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Journal of Bacteriology, January 2004, p. 287-295, Vol. 186, No. 2
0021-9193/04/$08.00+0     DOI: 10.1128/JB.186.2.287-295.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

The Methyltransferase from the LlaDII Restriction-Modification System Influences the Level of Expression of Its Own Gene

Lisa Lystbæk Christensen and Jytte Josephsen*

Department of Dairy and Food Science, Centre of Advanced Food Studies, The Royal Veterinary and Agricultural University, DK-1958 Frederiksberg C, Denmark

Received 14 July 2003/ Accepted 13 October 2003


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ABSTRACT
 
The type II restriction-modification (R-M) system LlaDII isolated from Lactococcus lactis contains two tandemly arranged genes, llaDIIR and llaDIIM, encoding a restriction endonuclease (REase) and a methyltransferase (MTase), respectively. Interestingly, two LlaDII recognition sites are present in the llaDIIM promoter region, suggesting that they may influence the activity of the promoter through methylation status. In this study, separate promoters for llaDIIR and llaDIIM were identified, and the regulation of the two genes at the transcriptional level was investigated. DNA fragments containing the putative promoters were cloned in a promoter probe vector and tested for activity in the presence and absence of the active MTase. The level of expression of the MTase was 5- to 10-fold higher than the level of expression of the REase. The results also showed that the presence of M.LlaDII reduced the in vivo expression of the llaDIIM promoter (PllaDIIM) up to 1,000-fold, whereas the activity of the llaDIIR promoter (PllaDIIR) was not affected. Based on site-specific mutations it was shown that both of the LlaDII recognition sites within PllaDIIM are required to obtain complete repression of transcriptional activity. No regulation was found for llaDIIR, which appears to be constitutively expressed.


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INTRODUCTION
 
Although a great number of restriction-modification (R-M) systems have been identified (27), relatively little about regulation of their expression has been determined. Tight regulation of the sequential expression of modification and restriction that prevents premature activity of the restriction endonuclease (REase) is essential as many R-M systems are encoded by mobile genetic elements that can be transferred to recipient cells having no prior protection from the cognate modification enzymes. Controlling the activity of m5C methyltransferases (MTases) is important as excessive methylation may lead to DNA mutations (2, 28). Also, having very high MTase activity at all times would decrease the efficiency of restriction of viral DNA.

There are different types of regulation for type II R-M systems. One group, exemplified by PvuII (31) and BamHI (8), is regulated at the transcriptional level by small so-called C-proteins; the genes that encode these proteins are usually located upstream of and in some cases partially overlap the REase gene. The C-proteins constitute a family of regulatory proteins that bind to a common operator sequence (C box) through a helix-turn-helix (HTH) motif (26, 32, 33). They function as transcriptional activators of the REase genes, as well as their own genes, and in some systems, such as BamHI, they have been found to down-regulate expression of the MTase genes as well and thus to have dual functions. A couple of examples of regulatory proteins not classified with the C-proteins mentioned above have been described. In Kpn2I the gene organization is R-M-C, and the direction of transcription of kpn2IC is opposite the direction of transcription of kpn2IM (15). C.Kpn2I was found to repress MTase expression but had no effect on expression of the REase, which apparently is constitutively expressed. C.Kpn2I is a small protein containing an HTH motif, but no known operator sequence could be identified upstream of kpn2IM. Supposedly, the initial overexpression of M.Kpn2I is sufficient to ensure methylation of the chromosomal DNA before R.Kpn2I is active. In contrast to this, C.EcoO109I is necessary for expression of the REase of EcoO109I but has no influence on expression of the MTase (13). C.EcoO109I presumably functions as a transcriptional activator, binding to an inverted repeat upstream of ecoO109IC and thus enhancing the expression of both its own gene and the cotranscribed gene ecoO109R. C.EcoO109I and its binding site exhibit no sequence similarity to the C-proteins and the C box, respectively. Another interesting mode of regulation was found for MspI (30), SsoII (10), and EcoRII (29). The MTases of these R-M systems contain N-terminal HTH motifs and have been found to interact directly with their own promoter sequences and to down-regulate the transcriptional activity. Inverted repeat sequences, which are not conserved, have variable lengths, and are present upstream of the MTase genes, have been proposed to be the binding sites for the corresponding MTases (10). Only in SsoII do the promoters of the divergently transcribed MTase and REase genes overlap, resulting in regulation of both genes; the expression of ssoIIM is down-regulated, and the expression of ssoIIR is up-regulated. Recently, a novel type of autoregulation was found for CfrBI (3). A single CfrBI recognition site is present within the overlapping promoter regions of the divergently transcribed MTase and REase genes. When the CfrBI recognition site is methylated, expression of the MTase gene is down-regulated and expression of the REase gene is up-regulated; the opposite occurs when the recognition site is unmethylated.

