 (Roussel et al., 1993), and two plasmids (108 kb and 169 kb) from L. plantarum (Mayo et a]., 1989).
In earlier days, the method used to detect the presence of Lactobacillus plasmids was dye-buoyant density centrifugation (Chassy et a]., 1976), but recently electrophoresis on agarose gel is routinely utilized. Because the agarose gel can reveal the plasmids in a range between 1.7 and 60 kb, the bands of larger plasmids hidden by chromosomes might not be observed and thus the numbers may be underestimated. The plasmids found are usually in a circular form, but two linear plasmids from L. gasseri, CNRZ222 (150 kb) and IP102991 (50 kb), have been identified (Roussel et al., 1993).
Studies of DNA hybridization and DNA sequences have shown that certain homology existed in some plasmids from the same strain, species, genus, and even intergenus. For example, plasmids pl and p3 from the same strain L. acidophilus 168s showed a high DNA homology (Damiani et al., 1987). pLJl and pLH2, pLH3, pLH4, from same species, L. helveticus shared 85 to 98% homologous sequences (Pridmore et al., 1994).
Within the same genus, some small plasmids from 20 strains of L. curvatus and L. sake were homologous with pLC2 from L. cuwatus LTH683 (Vogel et al., 1991). Moreover, pLP1-like plasmids could be found from L. curvatus, L plantarum, L. sake, and nonLactobacillus bacteria such as Carnobacterium spp., and Leuconostoc mesenteroides (Bringel et al., 1989). These phenomena strongly suggest that there is considerable plasmid recombination among Lactobacillus plasmids and/or horizontal transfer of Lactobacillus plasmids occurs among intraspecies, interspecies, and with other genera, or these plasmids have evolved from common ancestors. Interestingly, two plasmids, pPM.52 and pPM68, from L. johnsonii VPII 1088 could be of chromosome integration (Muriana and Klaenhammer, 1987).
3. PLASMID STRUCTURE AND REPLICATION
Although many Lactobacillus plasmids have been found, very little is known about their structure. Only 12 of them have been completely DNA sequenced. They are pLC2 from L. curvatus (Klein et al., 1993), pLJl (Takiguchi et al., 1989), pLH2, pLH3, and pLH4 from L. helveticus (Pridmore et al., 1994), pLABl000 from L. hilgardii (Josson et al., 1990), p53-2 from L. pentosus (Leer et al., 1992), pAl (Vujcic and Topisirovic, 1993), pC3Oil (Skaugen, 1989), pLB4 (Bates and Gibert, 1989), pLPl (Bouiaet al., 1989), and ~8014-2 from L. plantarum (Skaugen, 1989) (Table 1). In addition, pl from L. acidophilus was partially sequenced (Damiani et al., 1987).
They varied from 1.9 to 5.7 kb in size. The G + C content of the plasmids from L. acidophilus, L. curvatus, L. helveticus, L. plantarum, and L. reuteri was in the range of 33 to 42% (Table 1, Vescovo et al., 1981). The comparison of DNA sequences and putatively translated amino acid sequences of Lactobacillus plasmids show that pAl, pC3Oi1, pLAB1000, pLB4, pLC2, pLP1, p353-2 and p8014-2 have highly homologous DNA sequences with plasmids pC194 (Horinouchi and Weisblum, 1982a), pE194 (Horinouchi and Weisblum, 1982b), pSN2 (Khan and Novick, 1982) and pT181 (Khan and Novick, 1983) from other Gram-positive bacteria.
pC194, pE 194, pSN2, and pT18 1 amplify themselves by a model of rolling circle replication (RCR) and produce single-stranded DNA (ssDNA) as intermediates (Gruss and Ehrlich, 1989).
For the RCR model, there are at least three plasmid-borne elements involved:
(1) a gene (rep) encoding a replication initiation protein (REP), (2) a plus origin, the target site of REP that first nicks the positive strand of plasmid DNA for replication initiation, and then terminates the replication when a leading strand (ssDNA) is synthesized, and (3) a minus origin, characterized by a long, imperfect inverted repeat, which enables the conversion of the ssDNA intermediate into the double-stranded DNA (dsDNA) molecule.
The DNA fragment containing plus origin and rep is called the replicon. In addition, some other Gram-positive bacterial plasmids contain extra elements such as a specific cointegration site (RS,) at which plasmid recombination occurs (Gruss and Ehrlich, 1989), a DNA region directing countertranscript-RNA (CT-RNA), and an open reading frame (OW) encoding a repressor that accounts for controlling the copy number of plasmid.
On the basis of similarities in the structure of REP and plus origin, the RCR plasmids could be classified into four groups: pC194, pE194, pT181, and pSN2, which are regarded as typical representatives (Gruss and Ehrlich, 1989, Pouwels and Leer, 1993, Seery et al., 1993).
Generally, the Lactobacillus plasmids hypothetically consist of one or more ORFs, plus origin, inverted repeat sequences, minus origin, RSA, and CT-RNA (Figure 1).
