National Food Biotechnology Centre1,Department of Microbiology2, and Department of Food Science and Technology3, University College Cork, Cork, Ireland
Author for correspondence: Gerald F. Fitzgerald. Tel: +353 21 902730. Fax: +353 21 903101. e-mail: g.fitzgerald{at}ucc.ie
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ABSTRACT |
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Keywords: Lactococcus lactis, restriction, modification, magnesium transport, cobalt resistance
Abbreviations: abi, abortive infection; R/M, restriction/modification
The GenBank accession numbers for the sequences reported in this paper are AF153410AF153414.
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INTRODUCTION |
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R/M systems are classified into three groups, based on their molecular structure, sequence recognition, cleavage position and co-factor requirements. Representatives of all three groups have been found in lactococci. The most common are type II R/M systems. These generally consist of two separate enzymes, one responsible for restriction, the other for modification. They typically recognize a palindromic sequence of 48 nt and cut the DNA within this sequence. Several R/M systems have been found that resemble the type II systems, but cut the DNA outside the recognition sequence. These have been subdivided as type IIs systems. A lactococcal example of a type IIs R/M system is the LlaI system from the conjugative plasmid pTR2030 (OSullivan et al., 1995 ). This system also differs from other type II R/M systems because the restriction activity is encoded by three genes rather than one. Several type II R/M systems have been identified in lactococcal strains. As is common for most phage resistance mechanisms, all of these, with the exception of the ScrFI R/M system (Davis et al., 1993
), were found to be plasmid located. Type III R/M systems consist of two subunits (Mod and Res) that form one functional holoenzyme with both restriction and modification activity. The enzyme recognizes specific asymmetric sequences and cuts the DNA at a fixed distance, 2527 bp to one side of the recognition site. Recently, the first example of a lactococcal type III R/M system was identified and characterized (Su et al., 1999
).
The most complex R/M systems are those of type I R/M. These consist of three subunits, named Hsd for host specificity determinant, that can form one holoenzyme. One subunit, HsdS, determines the nucleotide specificity of the holoenzyme. The second subunit, HsdM, is required for modification of the recognition sequence whilst the third, HsdR, is required for restriction activity. Methylation can occur only in the presence of HsdS and HsdM and does not require ATP. Restriction, however, requires the presence of all three subunits, as well as ATP, and occurs at non-specific sites that can be more than 1 kb from the recognition sequence (Yuan, 1981 ). HsdS subunits consist of two variable domains separated and flanked by highly conserved sequences. It was shown that the first variable domain specifies the 5' DNA recognition sequence whereas the second specifies the 3' DNA recognition sequence, with a spacing of 68 nt between both (Fuller-Pace & Murray, 1986
). As a consequence of their structure, variable domains can be swapped between the DNA regions encoding HsdS subunits via crossing-over events, resulting in novel hsdS genes with altered specificities (Fuller-Pace & Murray, 1986
). Type I R/M systems have recently been shown to be widespread in lactococci and they could well be the most abundant R/M systems present in this group of bacteria (Schouler et al., 1998a
, b
). Several of these systems are plasmid located and the subunits may be located on one plasmid or on different plasmids (Forde et al., 1999
; Schouler et al., 1998b
) whilst others are chromosomally located (Schouler et al., 1998b
).
Lactococcus lactis subsp. cremoris UC509.9 is a prophage-cured derivative of strain UC509 which was originally isolated from a mixed starter culture (Arendt et al., 1994 ) and can be used as an indicator strain for the temperate phage Tuc2009. It harbours six cryptic plasmids, denoted pCIS1 to pCIS6. We found that phage propagated on a UC509.9 derivative which was cured of pCIS3 exhibited a 104-fold reduction in the e.o.p. when used for the infection of L. lactis UC509.9 in the presence of pCIS3. Here, the entire nucleotide sequence of pCIS3 is reported. It was found to encode an hsdS gene, typical of type I R/M systems. Two additional hsdS genes were identified, one located on the chromosome and the other located on a 4 kb plasmid. The presence of multiple hsdS genes in a single host provides great potential for genetic recombination between them with the consequential possibility of increasing phage resistance. Furthermore, an ORF was identified on pCIS3, which shows homology to a ubiquitous class of magnesium transporters, known as corA (Kehres et al., 1998
).