Until now, LlaI (22) and ScrFI (4) have been the only two lactococcal R-M systems whose regulation has been studied. LlaI consists of five genes, including a type IIS MTase gene and three genes responsible for the restriction activity; the fifth gene, llaIC, encodes a small protein which was found to be involved in posttranscriptional regulation of the restriction activity, possibly through mRNA stabilization (22). All five genes involved in the restriction and modification activity of LlaI are transcribed as a polycistronic operon. The chromosomally encoded type II R-M system ScrFI, which contains an REase gene (scrFIR) flanked by two MTase genes (scrFIBM and scrFIAM), has a more complicated transcription pattern (4). An open reading frame (ORF) having an unknown function, orfX, is cotranscribed with scrFIBM and scrFIR on a single mRNA, whereas scrFIAM is transcribed separately. M.ScrFIA contains an N-terminal HTH motif and was found to bind to its own promoter region and subsequently down-regulate the transcriptional activity. No binding to the promoter responsible for orfX, scrFIBM, and scrFIR transcription could be established; however, M.ScrFIA appeared to repress the activity of this promoter as well.

LlaDII (accession no. Y12707), isolated from Lactococcus lactis subsp. cremoris W39, was cloned and partially characterized previously (16). LlaDII is a type II R-M system that recognizes the palindromic sequence 5'-GC{downarrow}NGC-3'. In this study we investigated regulation at the transcriptional level of the REase and MTase genes. We show that expression of M.LlaDII is subject to autoregulation due to the presence of LlaDII recognition sites within its own promoter, while no regulation of R.LlaDII was observed.


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MATERIALS AND METHODS
 
Bacteria and growth media. L. lactis subsp. cremoris MG1363 (5) was propagated at 30°C in M17 medium (Oxoid Limited, Basingstoke, United Kingdom) supplemented with 0.5% (wt/vol) glucose (GM17). Cells were made competent and transformed by electroporation as described by Holo and Nes (7). When appropriate, chloramphenicol and erythromycin were added to a final concentration of 5 µg/ml and tetracycline was added to a final concentration of 2 µg/ml. For screening for promoter activity, GM17 agar plates containing the appropriate antibiotic(s) and 80 µg of 5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside (X-Gal) per ml were used.

Molecular cloning techniques. All plasmids and primers used in this study are listed in Tables 1 and 2, respectively. Plasmid DNA was isolated with QIAGEN plasmid purification kits (QIAGEN, Hilden, Germany) after treatment with lysozyme (20 mg/ml) at 37°C for 30 min. Pfu DNA polymerase (Promega, Madison, Wis.) was used for PCR amplification with plasmid pHW393 as the template. Plasmids pLLC11-pLLC20, pLLC26-pLLC28, and pLLC30 containing DNA upstream of llaDIIR or llaDIIM were constructed by inserting BamHI-digested PCR fragments into BamHI-digested plasmid pTRK390. The following primers were used: P.14 plus P.18 (pLLC11), P.14 plus P.17 (pLLC12), P.15 plus P.18 (pLLC13), P.15 plus P.17 (pLLC14), P.16 plus P.18 (pLLC15), P.16 plus P.17 (pLLC16), P.19 plus P.22 (pLLC17), P.19 plus P.21 (pLLC18), P.20 plus P.22 (pLLC19), P.20 plus P.21 (pLLC20), P.23 plus P.21 (pLLC26), P.24 plus P.21 (pLLC27), P.25 plus P.21 (pLLC28), and P.26 plus P.21 (pLLC30).


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  		TABLE 1. Plasmids used in this study


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  		TABLE 2. Primers used in this study

Plasmid pLLC32 was constructed by inserting an NcoI-HindIII-digested PCR fragment encoding llaDIIM (nucleotides [nt] 1370 to 2346, generated with primers P.29 and P.30) into plasmid pNZ44 digested with NcoI and HindIII. The MTase activity of pLLC32 in L. lactis MG1363 was verified, as jj50 bacteriophages (9) propagated on the strain were able to circumvent LlaDII activity. Plasmid pLLC6 was constructed by shotgun cloning of HindIII-EcoRI-digested pHW393 in plasmid pCI3340. The nucleotide sequences of all cloned fragments except pLLC6 and pLLC32 were verified by DNA sequencing performed with a CEQ dye terminator cycle sequencing kit and a CEQ 2000 DNA analysis system (Beckman Coulter, Chaska, Minn.).

