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Unité de Biochimie Bactérienne, UR 477, INRA, 78350 Jouy-en-Josas, France
Correspondence
Mireille Yvon
mireille.yvon{at}jouy.inra.fr
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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The GenBank/EMBL/DDBJ accession number for the pGdh442 plasmid sequence reported in this paper is AY849557.
Present address: Unité de Génétique des Génomes Bactériens, Institut Pasteur, 75724 Paris, France.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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; Salama et al., 1995; Teuber, 1995). Typically, Lactococcus strains possess an abundance of plasmid DNA. Many large L. lactis plasmids are self-transmissible or mobilizable by conjugative elements carried by the chromosome or plasmids of the host strain (Gasson et al., 1995). Through their natural transfer, these plasmids increase the probability of the gene moving between bacteria, and enable strains to acquire a wide variety of new genetic traits. They thus endow their hosts with many traits which offer a selective advantage to colonize specific biotopes. For example, the majority of dairy strains have adapted to growth in milk by acquiring plasmids that encode lactose catabolism (De Vos & Gasson, 1989), casein degradation (Kok, 1990), citrate utilization (Kempler & McKay, 1979) and oligopeptide transport (Yu et al., 1996). In addition, strains isolated from plant and animal environments, which contain a wide variety of cytotoxic compounds, have developed appropriate protection mechanisms (Putman et al., 2000), such as multidrug resistance, nisin resistance and heavy metal resistance (Dougherty et al., 1998; Liu et al., 1997; Perreten et al., 1997), and the genes for these mechanisms are often carried by plasmids.
Many of the properties carried by plasmids are of technological interest, and natural plasmids carrying both key industrial traits and a food-grade selectable marker have been the subject of considerable interest. In particular, heavy metal resistance is much sought after as a marker (Liu et al., 2002; O'Sullivan et al., 2001); this is because antibiotic resistance is an unsuitable marker for food micro-organisms due to the risk of promoting antibiotic resistance in the human intestinal microflora. The association of industrially significant properties and a food-grade selectable marker on a conjugative or mobilizable plasmid opens interesting perspectives for the construction of natural strains, and thus the development of novel and innovative products.
We have recently isolated a novel plasmid from a plant L. lactis strain (Tanous et al., 2005, 2006). This plasmid, named pGdh442, carries a transposon remnant containing genes which encode for two interesting properties applicable in the dairy industry. The first property is a glutamate dehydrogenase (GDH) activity which catalyses the reversible oxidative deamination of glutamate to 2-oxoglutarate and ammonia, mainly using NADP as the co-factor. This activity stimulates amino acid catabolism in LAB by supplying the 2-oxoglutarate required for amino acid transamination, which is the first step of amino acid conversion to aroma compounds (Rijnen et al., 2000; Tanous et al., 2002). Indeed, inactivation of the gdh gene in L. lactis has been shown to totally prevent the strain from degrading amino acids (Tanous et al., 2006). The second property is a cadmium/zinc resistance that can be used as a selectable marker. Moreover, we have demonstrated that this plasmid can be transferred by a mating procedure, indicating that it is either self-transmissible or mobilizable. However, pGdh442 appears to be unstable in a strain containing a protease/lactose plasmid, thus suggesting an incompatibility between the two plasmids (Tanous et al., 2006). It would be of great interest to the dairy industry to be able to transfer pGdh442 to strains capable of growing in milk, or to strains with a variety of amino acid catabolism profiles in order to diversify aroma production. Utilization of this plasmid for food applications requires its complete characterization. It is particularly necessary to identify its replication mode, as this might explain its incompatibility with other plasmids, the mechanism involved in its natural transfer, and other plasmid-encoded functions. It is also essential to ensure the absence of undesirable DNA sequences such as those for antibiotic-resistance genes, since certain lactococcal plasmids can harbour resistance to numerous antibiotics (Perreten et al., 1997).
