Laboratory of Microbial Gene Technology, PO Box 5003, Norwegian University of Life Sciences, N-1432 Ås, Norway
Correspondence
Morten Skaugen
morten.skaugen{at}umb.no
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ABSTRACT |
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Present address: Norwegian Defence Research Establishment, PO Box 25, N-2027 Kjeller, Norway.
Present address: Department of Chemistry, Biotechnology and Food Science, Agricultural University of Norway, PO Box 5003, N-1432 Ås-NLH, Norway.
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INTRODUCTION |
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LasX belongs to a group of regulators often referred to as the Rgg-like regulators (Skaugen et al., 2002), which are found exclusively in Gram-positive bacteria, and are reported to regulate expression of target genes by stimulating transcription initiation. Rgg was originally discovered in Streptococcus gordonii, where it was demonstrated to regulate the expression of the glucosyltransferase gene gtfG (Sulavik et al., 1992
; Sulavik & Clewell, 1996
). Since then, a number of Rgg-like regulators have been described: the Streptococcus oralis Rgg regulates the expression of the glucosyltransferase gene (gtfR) (Fujiwara et al., 2000
), RopB (also known as Rgg) regulates the expression of the virulence gene speB in Streptococcus pyogenes (Chaussee et al., 1999
; Lyon et al., 1998
), GadR regulates the expression of genes involved in glutamate-dependent acid resistance in Lactococcus lactis (Sanders et al., 1998
), and MutR regulates the expression of genes involved in biosynthesis of the lantibiotic mutacin II in Streptococcus mutans (Qi et al., 1999
). With the completion of the genome sequences of S. pyogenes (Ferretti et al., 2001
), Streptococcus pneumoniae (Tettelin et al., 2001
), S. mutans (Ajdi
et al., 2002
), Streptococcus agalactiae (Glaser et al., 2002
), Listeria monocytogenes and Listeria innocua (Glaser et al., 2001
), several additional rgg-like genes have been identified, suggesting that transcription regulation mediated by Rgg-like proteins may be common among Gram-positive organisms.
Although most of the known Rgg-like proteins appear to be associated with a single target gene, recent reports on the S. pyogenes Rgg indicate that this class of proteins may also have more global regulatory functions in some organisms (Chaussee et al., 2001, 2002
, 2003
).
The Rgg-like regulators described to date (Fujiwara et al., 2000; Lyon et al., 1998
; Qi et al., 1999
; Sanders et al., 1998
; Sulavik et al., 1992
; Sulavik & Clewell, 1996
) have in common a predicted N-terminal helixturnhelix motif, a structure known to be involved in the recognition and binding of DNA by many transcriptional regulators (Huffman & Brennan, 2002
; Wintjens et al., 1996
). However, there are no obvious common binding-site motifs in the promoter regions of the genes regulated by the Rgg-like proteins. Nevertheless, it was recently demonstrated that Rgg of S. gordonii can bind DNA upstream of gtfG (Vickerman et al., 2003
), and that RopB specifically binds within the ropBspeB intergenic region of S. pyogenes (Neely et al., 2003
).
In this report, we present results strongly suggesting that LasX, too, controls the transcription of lactocin S biosynthetic genes by binding to a specific DNA sequence within the promoter region of these target genes.
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METHODS |
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The amplification of DNA was performed in 50 or 100 µl reactions employing Taq polymerase (Qiagen) or the proofreading DNA polymerase Platinum Pfx (Invitrogen), according to the manufacturer's instructions. The thermocyclers employed in DNA amplification were PTC100 (MJ Research) and Mastercycler gradient (Eppendorf). DNA sequencing was performed using BigDYE sequencing premix (Applied Biosystems) and the ABI Prism 377 sequencer (Perkin Elmer/Applied Biosystems).
Construction of the LasX expression plasmid pER11.
The expression plasmid pER11 was constructed as follows: lasX was amplified from pLas7.5-1 (Skaugen et al., 1997) using the primers EA-X-NdeI-f and EA-X-XhoI-r (Table 2
), and the resulting amplicon was cloned into the vector pCR-Blunt II-TOPO, yielding pER1. The NdeIXhoI fragment of pER1 containing lasX was then subcloned into the NdeIXhoI-digested pET22b(+) expression vector to produce the LasX-expression plasmid pER11.
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Overproduction of LasX, and partial purification of His-LasX.