Northern blotting. Total cellular RNA was isolated from exponentially growing cells. Harvested cells were disrupted with glass beads (maximum diameter, 106 µm; Sigma-Aldrich) in a Fastprep FB 120 homogenizer (Thermo Savant, Holbrook, N.Y.) for 45 s. Total RNA was purified with an RNeasy mini kit (QIAGEN). RNA gel electrophoresis was performed as described by Pellé and Murphy (24), with minor modifications. RNA size standards from New England Biolabs (Beverly, Mass.) were included to estimate transcript sizes. Samples were transferred to a positively charged nylon membrane by standard diffusion blotting and were hybridized with the appropriate DNA probes. Hybridization probes were obtained by performing PCR with primers P.31 and P.32 for llaDIIR and with primers P.33 and P.34 for llaDIIM and were labeled with [{alpha}-32P]dATP (Amersham Biosciences, Little Chalfont, Buckinghamshire, England) by using the Multiprime DNA labeling system (Amersham Biosciences). Following hybridization, the membranes were scanned with a PhosphorImager (Storm system; Molecular Dynamics).

Primer extension. Total cellular RNA was isolated as described above for the Northern blot analysis. In addition, the RNA was treated with an RNase-free DNase set (QIAGEN). Five micrograms of RNA was mixed with 1 pmol of Cy5-labeled primer in hybridization buffer (50 mM potassium HEPES, 100 mM KCl; pH 7) in a 10-µl (total volume) reaction mixture. The mixture was denatured at 70°C for 5 min and immediately transferred to ice. Annealing was performed at 48°C for 15 min. Then 200 U of Moloney murine leukemia virus reverse transcriptase (Promega) and extension buffer (Promega) containing each deoxynucleoside triphosphate at a concentration of 200 µM in a 15-µl (total volume) mixture were incubated at 48°C for 1 h. Following ethanol precipitation the pellet was dissolved and loaded on a DNA sequencing gel next to a sequencing reaction mixture prepared with a Thermo Sequenase kit (Amersham Biosciences) and the same primer that was used for the extension reaction. The primer extension products were analyzed with an ALFexpress DNA sequencer (Amersham Biosciences) as described by Myöhänen and Wahlfors (20).

Measurement of promoter activity. ß-Galactosidase activity was determined by using exponentially growing cells. Cells were permeabilized with sodium dodecyl sulfate (0.1%) and chloroform. The assays were carried out essentially as described by Miller (19). The specific ß-galactosidase activity was calculated on the basis of the optical density at 600 nm of the culture and was expressed in Miller units. At least three independent assays were performed with duplicate cultures of each strain. The results of these measurements (at least six measurements) were used to calculate the average and the standard error.


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RESULTS
 
Transcriptional analysis of LlaDII. Sequence analysis of LlaDII (a schematic representation is shown in Fig. 1a) showed that both the REase and MTase genes contained putative consensus promoters, as shown in Fig. 1b and c. llaDIIM has previously been shown to express a functional MTase when it is cloned alone, thus establishing that there is a functional promoter in front of this gene (16). Furthermore, an inverted repeat is present in the intergenic region and may constitute a rho-independent transcriptional terminator, as suggested in Fig. 1d. The free energy of formation of the hairpin is only -6.2 kcal/mol (34), and considerable readthrough may therefore occur, especially when the competing hairpin with a free energy of formation of -15.4 kcal/mol is considered (Fig. 1d). However, collectively, these observations suggested that the two genes are transcribed separately. To establish the size of the transcripts from LlaDII, Northern blotting was performed. Total RNA was isolated from L. lactis MG1363 containing either plasmid pCAD1 encoding LlaDII or no plasmid. Hybridization with the llaDIIR probe resulted in identification of an approximately 650-nt transcript, which corresponded to llaDIIR being transcribed on an individual transcript (Fig. 2). A weak band corresponding to an approximately 950-nt transcript was also detected with the llaDIIR probe, which may have corresponded to an extended mRNA due to inadequate transcriptional termination or a longer processed transcript. A transcript larger than 1,500 nt would be expected if a dicistronic mRNA containing both llaDIIR and llaDIIM was synthesized. No transcripts were observed with the llaDIIM probe. As determined subsequently, this was most likely due to the very low level of expression of the MTase in a cell fully modified by LlaDII, in accordance with the results of the promoter fusion studies described below.