In this paper we present the complete sequence of the pGdh442 plasmid of L. lactis NCDO1867, obtained by shotgun sequencing, and we discuss the presence of IS elements, different aspects of plasmid replication and mobilization, and the diverse putative biological functions encoded by the plasmid. Finally, we confirm its presence in other GDH-positive L. lactis strains.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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). Lactococcus strains were grown at 30 °C in M17 medium (Difco Laboratories; Terzaghi & Sandine, 1975) supplemented with 0.5 % (w/v) glucose (M17glu). pGdh442 was prepared from L. lactis TIL504 using the Large Construct kit (Qiagen), according to the manufacturer's instructions, with one minor modification: before treatment with NaOH, the cultures were incubated with 30 mg lysosyme ml–1 at 37 °C for 1 h. Standard recombinant DNA techniques and gel electrophoresis were performed, as described by Sambrook et al. (2001). The oligonucleotides used in this study were synthesized by Eurogentec and are listed in Table 1. L. lactis electrocompetent cells were prepared and transformed as described by Holo & Nes (1989). Plasmid DNA was prepared according to the method of O'Sullivan & Klaenhammer (1993). PCR amplification was carried out on a Perkin Elmer DNA thermal cycler 2400 using Taq polymerase (Qbiogen). For Southern blot analysis, plasmid DNA from TIL504 and TIL507 [transconjugant obtained by Tanous et al. (2006)] was digested with SacI, and a mixture of PCR fragments obtained with nahF/nahR, 18F/19R, 12F/13R, 10F/11R and 4F/6R primers was used to prepare a DNA probe with an ECL kit (Amersham). Hybridization was performed as recommended by the supplier. For Northern blot analysis, total RNA was extracted from cells grown in M17glu to OD600 0.7, as described by Chomczynski & Sacchi (1987) using TRIzol/chloroform/isoamyl alcohol reagent (Invitrogen). PCR fragments obtained with dldF/dldR, orf30F/orf30R, orf31F/orf31R, orf44F/orf45R and gdh13/gdh18 primers were used to prepare the DNA probes with an ECL kit. For RT-PCR, RNA samples were treated with DNase I (Ambion) as recommended by the manufacturer, and cDNA synthesis was generated from 5 µg RNA by using M-MLV reverse transcriptase (Invitrogen) according to the manufacturer's protocol. PCR amplification was carried out with primers dldF/dldR, orf30F/orf30R, orf31F/orf31R, orf44F/orf45R and gdh13/gdh18.
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Plasmid DNA sequencing, assembly and annotation.
pGdh442 was partially sequenced by GENOME express. Purified pGdh442 DNA was sheared by nebulization with a Hydroshear (GeneMachine). The sheared DNA was treated with T4 DNA polymerase (Appligene) to create blunt ends, and DNA fragments were size-fractionated by gel electrophoresis. After purification with the Nucleospin Extract kit (Macherey-Nagel), the DNA fragments were cloned into the dephosphorylated SmaI site of the pUC18 vector (Stratagene). The ligation mixture was used to transform the Escherichia coli ElectroTen-Blue strain (Stratagene). Clones were selected on SOB agar plates supplemented with 100 µg ampicillin ml–1. IPTG and X-Gal were added to final concentrations of 0.1 and 120 µg ml–1, respectively, for blue–white colour selection. Positive colonies were picked into a 96-well growth plate.
Plasmid DNA was extracted from these colonies, purified by an alkaline-lysis procedure (Utterback et al., 1995), and sequenced with a 3730XL automatic sequencer (Applied Biosystems) using the PRISM Ready Reaction Dye Deoxy Terminator Cycle Sequencing kit (Applied Biosystems). A total of 960 sequencing reactions with a mean edited read length of 552 bp were successful. Shotgun sequences were assembled using a Phrap assembler (Gordon et al., 1998) into three contigs totalling approximately 62.5 kb, with a minimum coverage of two times and for most of the sequence a coverage of four times. Gaps in the pGdh442 molecule were closed by primer walking, using oligonucleotides designed to contig ends and dye terminator chemistry. Final assembly and editing of the pGdh442 plasmid resulted in a circular molecule of 68 319 bp. The gene-finding algorithm Prokov (based on the Markov model; www.genostar.org) was used to identify ORFs. These ORFs were translated and searched against publicly available archives [a non-redundant protein database of GenBank proteins, SWISS-PROT and cluster of orthologous groups (COG)] using the BLAST-P algorithm (Altschul et al., 1997). IS elements were identified by comparison with the IS database (www-is.biotoul.fr) belonging to the CNRS.