FIGURE 1. Comparison of Lactobacillus plasmids. (Symbols: black solid box, open reading fram (ORF); M, minus origin: P, puls origin (classic structure of rolling circle replication): R, RSA site: CTRNA, the region coding countertranscript RNA. Inverted repeat sequences are not shown [see context]) .
The map of pLAB loo0 contains all of these elements, whereas other plasmids do not. For instance, CT-RNA and RS,, located just before the ORF responsible for recombinatiodmobilization, were not deduced for most plasmids and classic RCR plus origin sequences could not be depicted on pLH2, pLH3, pLH4, and pLJ1.
Lactobacillus plasmid plus origins that are potential to have a hairpin structure could be classified into three groups: pC194 family, pE194 family (Figure 2), and others. Plasmids pAl, pLB4, pLC2, and pA2 from L. plantarum (Vujcic and Topisirovic, 1993) belong to pE194 family with a typical sequence of 5’-GTACTACGACC-3’.
Meanwhile, the pC194 family includes pC30i1, pLAI31OO0, pLP1, p353-2, and ~8014-2 with a conserved sequence of S-TC?TATC?TGATAC-3’, which is very similar to that of plus origin of single stranded phage 9x174 (5’-CCCCAACITG& TAT-3’). Because the bond between G and A is cut during $XI74 replication initiation, the same nucleotides G-A in all pC194 family plasmids are proposed as a nick site when REP protein recognizes plus origin and initiates replication. These two types of plus origins can coexist in the same Lactobacillus species.
For example, pAl, pLB4 (pE194 group), and pC3Oi1, pLP1, p8014-2 (pC194 group) were all isolated from L. plantarum. It suggests that (1) Lactobacillus provides an environment for both types of plasmids to replicate, or the factors required for the replication are very similar or identical. However, the plus origins of pLH2, pLH3, pLH4, and pLJl did not belong to either pC194 or pE194 family, and all these plasmids derived mainly from L. helveticus, which does not seem to contain pC194 or pE194 family plasmids. It might imply that (1) these plasmids may be duplicated by other replication mechanisms and (2) different Lactobacillus species use different mechanisms for plasmid replication. However, it is difficult to reach a valid conclusion illustrating the mechanism of Lactobacillus plasmid replication.
The sequences of Lactobacillus plasmids are supposed to contain at least three kinds of ORFs. Compared with the amino acid sequences with those of other ssDNA plasmids, the ORFs of Lactobacillus plasmids could encode proteins responsible for replication, copy number control, and recombination/mobilization.
In Figure 1, each plasmid possesses an ORF for REP synthesis. Within the same group of ssDNA plasmids, REPs showed an extensive homology (Gruss and Ehrlich, 1989). There was also a conserved region (N'-ETAKYEVKSAD-C') found in REPs from pC 194 family and $X 174. The Y (tyrosine residue) is regarded as an active site linking with DNA when replication initiates at the plus origin as seen in $X174 REP (Van Mansfeld et al., 1986).
The occurrence of the conserved region suggests that the mechanism of replication initiation among these plasmids could be very similar. However, the conserved sequence mentioned did not exist in pLH3, pLH4, and pLJl (Pridmore et al., 1994; Takiguchi et al., 1989).
This might infer that the replication of these plasmids utilizes new REPs and different replication mechanisms. The function of REP is of trans-acting, which means that a plasmid or a vector without rep can replicate in cells where REPs are available.
For example, vector pULP6 lacking rep did not replicate in Lactobacillus but replicated in the host with a rep-containing plasmid pLPl (Bringel et al., 1989).
The amino acid sequences of REP of pLH2 and pLC2 have shown over 80% homology (Pridmore et al., 1994), but the plus origin structure of pLC2 (pE194 group) was not found in pLH2. This suggests that REP could initiate replication at more than one type of plus origin.
The minimal sizes of some Lactobacillus plasmid replicons have been determined. The replicons of pLAB1000, pLC2, pLJ1, and of a 7-kb plasmid showed the sizes of 1.5 kb, 1.1 kb, 1.5 kb, and 1.6 kb, respectively (Cocconcelli et al., 1991a; Hashiba et al., 1992; Josson et al., 1990; Klein et al., 1993). These results are similar to the sizes of other bacterial plasmid replicons (Josson et al., 1990).
Nevertheless, pLUL63 1 from L. reuteri had a replicon larger than 2.4 kb in size (Ahme et al., 1992b). Two domains from Lactobacillus plasmids are supposed to control the copy number of plasmids. One of them encoding repressor protein could be derived from pAl (repA) and pLB4 (repA), whose amino acid sequences are analogous to each other and to other ssDNA plasmids such as PUB110 and pADB201, pE194, pLS 1 (Vujcic and Topisirovic, 1993; Bates and Gilbert, 1989). Another domain CT-RNA derives from pLABlOOO and p353-2 (Josson et al., 1990; Pouwels etal., 1994). In p353-2, two CT-RNAs with the sizes of 75 and 250 nucleotides were transcribed from the 5’ untranslated region of the rep gene. These two RNAs are presumably synthesized from the same promoter but terminated at two different sites in opposite direction to the rep gene.