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  		FIG. 1. (a) Schematic representation of LlaDII, showing the two ORFs encoding llaDIIR and llaDIIM. The position of the putative rho-independent terminator is indicated. (b) Sequence of the llaDIIR promoter. The unusual translational start codon (TTG) is indicated by a bent arrow below the sequence. The previously determined transcriptional start site (A715) is indicated by a bent arrow above the sequence. (c) Sequence of the llaDIIM promoter and the site-specific mutations within the promoter used for analysis of the importance of methylation in promoter activity. The mutations are underlined. LlaDII recognition sites (5'-GCNGC-3') are indicated by facing arrows. The translational start is indicated by a bent arrow below the sequence. The bent arrows above the sequence indicate the two alternative transcriptional start sites (A1364 and A1367) determined in this study. (d) Possible transcriptional terminator downstream of llaDIIR and competing hairpin. The free energy of formation ({Delta}G) for each of the two RNA secondary structures is shown. The numbers above the sequences indicate the positions in the LlaDII sequence. RBS, ribosome binding site.



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  		FIG. 2. Northern blot analysis of RNA isolated from L. lactis MG1363 (lane 1) or from L. lactis MG1363 containing pCAD1 (lane 2). A DNA probe corresponding to llaDIIR was used. Sizes are indicated on the left.

Identification of promoters within LlaDII. In order to identify promoters for the LlaDII genes, DNA fragments of various lengths containing the putative promoters of llaDIIR and llaDIIM were generated by PCR and cloned in the promoter probe vector pTRK390 containing a promoterless lacZ gene. The resulting plasmids were designated pLLC11 to pLLC20, and an overview of the cloned promoter-containing fragments and measured specific ß-galactosidase activities is shown in Fig. 3a and b (- MTase column). Generally, both the PllaDIIR and PllaDIIM fusions containing the putative ribosome binding sites and the translational start codon of the genes expressed ß-galactosidase at higher levels than the constructs that did not contain the putative ribosome binding sites and the translational start codon (e.g., 521 and 31 Miller units for pLLC11 and pLLC12, respectively, and 5,645 and 172 Miller units for pLLC17 and pLLC18, respectively). This was probably due to increased mRNA stability as two translational stop codons are present upstream of the lacZ start codon, thus ruling out translational fusion as the cause of increased activity. However, all promoter fusions containing 328- to 60-bp fragments for llaDIIR (pLLC11 to pLLC16) and 217- to 60-bp fragments for llaDIIM (pLLC17 to pLLC20) expressed ß-galactosidase, clearly showing that they contained active promoters. Notably, when the highest specific ß-galactosidase activities obtained were compared, the values obtained for PllaDIIM were generally 5- to 10-fold higher than the values obtained for PllaDIIR (e.g., 5,645 Miller units for pLLC17 compared to 521 Miller units for pLLC11). Interestingly, the specific ß-galactosidase activities for the PllaDIIR fragments increased twofold with truncation from nt 618 to nt 673, as observed when the effects of plasmids pLLC13 and pLLC15 (528 and 1147 Miller units, respectively) were compared. The reason for this is not clear.



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  		FIG. 3. LlaDII promoter fusions. (a and b) DNA fragments upstream of llaDIIR (a) and llaDIIM (b) cloned in pTRK390. (c) Site-specific mutations within PllaDIIM (pLLC20). (d) Vector control with no insertion. Specific ß-galactosidase activity was determined in the absence of MTase (- MTase), in the presence of constitutively expressed M.LlaDII (+ pLLC32), and in the presence of M.LlaDII expressed from the wild-type promoter (+ pEE1) and is expressed in Miller units; unless indicated otherwise, the values are averages ± standard errors of three independent assays performed with duplicate cultures. A solid arrowhead indicates an LlaDII recognition site. The positions of the -35 sequence, the -10 sequence, and the ribosome binding site (RBS) are indicated above the DNA fragments. The numbers are the positions in the LlaDII sequence. n.d., not determined. An asterisk indicates that the value is the average of two independent assays.