The plasmid pET22b(+) (Novagen) and the lasX-expression plasmid pER11 were used to transform strain BL21-SI, in which the T7 RNA polymerase is under the control of the NaCl-inducible proU promoter. The resulting strains were grown to mid-exponential phase (OD600 approx. 0·5) in LBON medium supplemented with 400 µg carbenicillin ml1. IPTG and NaCl were added to final concentrations of 0·5 mM and 0·3 M, respectively, and incubated at 30 °C for a further 4 h. The cells were harvested by centrifugation at 3000 g for 15 min at 4 °C, then resuspended in buffer A (20 mM Tris/HCl, pH 8·4; 0·3 M NaCl; 0·1 % Tween 20; 10 %, v/v, glycerol; 10 mM imidazole), and lysed using a French press. The resulting homogenate was centrifuged at 12 000 g at 4 °C for 30 min, in order to remove insoluble cell debris. The crude soluble protein extract was subsequently loaded onto a His-Bind Quick column (Novagen) equilibrated with buffer A. After the column had been washed with 25 ml buffer A and 25 ml buffer B (20 mM Tris/HCl, pH 8·4; 0·3 M NaCl; 0·1 % Tween 20; 10 % glycerol; 20 mM imidazole), the protein was eluted with buffer C (20 mM Tris/HCl, pH 8·4; 0·3 M NaCl; 0·1 % Tween 20; 10 % glycerol; 0·5 M imidazole). Fractions of the partially purified protein extracts were stored in 50 % glycerol at 20 °C.
Protein samples were analysed by SDS-PAGE using 12·5 % polyacrylamide gels, according to standard procedures (Laemmli, 1970). The Rainbow protein molecular-mass marker (Amersham Pharmacia Biotech) was used as a molecular size marker. Protein concentrations were determined using the BCA protein assay (Pierce).
Electrophoretic mobility-shift assay with LasX-H6.
DNA binding of partially purified LasX-H6 was analysed using electrophoretic mobility-shift assays (EMSAs) (Fried, 1989). The primers used to generate the double-stranded DNA fragments used in the EMSAs are listed in Table 2
. The intergenic region of lasAlasX was amplified by PCR with the primers AX-f1 and AX-r1, and the negative control fragment was amplified from the lasM gene (Skaugen et al., 1997
) using the primers M-f1 and M-r1 (Table 2
). In both cases, pLas7.5-1 was used as a template. DNA probes P1P8 (Fig. 1
) were generated by annealing the primers BS1 or BS2 (Table 1
) to the 3' end of the synthetic single-stranded oligonucleotides P1, P4, P5, P8, and P2, P3, P6, P7, respectively (Fig. 1
), and carrying out a primer-extension reaction using the Klenow enzyme (Sambrook & Russell, 2001
). Annealing the single-stranded synthetic oligonucleotides P9P19 (see Fig. 5
) to their complementary strands generated the double-stranded DNA probes P9P19. The double-stranded DNA probes were end-labelled (Sambrook & Russell, 2001
) with 32P by using T4 polynucleotide kinase (Fermentas) and [
-32P]ATP (Amersham Pharmacia Biosciences).
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Reporter-plasmid construction.
A 3 kb fragment containing the E. coli lacZ gene was isolated from pCH-Z (C. Halvorsen & M. Skaugen, unpublished) by digestion with the enzymes AgeI and XbaI, and subsequently ligated into the XmaIXbaI-digested pELS100 (M. Skaugen, unpublished), yielding the expression vector pER30.
Using pUC7.5-1 (Skaugen et al., 1997) as the template, and the primers P1 (Fig. 1
) and 221002-rX (Table 2
), the complete lasAX intergenic region was amplified. This amplicon, designated RepAmp1, was purified and used as a template for a second PCR reaction, this time using either of the primer pairs F1-X221002-rP or F1-P221002-rX. Following restriction enzyme digestion, the PCR products were ligated to PstIXhoI-digested pER30, giving rise to pER301P and pER301X (see Table 1
and Fig. 7
), respectively. The plasmids pER302PpER308P were constructed in a similar manner, using RepAmpI as the initial template, and replacing P1 with one of the forward primers P2P8.
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RESULTS |
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The presence of the N-terminal HTH motif of LasX, and the phenotype of the lasX mutants (Skaugen et al., 2002), suggested that LasX may regulate the transcriptional activities of the PlasXY and PlasAW promoters by binding DNA sequences in the intergenic region. To test this hypothesis, EMSAs were carried out. A 289 bp PCR-amplified DNA fragment, which spans the entire 180 bp intergenic lasAlasX region, as well as part of the 5' end of the lasX gene, and part of the 5' end of the lasA gene (Fig. 1a
, AX probe), was incubated with partially purified LasX-H6. As can be seen from Fig. 2
, the mobility of the DNA fragment was shifted in the presence of partially purified LasX-H6. To verify that this mobility shift was due to the DNA probe forming a complex with LasX-H6, and not another component of the partially purified LasX-H6 extract, the DNA fragment was incubated with protein extract from cells harbouring the expression vector without the insert. As can be seen from Fig. 2
, no shift in the mobility of the DNA fragment was observed. In order to verify the specificity of LasX-H6 binding activity, an EMSA was carried out using a 289 bp PCR fragment arbitrarily chosen from within the lasM gene. No mobility shift of the lasM fragment was observed after incubation with partially purified LasX-H6 (Fig. 2
). Taken together, these results demonstrate that LasX is a DNA-binding protein, and that a binding site is located on the 289 bp fragment covering part of the 5' end of lasX, the intergenic region and part of the 5' end of lasA.