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METHODS |
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DNA manipulations.
Plasmid DNA was isolated from E. coli and L. lactis by using the Concert rapid plasmid isolation kit (Gibco-BRL). For L. lactis this was preceded by a 30 min incubation at 37 °C in solution G1 in the presence of 2 mg lysozyme ml-1 and 10 U mutanolysin ml-1 to obtain clear lysates. Restriction enzymes and T4 DNA ligase were purchased from Boehringer and used according to the suppliers instructions. Oligonucleotides were synthesized using an Applied Biosystems 391 DNA synthesizer. The CaCl2 transformation method was used for E. coli (Dagert & Ehrlich, 1979 ) whilst L. lactis was transformed by electrotransformation as described by Wells et al. (1993)
. DNA fragments generated by PCR were subcloned in pCR2.1-TOPO TA using the TOPO TA cloning system (Invitrogen) according to the manufacturers instructions.
Propagation of Tuc2009.
Propagation of phage was performed by growing the indicator strain to an OD600 of approximately 0·20·3 at which point CaCl2 was added to a final concentration of 10 mM, prior to the addition of phage. Incubation was subsequently continued until lysis occurred.
Bacteriophage assays.
Plaque assays were carried out as described by Lillehaug (1997) . Briefly, M17 double-layer agar plates, containing 0·5% glucose, 0·5% glycine and 10 mM CaCl2, were prepared with 1% agar for the bottom layer and 0·4% agar for the top layer. Media were sterilized by boiling for 5 min in a microwave oven. Plates contained approximately 30 ml 1% agar and were overlaid with 2·5 ml 0·4% agar, supplemented with 100 µl indicator strain (OD600=0·60·8) and appropriate dilutions of freshly prepared phage lysates.
Subcloning of pCIS3 and sequence analysis.
Plasmid pCIS3 was isolated from an agarose gel and subjected to digestion with a number of endonucleases. A single linear DNA fragment was obtained upon digestion with the endonucleases EcoRI, BamHI and PstI. The latter was used for cloning of the plasmid into the PstI site of pBluescript SK+, generating pBIS3. The plasmid was subcloned by ligation of a partial Sau3A digest with a BamHI digest of pBluescript SK+. The ligation mixture was used to transform E. coli XL-1 Blue and transformants were selected on plates containing X-Gal. Plasmid DNA from individual white colonies was isolated, checked for the presence of inserts and used for sequence analysis that was performed with an Applied Biosystems 373A automated DNA sequencer. Assembly of sequences was done with the SEQMAN program of the DNASTAR software package. Database searches were performed with the programs BLASTP, BLASTN and TBLASTN (Altschul et al., 1997 ). Finally, oligonucleotides flanking the PstI site were used to generate a PCR fragment using pCIS3 as a template. This fragment was sequenced in order to confirm that the PstI site was unique and not closely linked to another PstI site.
PCR.
PCR amplifications were performed by using the mastermix supplied by Qiagen with a Gene Amp PCR system 2400 thermal cycler (Perkin-Elmer Cetus). To obtain reproducible results with colony PCR for L. lactis, 1 U mutanolysin per reaction was added to the PCR mix. An initial incubation step of 15 min at 37 °C prior to the normal PCR cycle was added to the program and found to be sufficient for reliable results. Addition of minor amounts of lysozyme did not improve the results, whereas the addition of lysozyme in excess of 100 ng ml-1 actually inhibited the PCR reactions.
Southern hybridizations.
Southern transfers were carried out as described by Sambrook et al. (1989) using Hybond-N+ membranes (Amersham) as carrier. Probe labelling, hybridization and detection of complementary DNA were conducted using the ECL gene detection system (Amersham) as recommended by the manufacturer.
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RESULTS |
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As can be seen from Fig. 1, pCIS1 appears smaller when isolated from UC509.935 than when isolated from UC509.9. This is possibly due to some deletion event. Endonuclease treatment showed that pCIS1 from UC509.935 is approximately 100 bp smaller than pCIS1 isolated from UC509.9.