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RESULTS AND DISCUSSION |
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) to facilitate its purification. This plasmid of 68 319 bp (Fig. 1) has a G+C content (35.1 %) close to that of the chromosomal DNA of L. lactis subsp. lactis IL1403 (35.4 %) (Bolotin et al., 2001), while 67 % of L. lactis plasmids have a G+C content lower than 34 % (Siezen et al., 2005). Detailed analysis of the sequence revealed 67 ORFs, which are listed in Table 2. Twenty ORFs encoded IS elements that may have mediated many transposition events and segment acquisition in pGdh442. In particular, we identified five gene clusters flanked by IS elements that could have resulted from gene-block transfers from L. lactis (ORFs 22–23, ORFs 55–64), Pediococcus (ORFs 29–31), Streptococcus (ORFs 43–53) and Lactobacillus (ORFs 10–12). Moreover, we had previously identified a Tn3-like transposon remnant (ORFs 36–40) that may originate from Streptococcus (Tanous et al., 2005). These observations suggest that gene transfers have occurred among Lactococcus species as well as between genera living in the same biotope, such as Streptococcus, Lactobacillus and Pediococcus, as previously reported for other L. lactis plasmids (Dougherty et al., 1998; O'Sullivan et al., 2001; Perreten et al., 1997). These four LAB genera are present in both plant and dairy niches (Lin et al., 1992; Mundt & Hammer, 1968; Mundt et al., 1969; Salama et al., 1995), probably because they can transfer from forage plants and meadow grasses to milk via cattle, and vice versa. For L. lactis subsp. lactis, plant-derived strains are thought to be the natural source of milk-derived strains (Salama et al., 1995).
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). The IS elements identified on pGdh442 belonged to three IS families, according to the classification of Mahillon and Chandler (1998). Firstly, five IS elements (ORFs 8–9, 13–14, 25–26, 41–42 and 66–67) exhibited characteristic features of the IS3 family, which is widespread in L. lactis. They contained two consecutive and partially overlapping reading frames that could generate a fusion protein by programmed translational frameshifting (Chandler & Fayet, 1993). Secondly, eight IS elements, four of them truncated (ORFs 4, 6, 18, 28, 32, 39, 54 and 65) belonged to the IS6 family, which is also widespread in L. lactis. Finally, one truncated ISLpl1 (ORF 19) from the IS30 family was identified on pGdh442, and has previously been reported in plasmids of Lactobacillus, Pediococcus and Oenococus (Nicoloff & Bringel, 2003). The large number of IS elements located on this plasmid was indicative of the overall plasticity of the pGdh442 structure.
Replication and maintenance system
The region involved in replication and the maintenance system in pGdh442 was made up of one replication origin (ori), one replication initiation protein [RepA (ORF 1)] and two ORFs (ORFs 2 and 3) encoding ParA and ParB, which are involved in the partition mechanism. This region, including the ori site, repA, parA and parB was closely identical (95 % identity) to the replication and maintenance region of the lactococcal protease/lactose pSK08 plasmid (accession no. AF300944) identified in L. lactis ML3. The region upstream of repA contains motifs characteristic of the replication origins of lactococcal theta-type replicons (Fig. 2) (Gravesen et al., 1995; Seegers et al., 1994; Foley et al., 1996). These include an AT-rich region that may be the recognition site for host-encoded functions involved in replication (Seegers et al., 1994), a 21 bp sequence directly repeated three and a half times, which interacts directly with the RepA protein to initiate replication, and two IR sequences, IRa and IRb, of 10 and 7 bp, respectively. These IRs overlap the putative –10 and –35 regions of the repA promoter and probably serve as a RepA binding site to regulate its own transcription (Foley et al., 1996). In addition, the pGdh442 replicon contained an 18 bp sequence directly repeated twice between the IR sequences and the DR sequence, while a similar 10 bp tandemly repeated DR sequence is located upstream of the AT-rich region in pCI305 (Foley et al., 1996). The presence of ParA and ParB suggested that pGdh442 is endowed with a putative active partition mechanism that counteracts plasmid loss at cell division, resulting in the marked stability of this plasmid, as reported in Gram-negative bacteria and in L. lactis pCI2000 (Kearney et al., 2000). This maintenance system is uncommon in L. lactis and has only been found on large plasmids (pSK08, pCI2000). It is generally encoded by low-copy-number plasmids, which is in agreement with the low copy number previously reported for pGdh442 in NCDO1867 (Tanous et al., 2005).