Regulation of expression. The presence of two LlaDII recognition sites within the promoter region of llaDIIM raised the question of whether these sites have an effect on expression of M.LlaDII and, less likely, R.LlaDII. The different promoter fusions were cotransformed with plasmid pLLC32 or pEE1 into L. lactis MG1363. pLLC32 contained the llaDIIM gene constitutively expressed from the P44 promoter in plasmid pNZ44, whereas pEE1 contained the wild-type MTase gene. Using the constitutively expressed M.LlaDII gene instead of the wild-type gene should preclude any anomalies resulting from having the MTase promoter present twice in the experiments. The results of ß-galactosidase assays with cells containing promoter fusions and pLLC32 are shown in Fig. 3a and b (+ pLLC32 column). No significant effects were observed for the PllaDIIR fusions (Fig. 3a), whereas the specific ß-galactosidase activities obtained for the PllaDIIM fusions were reduced to the background level (Fig. 3b). When the wild-type llaDIIM gene on plasmid pEE1 was present instead of pLLC32, significantly higher specific ß-galactosidase activities were found for the MTase promoter fusions (measured for pLLC19 and pLLC20) (Fig. 3b, + pEE1 column). The PllaDIIM activity was reduced only 10-fold compared to the full activity in the absence of the MTase. As with pLLC32, no significant changes in specific ß-galactosidase activities were observed with pEE1 for PllaDIIR (measured for pLLC13 to pLLC16) (Fig. 3a, + pEE1 column).

Not surprisingly, M.LlaDII seemed to have no effect on regulation of llaDIIR. During the original cloning of LlaDII, 744 bp of apparently noncoding DNA was found upstream of llaDIIR (16). A new cloning analysis of LlaDII containing only 272 bp upstream of llaDIIR (plasmid pLLC6) resulted in a fully functional R-M system exhibiting levels of bacteriophage resistance similar to those observed for the original clone (results not shown), substantiating the conclusion that no sequence elements relevant for expression of LlaDII are present from nt 1 to 471. A search for ORFs within or in the vicinity of the LlaDII sequence (nt 472 to 2355) resulted in identification of four small ORFs encoding 34 to 50 amino acids (results not shown). No sequence similarity with known regulatory C-proteins or any other proteins was found, and no consensus promoter elements could be identified for these additional ORFs.

Site-specific mutations within the promoter of llaDIIM. To verify that the LlaDII recognition sites are directly responsible for regulation of llaDIIM and to determine whether both sites are needed to repress transcription, a number of site-specific mutations were constructed within the PllaDIIM fragment found in pLLC20 comprising nt 1318 to 1377 (Fig. 1c). Single point mutations were introduced into either one (pLLC26 and pLLC27) or both (pLLC28) of the LlaDII recognition sites. The double mutation in pLLC30 (T1331C T1332C) destroyed the -35 region while leaving the LlaDII recognition sites intact. Specific ß-galactosidase activities for pLLC26 to pLLC28 and pLLC30 are shown in Fig. 3c. In the absence of an active MTase, the specific ß-galactosidase activities induced by the mutated promoters were approximately the same (within twofold) as the specific activities for the wild-type promoter (pLLC20). When the -35 region was impaired in pLLC30, a 10-fold-lower specific ß-galactosidase activity was observed, which was even further down-regulated when pLLC32 was present, as expected.

In the presence of the constitutively expressed MTase on pLLC32, the specific ß-galactosidase activity of the wild-type promoter fusion pLLC20 decreased approximately 100-fold. When a single point mutation was introduced (pLLC26 and pLLC27), the specific ß-galactosidase activity decreased approximately 10-fold, whereas when both recognition sites were destroyed (pLLC28), no decrease in the expression level was observed. This shows that a single LlaDII recognition site is not sufficient to obtain complete down-regulation of promoter activity.