DNA sequence analysis
The entire nucleotide sequence of pCIS3 was determined. It consists of 6159 bp with a total G+C content of 35·9 mol%. Four ORFs could be identified (Fig. 2, Table 2
). The first ORF, designated rep, shows high similarity to a family of replication genes that are commonly found in lactococcal strains and encode the replication-initiation protein. The highest level of similarity was found to the rep gene of pCI305. Similar to other members of this family of replicons, rep is preceded by a 22 bp sequence, repeated three and a half times (nt 314390). This repeated region is 100% identical to that of pCI305. rep is followed by an ORF of unknown function, designated orfX. Directly downstream of orfX, a third ORF is found, which shows high levels of homology to hsdS genes of type I R/M systems. Alignment of the deduced amino acid sequence of this gene with the hsdS-encoded proteins of other lactic acid bacteria clearly shows the conserved domains, separated by variable domains (Fig. 3a
). The N-terminal constant region is repeated at the 3' end of the middle region, while the conserved C-terminal domain is repeated at the 5' end of the middle region. rep, orfX and hsdS are organized in an operon-like structure with the last two codon sequences of rep overlapping the first two of orfX and the last three codon sequences of orfX overlapping the first three of hsdS. No putative promoter sequences could be identified directly upstream of orfX and hsdS. Each is, however, preceded by near-consensus ribosome-binding sites.
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Natural stacking of hsdS genes in L. lactis UC509.9
Recombination between the variable domains of different hsdS genes in a single strain is likely to play a role in ensuring continued dynamics in phage defence. In addition, plasmids can be mobilized between strains and if the conjugated plasmid carrying an hsdS gene is introduced into a strain that already carries a different hsdS locus, the unique structure of these genes allows recombination to occur, resulting in novel hsdS genes with altered specificities (Fuller-Pace et al., 1984 ). This also implies that one strain could carry two or more hsdS genes. To determine if this is the case in L. lactis UC509.9, two oligonucleotides were designed, based on the conserved domains that flank the variable domains of hsdS genes (SC1, 5'-GATTGGGAAGAGCGTAAGTT-3' and SC2, 5'-AACTTACGCTGATGAAGA-3'). These were used for PCR amplification of total DNA isolated from UC509.935. A product of the expected size was obtained and subcloned into pCR2.1-TOPO TA. Eight individual colonies were checked for inserts and used for sequence analysis. Three variable domains, designated V3, V4 and V5, differing from those of pCIS3, could thus be identified, indicating the presence of at least two additional hsdS genes in this strain. The alignment of the protein products specified by V3, V4 and V5 is presented in Fig. 3(b)
. The combined PCR products were used for Southern analysis. This confirmed the presence of two additional hsdS genes, one of which was found to be chromosomally located, whilst the other was located on pCIS1, the smallest plasmid present in UC509.9 (approx. 4 kb; Fig. 4b
, lane 4).
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To determine the orientation of the chromosomally encoded hsdS gene with respect to the hsdM gene, PCR reactions were performed with several combinations of the oligonucleotides M5, M6, SC1 and SC2. A product with a size of approximately 1·3 kb was obtained with the combination of M5 and SC2. All other combinations with the M and SC oligonucleotides failed to give a product. This indicates that the hsdS gene is located directly downstream of the hsdM gene in an orientation similar to that in L. lactis IL1403. The close proximity of hsdM to hsdS on the chromosome was confirmed by Southern hybridization where a common band of 1·2 kb was observed when either part of the hsdM gene or a combination of variable domains from all hsdS genes was used as a probe (Fig. 4a and 4b
, lanes 2 and 4, respectively).
Effectiveness of HsdS subunits
The effect of the HsdS subunit of pCIS3 on the e.o.p. of phage Tuc2009 on UC509.9 and phages bIL66 and 952 on IL1403 was studied. For this purpose pCIS3 was genetically marked with the erythromycin-resistance gene of pGKV210 to create pCIS31.1 (see Methods). This plasmid was introduced into L. lactis strains UC509.93 and IL1403. Phage was then isolated from UC509.93 or IL1403 and used to infect both strains in the presence or absence of pCIS31.1 (Table 3). Whereas the presence of pCIS31.1 resulted in a 104-fold decrease in the e.o.p. for Tuc2009 on UC509.93, this effect was markedly less (only a 10-fold decrease) for bIL66 and 952 on IL1403. These values for IL1403 were similar to those observed by Schouler et al. (1998b)
with the hsdS genes from pIL7 and pIL261 in conjunction with phage bIL67.