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). pLP712 is a protease/lactose plasmid isolated from L. lactis NCDO763 (a strain derived from NCDO712), which is very similar to pSK08 (Gasson, 1983). The identity of the ori site, RepA, ParA and ParB of pGdh442 with those of pSK08, and presumably with those of pLP712, explains the incompatibility between these two plasmids. Indeed, Foley et al. (1996) have demonstrated the incompatibility of two plasmids with the same ori site by providing three and a half copies of the 22 bp sequence of the pCI305 ori site in trans on a compatible vector, which leads to the loss of pCI305 from the cell population. In addition, partition-mediated incompatibility occurs when two plasmids introduced into the same cell carry identical centromere-like sites, which are indistinguishable from each other during the partitioning process, as previously reported in the Staphylococcus pSK1 plasmid (Simpson et al., 2003). The incompatibility of pGdh442 with pSK08-type plasmids will probably prevent the transfer of pGdh442 in most strains capable of growing in milk, since out of 15 tested prtP-containing strains, 12 contained the same replication and partition genes as pSK08, as revealed by PCR amplification with specific primers (Table 1
) derived from the ori, repA, parA and parB sequences of pSK08 (data not shown). This incompatibility could explain why the majority of strains exhibiting GDH activity carried by pGdh442 have been isolated from a plant or animal environment, while this activity has only rarely been detected in L. lactis strains with a history in dairy fermentation (Tanous et al., 2002). However, pGdh442 is probably compatible with the two plasmids that separately carry the genes responsible for lactose-fermenting ability and casein utilization in L. lactis SK11, because of the very low homology of their iterons (Siezen et al., 2005).
Plasmid mobilization
We had previously demonstrated that pGdh442 could be transferred from a L. lactis derivative strain of MG1363 (TIL504) as a donor strain to another L. lactis strain via a mating procedure (Tanous et al., 2006). However, no gene encoding conjugative function was present in pGdh442, indicating that pGdh442 is not self-transmissible but rather is mobilizable by the sex factor of MG1363 that encodes conjugative functions (Gasson et al., 1995), as reported elsewhere for many L. lactis plasmids (Lucey et al., 1993).
The mobilization of pGdh442 by the NCDO712 sex factor may occur either by conduction or via donation mechanisms (Steele & McKay, 1989). Conduction can be mediated by IS6 elements of a mobilizable plasmid, and leads to the formation of a cointegrate structure between the sex factor and the plasmid. This type of mechanism has been demonstrated for mobilization of the pLP712 plasmid by the NCDO712 sex factor (Gasson, 1990). The presence of numerous IS elements belonging to the IS6 family on pGdh442 and a transfer frequency (1x10–7 transconjugants per recipient) similar to that of pLP712 (Gasson et al., 1992) suggest a similar mechanism. However, attempts to recover cointegrate plasmids in transconjugants proved unsuccessful. Southern analysis of SacI digests of the plasmid in TIL504 and the transconjugant plasmid in TIL507 indicated that no significant amount of extra DNA was present in the latter: the sizes of the plasmid fragments were similar in both strains (Fig. 3). Also, the sizes of the fragments amplified by PCR with specific oligonucleotides of pGdh442 were similar in both strains (Fig. 4). However, plasmid recombination may have occurred during transfer, and cointegrate structures could have been resolved by Tn3 resolvase, as suggested by Gasson (1990).
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). pGdh442 is predicted to encode two putative mobilization proteins, MobDEI (136 aa) and MobC (122 aa), which share 94 and 90 % identity with those of pAH82 and pSK11A, respectively, and belong to the relaxase family. However, mobilization proteins previously found in L. lactis have generally been longer (around 550 aa), and the presence of a truncated ISLpL1 immediately downstream of the putative MobDEI end suggests that this protein may be truncated in pGdh442. In this case, the mobilization protein used during the transfer of pGdh442 could be supplied in trans by the sex factor of MG1363. The OriT site, which is reported to be the only cis-acting site required for DNA transfer of the mobilizable plasmid, was not identified upstream of the mob gene, as if has generally been reported for mobilizable plasmids (Lanka & Wilkins, 1995) or elsewhere on the plasmid. The absence of the oriT site and the transfer frequency (1x10–7 transconjugants per recipient) which was lower than the transfer frequency via donation, (>1x10–5) (van Kranenburg & de Vos, 1998; Wang & Macrina, 1995), strongly suggest that mobilization occurs via a conduction mechanism. In any case, pGdh442 is actually mobilized by the conjugative element of MG1363, indicating that this plasmid can be naturally transferred from strains containing a chromosomal sex factor element, or a self-transmissible plasmid carrying conjugative elements.