Determination of the transcriptional start site for llaDIIM. The transcriptional start site was previously determined for llaDIIR (16); however, attempts to determine the start site for llaDIIM were unsuccessful (Annette Madsen, personal communication). This was probably due to repressed expression of the MTase, leading to very small amounts of mRNA for llaDIIM, as observed for the Northern blots in this study. As the promoter fusion in pLLC17 had high transcriptional activity in the absence of M.LlaDII, this clone was chosen for primer extension. Total RNA was isolated from L. lactis MG1363 containing pLLC17, pLLC17 plus pLLC32, or pTRK390 as a control. Primer extension was performed with two different Cy5-labeled primers (P.42 and P.43 [Table 2]) annealing to the lacZ gene within pTRK390 and located approximately 50 bp apart. The results of primer extension of RNA isolated from L. lactis MG1363 containing pLLC17 with primer P.43 are shown in Fig. 4. Two different transcriptional start sites, at A1364 and A1367, corresponding to 28 and 25 nt upstream of the ATG translational initiation codon, respectively, were identified with both primers. As expected, no extension products resulted from primer extension of RNA isolated from L. lactis MG1363 containing pLLC17 plus pLLC32 or pTRK390.



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  		FIG. 4. Primer extension mapping of the llaDIIM promoter. The extension reaction was performed with RNA isolated from L. lactis MG1363 harboring pLLC17. The dotted lines indicate the positions of the two extension products corresponding to two alternative transcriptional start sites, A1364 and A1367.


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DISCUSSION
 
From the sequence encoding LlaDII the two tandemly arranged genes, llaDIIR and llaDIIM, were expected to be transcribed from individual promoters. This hypothesis was previously partially substantiated by the observed MTase activity of plasmid pEE1, which does not contain the promoter upstream of llaDIIR. In this study the existence of two separate promoters within the llaDII gene cluster was established by analysis of the transcriptional pattern. In the SalI type II R-M system the two genes, which are arranged with the REase gene preceding the MTase gene, as observed for LlaDII, are cotranscribed; however, the MTase is also transcribed from an intrinsic promoter, supposedly ensuring that the MTase is active prior to the REase when the genes are transferred to a new host cell (1). A similar transcription pattern could have been expected for LlaDII; however, Northern blotting revealed no transcript large enough to cover both genes. A faint band at 950 nt could indicate a processed form of a longer transcript covering both genes. If this is the case, the long transcript is very unstable. However, production of such a transcript might help produce larger amounts of MTase fast by introduction of the R-M system into a new host. The only clearly identifiable transcript corresponded to llaDIIR because the level of expression of llaDIIM was too low to detect in mRNA isolated from cells harboring the fully implemented R-M system. The size of the llaDIIR transcript correlates well with the size expected from termination at the proposed rho-independent transcriptional terminator. Use of one of the promoter fusions, pLLC17, which is known to sustain high transcriptional activity of ß-galactosidase in the absence of M.LlaDII, allowed determination of the 5' end of the llaDIIM transcript. The primer extension analysis showed that there are two alternative transcriptional start sites within the expected distance from the -10 consensus region. Thus, the promoter for the MTase gene was established both by promoter fusions and by primer extension analysis.

The boundaries of the two promoters within LlaDII were examined by cloning DNA fragments of different lengths in the promoter probe vector pTRK390. The results showed that fragments as small as 60 bp for both promoters still induced expression of ß-galactosidase. When the fragments containing PllaDIIR were shortened from the 5' end from nt 618 to 673, twofold-higher specific ß-galactosidase activity was observed. Although the effect of deletion is small, it could be speculated that the area between nt 618 and nt 673 contains an operator site for a repressor of gene expression. Repressors usually bind within the promoter region, thereby inhibiting the initiation of transcription; however, cooperative binding of repressor multimers to operators, one of which is located at a remote site that results in looping of the intervening DNA, has been described in several cases (17). In this scenario, llaDIIR would contain operator sites both within the promoter and within the upstream region covering nt 618 to 673. There is at this point no evidence for the existence of such a regulation scheme for llaDIIR, and no repressor protein or potential operator sites have been identified. In the cases of gene regulation by a third independent protein that have been described, the gene is usually located close to or overlapping the REase and MTase genes. A number of small ORFs were identified within or close to llaDIIR and llaDIIM, but none of these seem to encode functional genes. However, our knowledge of gene regulation within R-M systems is still very limited; therefore, a regulatory role for these ORFs cannot be ruled out.

The specific ß-galactosidase activities of the promoter fusions were also determined in cells containing plasmid pEE1 expressing the LlaDII MTase from the wild-type promoter or plasmid pLLC32 expressing the LlaDII MTase constitutively. The results showed that the expression of llaDIIR was not influenced by the presence of M.LlaDII, while the expression of llaDIIM was reduced 10- to 1,000-fold. The effect of the constitutively expressed MTase on the down-regulation of llaDIIM promoter activity was significantly more severe than the effect of the wild-type MTase. This was most likely because the M.LlaDII activity in cells harboring the constitutively expressed gene was much higher than the M.LlaDII activity in cells expressing llaDIIM from the wild-type promoter, the activity of which was itself down-regulated. Also, the constitutively expressed gene was present on a high-copy-number plasmid, pNZ44, in contrast to the wild-type gene on pCI3340, a medium-copy-number plasmid.