The CT-RNA interacting with a loop near the 5’ untranslated region of the RNA of rep could cause another loop to be formed. This conformation change in the RNA of rep might affect the expression of rep, and in turn result in lowering plasmid replication. This mechanism, called transcription attenuation, of controlling the copy number of plasmids is similar to that of other ssDNA plasmids such as pC194, pE194, pT181, and pUBllO. The mob of pLAB 1000, and pAl (not complete), and the preA of pLB4 are acknowledged to encode recombinatiodmobilization proteins that theoretically are able to mobilize non-conjugative plasmids that fused at a specific cointegration site, RSA (Bates and Gilbert, 1989; Josson et al., 1990; Vujcic and Topisirovic, 1993) but there is no further evidence to support this statement. Certain homology existed in the N-terminal amino acid sequences of recombination/ mobilization proteins between pAl (53.8 to 72.5%), pLAB 1000 (59.9%), pLB4, and other ssDNA plasmids, pE194, pGl2, pMV158, pT18 I, and PUB 1 10 (Josson et al., 1990). RSA contains an inverted repeat and is located upstream of the ORF of recombinatiodmobilization and overlaps its Pribnow box.
The comparison of DNA sequence shows that the DNA sequence of Lactobacillus plasmids RSA (5’-(AG)TAA(AG)- TATAGTGGGTTATACTTTAC-3’) is highly similar (56.8 to 80%) to those of other ssDNA plasmids (Bates and Gilbert, 1989; Josson et al., 1990). Minus origins contain imperfect inverted repeats to form palindromic structures (Gruss and Ehrlich, 1989).
Although three groups of minus origins were designated: palA, paZT, and paw (Pouwels and Leer, 1993), these minus ongin structures could not be deduced from Lactobacillus plasmids already discussed. However, a highly conserved region (74 to 98% similarities in a region from 82 to 89 bp) was found from Lactobacillus plasmids, pC3Oi1, pLB4, pLC2, pLJ1, pLP1, p353-2, and p8014-2. These minus origins from Lactobacillus plasmids were found to be shorter than those of other ssDNA plasmids (130 to 220 bp) (Gruss and Ehrlich, 1989). This may form a new type of minus origin (Klein etal., 1993; Leer et al., 1992) and represent further evidence to support the fact that Lactobacillus plasmids exploit different mechanisms for replication. Like other ssDNA plasmids, the minus origin of Lactobacillus plasmid is also related to replication efficiency.
For example, a pLAB 1000 derivative plasmid containing no minus origin resulted in accumulation of ssDNA with little dsDNA (Josson etal., 1990). When the minus origin (palA) of pC 194 was cloned into a Lactobacillus vector pH2012 lacking the minus ongin, the palA inserted in both orientations increased the stability of recombinant vectors in L. casei (Shimizu-Kadota et al., 1991).
These results indicate that (1) the minus origin of pC 194 is not host-specific, (2) the minus origin of other Gram-positive bacteria can function in Lactobacillus as plus origin does, and (3) the minus origin that functions in LactobacilEus casei is not orientation dependent, which contradicts a theory that all minus origins are orientation dependent (Gruss and Ehrlich, 1989). In addition to the DNA regions discussed previously, other kinds of imperfect repeat sequences were found in noncoding regions of Lactobacillus plasmids: pC30i1, pLC2, pLH2, pLH3, pLH4, pLJ1, pLP1, and p8014-2 (Leer et al.; 1992, Pridmore et al., 1994). For example, a typical sequence of 5’-CCTGCCT(C)ACGGCGAGT-3’ was found twice in pLH2 and once in pLC2, pLH3, pLH4, and pLJ 1. Similar structures are also present in pC30i1, pLPl, and p8014-2. pC3Oil possessed 14 17 bp-imperfect directed repeats, pLPl 13 17 bp-direct repeats, p8014-2 1 17 bp-direct repeat and 1 18 bp-direct repeat. These sequences are very similar but not identical. The real function of the repeats is still unknown, although they may be related to incompatibility of the plasmids (Leer et al., 1992). The ssDNA intermediates detected from the replications of plasmids pAl, pLAB 1000, pLC2, p353-2, and vector pPSC22 (based on a 7-kb plasmid from L. plantarum) (Cocconcelli et al., 1991a; Josson et al., 1990; Klein et al., 1993; Leer et al., 1992; Vujcic and Topisirovic, 1993) strongly supports the fact that these small Lactobacillus plasmids belong to RCR plasmids. Although some small Lactobacillus plasmids have certain characteristics of RCR plasmids, such other Gram-positive bacterial plasmids, certain evidence discussed above to show that Lactobacillus plasmids might utilize other mechanisms than RCR for replication. Furthermore, the Lactobacillus plasmids discussed previously had sizes ranging from 1.9 to 5.7 kb, but certain numbers of Lactobacillus plasmids were larger than 6 kb. Whether these larger Lactobacillus plasmids replicate by RCR mechanism by theta-type replication, or other mechanisms are not known.