Plasmid-encoded functions
In addition to the transposases and proteins involved in plasmid replication and mobilization, putative biological functions could be assigned to 32 of the 67 ORFs of the plasmid. The majority of these genes displayed the highest degree of homology with genes located on other L. lactis plasmids, but some have never been found in L. lactis and share close identity with genes in Pediococcus, Lactobacillus and Streptococcus. The most interesting functions are presented below.
Oligopeptide transport system (Opp).
ORFs 55–60 shared 
98 % identity with the six genes encoding the Opp and the endopeptidase PepO of L. lactis. As in the chromosomal DNA of L. lactis subsp. lactis SSL135 (Tynkkynen et al., 1993), the six genes oppDFBCApepO form part of an operon in pGdh442. We identified two putative promoters immediately upstream of oppD (ORF60) and oppA (ORF56), and one unique terminator site downstream of pepO (ORF55). The sequence alignment of the region upstream of the opp operon with the sequenced upstream region of oppD (4.1 kb) in the chromosomal DNA of L. lactis subsp. cremoris MG1363 (AJOO6750) revealed 98.4 % identity. This region includes genes predicted to encode components of cation ATPases (ORFs 64 and 63), storage proteins related to the DNA binding protein stationary phase (ORF 62) and a transcriptional regulator FNR-like protein (ORF 61). ORFs 63, 62 and 61 constituted an operon named the flp operon (Gostick et al., 1999). The whole region including ORFs 55–64 in pGdh442 was flanked by two truncated ISS1-like elements (IS1297 and IS946 of the IS6 family), suggesting that this segment had been transferred from the chromosomal DNA of L. lactis as a gene block. The opp gene cluster flanked by iso-ISS1-like elements has already been found on the pLM3001, pJK430 and pSK11L plasmids of L. lactis subsp. lactis C2 and C20, and subsp. cremoris SK11, respectively (Siezen et al., 2005; Yu et al., 1996), further suggesting that the opp gene cluster may be associated with a transposon.
The Opp system plays an essential role in the nitrogen nutrition of L. lactis, especially in milk cultures. It allows the strain to transport and utilize oligopeptides released from casein degradation by the cell wall protease, and so leads to rapid milk coagulation (Tynkkynen et al., 1993). The presence of the Opp system on pGdh442 should improve the growth of host strains in milk when associated with a protease-positive strain, since pGdh442-containing strains are generally protease negative because of their incompatibility with protease/lactose plasmids. Besides these nutritional functions, the Opp system may also be involved in the regulation of several cellular processes, such as the sensing, through transport, of extracellular signalling molecules required for competence, as demonstrated in Bacillus subtilis (Lazazzera et al., 1999), and in the induction of conjugation, as in Enterococcus faecalis (Leonard et al., 1996).
Heavy metal or multidrug resistance.