The presence of two LlaDII recognition sites in the intergenic region, one of which partially overlapped the -35 region of PllaDIIM, suggested the possibility that these sites influence the promoter activity. The direct involvement of the two recognition sites in the down-regulation of transcriptional activity was verified by site-specific mutational analysis. When either of the two sites was mutated, the repression of llaDIIM gene expression was only partial. When both were destroyed, no repression was observed, showing that both recognition sites are required for full repression.

This study provided evidence that M.LlaDII regulates the expression of its own gene. Further studies are needed to establish whether methylation of the LlaDII recognition sites within the promoter region is the actual cause of the observed regulation of expression of llaDIIM in hindering the interaction of the RNA polymerase with the promoter, as hypothesized for cfrBIM (3), or whether the binding of the MTase itself is sufficient. In any case, the regulation of llaDIIM appears to differ from the regulation of MspI (30), SsoII (10), and EcoRII (29), in which inverted repeat sequences with no relation to recognition sites were identified as potential binding sites for the MTases upstream of their own genes. Incidentally, the two recognition sites within PllaDIIM constitute an inverted repeat (Fig. 1c), a feature that seems to be merely coincidental. Our investigations did not reveal any regulation of the REase gene on the transcriptional level; the gene must therefore be considered constitutively expressed or may be translationally regulated. However, when LlaDII is introduced into a new host cell, some measures must be taken to ensure that the REase is not active before the chromosomal DNA has been fully methylated. A number of conditions may be considered relevant in accordance with this, as follows. (i) REases of type II R-M systems usually function as homodimers, although one example of a homotetrameric REase has been described for SfiI (21). The dimerization or possibly multimerization step may permit a delay in restriction activity compared to methylation. (ii) The promoter study showed that the activity of PllaDIIR is 5- to 10-fold lower than that of PllaDIIM. This initial overexpression of MTase compared to expression of the REase could ensure methylation of the cell prior to restriction. (iii) R.LlaDII has an unusual initiation codon, UUG. UUG has been found to be an inefficient initiation codon compared to the usual AUG codon (25) and thus may be an effective way of ensuring low expression of a gene at the translational level. The transcriptional activity of llaDIIR may be up to 10-fold lower than that of llaDIIM (prior to methylation), but with the noncanonical inititation codon the expression of R.LlaDII compared to the expression of M.LlaDII may be even lower.

Gene expression of R-M systems influenced by methylation has previously been described only for CfrBI (3). In CfrBI a single recognition site present in the intergenic region of the divergently transcribed genes regulates both the MTase and REase genes. Other R-M systems are, however, expected to employ the same type of control over gene expression; e.g., the LlaDII isoschizomer Bsp6I (14) has a sequence that is highly related to the sequence of LlaDII and also contains two recognition sites in the promoter region of the MTase. FokI is another example. The MTase and REase genes are cotranscribed from a promoter upstream of fokIM (12). Two FokI recognition sites are present just downstream from the -35 and -10 sequences, which may be involved in regulation of transcription. In the intergenic region there is a third FokI recognition site in an inverted repeat forming a stem-loop structure within the dicistronic mRNA that had to be removed to overproduce R.FokI (11). No regulatory role can be expected from methylation of this intergenic recognition site.


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ACKNOWLEDGMENTS
 
This research was supported by the Programme Committee for Nutrition and Food Research (FELFO) under FØTEK 3.

We thank Todd R. Klaenhammer and Douwe van Sinderen for providing plasmids pTRK390 and pNZ44, respectively. We are grateful to Karin Hammer for valuable suggestions concerning the manuscript.


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Dairy and Food Science, The Royal Veterinary and Agricultural University, Rolighedsvej 30, DK-1958 Frederiksberg C, Denmark. Phone: 45 35 28 32 32. Fax: 45 35 28 32 14. E-mail: jyj{at}kvl.dk. Back


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Journal of Bacteriology, January 2004, p. 287-295, Vol. 186, No. 2
0021-9193/04/$08.00+0     DOI: 10.1128/JB.186.2.287-295.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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