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Absence of corA results in increased cobalt resistance
ORF4 of pCIS3 encodes a protein with similarity to CorA proteins identified in many prokaryotes (Kehres et al., 1998 ), where they function as the dominant Mg2+-uptake system. ORF4 is similar in size to other corA genes and its deduced protein product contains three putative transmembrane domains (Fig. 5
). To assess the function of corA, its effect on the cobalt tolerance of the host strain was determined. For this purpose, both UC509.93 and UC509.9 were streaked on plates with increasing amounts of CoCl2. UC509.93 (lacking pCIS3) was capable of growing at concentrations up to 5 mM CoCl2, whereas UC509.9 could only grow to a maximum of 1 mM CoCl2 (Fig. 6
). Introduction of pCIS31.1, from which corA was deleted (see Methods), into UC509.93 restored phage resistance, but failed to increase cobalt resistance.
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DISCUSSION |
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The presence of mobilization functions on lactococcal plasmids allows for transfer of plasmids (and the traits they encode) between different strains. When a plasmid that carries an hsdS gene is transferred to a strain that already carries such a gene, crossover events can occur, resulting in novel HsdS subunits with altered specificities. It can be envisaged that it is advantageous for a strain to carry more than one copy of the hsdS genes in order to achieve a high level of variation, resulting in increased phage resistance.
PCR analysis indicated that the chromosomally located hsdS gene lies downstream of the hsdM gene in an orientation similar to that found for IL1403 (Schouler et al., 1998b ). Based on the typical organization of type I R/M systems found in lactococci to date, it can be expected that the hsdR gene is located upstream of hsdM.
The effectiveness of two hsdS genes was evaluated for strains UC509.9 and IL1403. The effect on the e.o.p. of the genes that were tested was significantly greater in the UC509.9 host than in IL1403. The values that were obtained with pCIS3 and pCI65st for IL1403 were similar to those observed by Schouler et al. (1998a) for pIL7. Since the hsdM genes of IL1403 and UC509.9 are nearly identical, it can be speculated that this difference in effectiveness could be related to the activity of HsdR. It can not, however, be excluded that phage Tuc2009 has more recognition sites for the pCIS3 and pCI65st subunits than bIL66 and phage 952.
Three other genes were identified on pCIS3. The rep gene, encoding the replication-initiation protein, is located on a large family of theta replicating plasmids that is very common in lactococci (Seegers et al., 1994 ). A 22 bp sequence, repeated three and a half times, preceding this gene is thought to be the major compatibility factor (Foley et al., 1996
; Gravesen et al., 1997
). Indeed, comparison of this sequence from pCIS3 with sequences in the nucleotide database revealed that this region was 100 % identical to that of pCI305 (Hayes et al., 1991
). This latter plasmid was used for the construction of pFDi18, which carries the supB gene (Dickely et al., 1995
), which explains why pCIS3 was lost from the strain when pFDi18 was introduced and provides further evidence for the role of the repeated fragment in compatibility.
The first codons of the second gene, named orfX, overlap the last codons of rep. Similar genes are found in most plasmids of this family. To date no function has been attributed to this gene.
The amino acid sequence of the fourth gene that could be identified showed similarity to a group of magnesium-transporter proteins, commonly indicated as CorA. These genes have been shown to be ubiquitous in eubacteria and to encode their dominant Mg2+-uptake system (Smith & Maguire, 1998 ). Loss of the plasmid resulted in an increase in cobalt resistance, indicating it has a similar function in UC509.9. However, the absence of corA did not result in any other visible effects on growth under the conditions used. Possible explanations for this could be the presence of another corA gene on the chromosome or the presence of additional Mg2+-transporter proteins. To our knowledge this is the first example of such a gene located on a plasmid in bacteria.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 17 June 1999;
revised 19 October 1999;
accepted 2 November 1999.