We had previously reported the presence in pGdh442 of two genes, cadA and cadC (ORFs 37–38), that confer cadmium and zinc resistance on pGdh442 host strains (Tanous et al., 2005, 2006). These genes are identical to those found in another L. lactis plasmid (pAH82; O'Sullivan et al., 2001)
In addition to cadmium and zinc resistance genes, plasmid pGdh442 contains two ORFs (ORFs 31 and 30) predicted to encode a multicopper oxidase that might be involved in copper resistance, and a multidrug permease that might be involved in antibiotic resistance. However, these genes were not expected to be translated in L. lactis because of the absence of a Lactococcus RBS upstream of the initiator codons. Moreover, we did not detect transcription of these genes by Northern hybridization and RT-PCR against RNA extracted from a M17glu culture, while the gdh gene was expressed under the same conditions. Finally, functional analysis confirmed that these two genes did not result in copper resistance and antibiotic resistance in L. lactis. ORF31 encodes a 520 aa protein that shares 97 and 99 % identity with the multicopper oxidases of Pediococcus pentosaceus and Lactobacillus casei, respectively, but only 26 % identity with the copper oxidase (LcoC) of pND306 of L. lactis subsp. lactis LL58-1 that is responsible for the enhanced copper resistance of the strain (total inhibition at 4.5 mM) (Leelawatcharamanas, 1997; Liu et al., 2002). Although L. lactis NCDO1867 was moderately copper resistant (total growth inhibition at 1 mM CuSO4), the transfer of pGdh442 to other strains did not endow the host strains with enhanced copper resistance (total growth inhibition for all strains at 0.75 mM CuSO4). In fact, although genes encoding copper resistance have sometimes been reported on plasmids in L. lactis (Leelawatcharamanas, 1997; Siezen et al., 2005), they are generally chromosomally located. ORF30 encodes a protein of 457 aa that shares 98 and 99 % identity with major facilitator family permeases of P. pentosaceus and L. casei respectively, 35 % with a multidrug transporter in Listeria innocua (LmrP), but only 20 % with LmrP in L. lactis. In the last species, LmrP is associated with resistance to a broad spectrum of antibiotics belonging to the classes of macrolides, lincosamides, streptogramins and tetracycline (Perreten et al., 2001; Putman et al., 2001; van Veen et al., 1999), while the NCDO1867 strain containing pGdh442 was sensitive not only to erythromycin (macrolide) and tetracycline but also to chloramphenicol and streptomycin.
These two ORFs probably result from gene transfer from Pediococcus or L. casei, since they exhibit high levels of identity (97 and 98 %) with genes from those bacteria. These two ORFs are also next to each other in the chromosomes of Pediococcus and L. casei, in which they have the same G+C content (49.9 and 49.2 %) as in pGdh442. Moreover, these ORFs are flanked by two copies of functional IS946 (IRs at each extremity) in pGdh442, suggesting that they form part of a putative composite transposon.
D-ldh.
orf 20 is predicted to encode a D-LDH of 559 aa that is practically identical to the protein encoded by the dld gene carried by pSK11P in L. lactis SK11 (Siezen et al., 2005). The presence of a putative promoter and a short sequence homologous to L. lactis RBS upstream of the initiator codon suggested that the gene is functional in L. lactis. This putative FAD-dependent D-ldh differs from the nadh-dependent D-LDH of 320–350 aa which is generally found in L. lactis and other Gram-positive bacteria. It may result from a horizontal gene transfer from Gram-negative bacteria, because it displays 50 % identity with D-LDH of Gram-negative bacteria such as E. coli. Siezen et al. (2005) have suggested that it could function in D-lactate utilization under aerobic conditions, but we did not detect FAD-dependent D-LDH activity in pGdh442-containing strains (TIL442 and TIL507), and these strains were not capable of growing in M17 containing D-lactate as carbon source. In fact, Northern hybridization and RT-PCR of RNA extracted from an exponential M17glu culture of TIL442 indicated that the gene was not expressed under these conditions.
In addition to these three gene clusters predicted to encode interesting and well-known biological functions, pGdh442 contained three other gene clusters which to the best of our knowledge have never before been found in L. lactis and probably result from horizontal transfers. The first cluster included ORFs 10, 11 and 12, which shared the highest degree of homology (81–99 % identity) with transport proteins of Lactobacillus plantarum. However, as for ORFs 29, 30 and 31, we did not detect a putative L. lactis RBS upstream of the initiator codons. The second cluster was made up of three genes (ORFs 15, 16 and 17) that display 41–45 % identity with the potassium and sodium transport systems of clostridia, but no clear L. lactis RBS was detected upstream of these ORFs. Finally, ORFs 43–53 constituted a cluster of genes displaying the highest degree of homology (50–74 % identity) with streptococcal genes. Four of these genes are predicted to encode ribonucleotide reductases that supply the deoxynucleoside triphosphate (dNTP) substrates for DNA replication. A putative L. lactis RBS was present upstream of the initiator codons of these four genes, but we did not detect gene transcription by Northern hybridization and RT-PCR of RNA extracted from a M17glu culture.
Most of these genes that probably result from horizontal transfers do not seem to be expressed in L. lactis growing in rich medium. However, it remains to be verified whether some of them are expressed under other conditions and what functions they encode, in order to determine the advantages to the bacteria of acquiring and conserving these genes.
Distribution of pGdh442 in L. lactis
Fifty L. lactis strains with different genomic profiles from the INRA and Génoferment collections, isolated from dairy (36 strains) or non-dairy environments (14 strains), were tested for the presence of the gdh gene. Only five L. lactis subsp. lactis strains (NCDO1867, 2146, 2054, 2633 and S432) contained the gdh gene, as revealed by PCR amplification with two different primer pairs (gdh13/gdh18 and gdh15/gdh17) derived from the gdh sequence. Four gdh-positive strains were isolated from plants and animals, and one (NCDO2054) was isolated from milk. The five gdh-containing strains were tested for the presence of the pGdh442 plasmid by comparing the fingerprints that were obtained by PCR amplification with oligonucleotides distributed throughout the sequence of pGdh442 (see Fig. 1). The fingerprints obtained with four GDH-positive strains (including NCDO1867 used as a control) (Fig. 4) were identical, while no amplification was obtained with the plasmid-free strain MG1363 (Fig. 4) and other GDH-negative strains (data not shown), indicating that these four GDH-positive strains contained the same plasmid. For strain S432, PCR amplification was obtained with only three (2F/3R, 10F/11R and 18F/19R) out of the seven primer pairs used for the fingerprint, but amplification was also obtained with the primers derived from the ori, repA, parA, parB and nah sequences (data not shown). Moreover, Southern hybridization with gdh as a probe showed that the gdh gene was located on a plasmid of a size similar to that of pGdh442. These results suggested that the plasmid carrying gdh in strain S432 is slightly different from pGdh442, maybe because of differences in gene sequence, or rearrangements or further transposition events.
As expected from the incompatibility of pGdh442 with pSK08-type plasmids, most protease-positive strains (14 out of 15) did not contain the gdh gene, and four out of five strains containing pGdh442 were protease negative. However, strain S432, isolated from a meadow in Normandy, France, contained both gdh and prtP genes. Southern hybridization with prtP as a probe revealed that prtP was carried by a plasmid larger than pSK08 and of a size similar to that of pSK11P from L. lactis subsp. cremoris SK11 (Siezen et al., 2005). Moreover, like pSK11P, the plasmid carrying prtP in S432 probably does not carry the lactose operon, since a strain (S436) derived from S432 still contained prtP and gdh genes, although it did not grow in M17 with lactose. These observations suggest that in S432, prtP is carried by a pSK11P-type plasmid that is compatible with a pGdh442-type plasmid as a consequence of having a different replication system (Siezen et al., 2005).
Conclusion
The sequence analysis of pGdh442 encoding GDH shows that this plasmid does not carry genes that would trigger concern over its presence in human food. It does not encode antibiotic resistance, but it does endow the host strain with cadmium and zinc resistance which can be used as a selectable marker. Moreover, it can be transferred by a natural mechanism, since it is mobilizable by conjugative elements such as the 712-type sex factor that is widely distributed in the chromosome of L. lactis and in certain large lactococcal plasmids. Its incompatibility with pSK08-type plasmids harbouring genes for casein and lactose utilization is due to the identity of the replication and partitioning systems of these plasmids. Therefore, the lack of a similar replication and partitioning system in recipient strains should be verified to ensure the success of plasmid transfer.
The complete sequence of this plasmid, isolated from a plant strain, complements sequences of the 30 or so lactococcal plasmids (mainly isolated from dairy strains) that have already been published in databases (Siezen et al., 2005).
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ACKNOWLEDGEMENTS |
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!2536). We are also grateful to Danisco and the Fromageries Bel for their financial support. We would like to thank Veronique Monnet for her critical reading of the manuscript. We thank the laboratories that provided the strains for the Génoferment collection, especially Le Laboratoire de Microbiologie de l'Université de Caen Basse-Normandie, Caen, France, which provided strain S432 (UCMA 5713), and Le Laboratoire de Microbiologie et Génétique Moléculaire de l'Université Paul Sabatier – CNRS, Toulouse, France, which assembled and characterized the Génoferment collection. Génoferment is a National Food Research Programme funded by the French Research Agency (ANR). Edited by: M. Kleerebezem
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Received 6 September 2007;
revised 12 January 2007;
accepted 29 January 2007.
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