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In order to define a minimum binding site for LasX binding, DNA probes P10P19 (Fig. 6a) were synthesized, and subjected to EMSA analysis. As shown in Fig. 6(b), a
mobility shift was observed for the probes P12, P13, P14 and P19, thereby localizing the binding site of LasX to within a 19 bp region situated directly upstream of the PlasAW 35 element. The reason for the weak complex formation observed for P14 is unclear.
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-Galactosidase expression from the PlasAWlacZ fusion construct (pER301P), in the absence of LasX (pMS6607c), was close to background expression levels (pER30/pMS6607c). In the presence of LasX (pELA50),
-galactosidase activity was 114-fold higher than in the control (pER30/pELA50). These results are in agreement with the previously reported observation (Rawlinson et al., 2002
) that LasX positively activates PlasAW, and also indicates that the 5' UTR is important for the LasX-stimulated transcription of lasAW.
For the PlasXYlacZ fusion (pER301X) in the absence of LasX (pMS6607c), the -galactosidase activity was approximately 2·5 times above background level. In the presence of LasX (pELA50), this activity was reduced approximately twofold. Again, this is consistent with the results obtained in the previous study (Rawlinson et al., 2002
), indicating that PlasXY is a weak constitutive promoter, which is downregulated by LasX.
The P2P8 fragments were modified as described above for P1, cloned into pER30 in the reverse PlasAWlacZ orientation, and the clones assayed for -galactosidase activity in the presence or absence of LasX (Fig. 7
). When all three repeats were substituted (pER302P), no promoter activity above background was detected in the presence or absence of LasX (pELA50 and pMS6607c, respectively). When the middle repeat was present, the substitution of the left and right repeats (pER303P and pER305P, respectively) resulted in a moderate reduction of LasX-dependent increase in promoter activity. In contrast, when the middle repeat was substituted, LasX-independent expression was observed for all configurations (pER304P, pER306P and pER308P).
Identification of conserved nucleotides within the promoter region of genes regulated by Rgg-like regulators
An alignment of the lasXlasA intergenic region with the corresponding region of the gtfG promoter region of S. gordonii demonstrated that the 19 bp LasX-binding site overlaps with a 36 bp sequence shown to be essential for Rgg-dependent stimulation of transcription from the gtfG promoter. The percentage of identical residues was slightly higher in the overlap compared to the entire alignment (37 versus 33 %), suggesting some degree of conservation of the 38 to 56 region (not shown). When the corresponding promoter segments from the Streptococcus oralis gtfR gene, the S. mutans mutacin II and mutacin 1140 mutA genes, and the L. lactis gadR gene were included (Fig. 8a), the number of identical residues in the 74 nt alignment was reduced to three, all of which are situated within the region corresponding to the proposed LasX binding site.
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DISCUSSION |
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The inactivation of lasX leads to a strong reduction in the levels of lasA (and lasAW) mRNA (Skaugen et al., 2002), suggesting that the primary function of LasX is the stimulation of transcription from PlasAW. In cases where the issue has been addressed experimentally, it has been demonstrated that other Rgg-like proteins have similar functions. Consistent with their proposed function as regulators, the Rgg-like proteins all possess an N-terminal helixturnhelix motif, which is a structure demonstrated to facilitate specific DNA binding for a number of other proteins involved in regulation of transcription. DNA binding has been demonstrated for two of the Rgg-like regulators: Rgg of S. gordonii (Vickerman et al., 2003
), and RopB of S. pyogenes (Neely et al., 2003
). Although the Rgg proteins are clearly functionally as well as structurally related, the corresponding target promoters have no obvious common features to suggest conservation of a (putative) Rgg binding site. In the target promoters of the mutacin II (Qi et al., 1999
), gtfG (Sulavik et al., 1992
; Vickerman et al., 2003
), speB (Lyon et al., 1998
; Neely et al., 2003
) and gadR (Sanders et al., 1998
) systems, inverted repeat structures upstream of the promoters have been proposed as candidate binding sites. The most conspicuous structures in the proposed las regulatory region are the two identical heptanucleotide (TTATCCC) motifs in the 40 to 70 region relative to the lasA transcription initiation point. In addition, a third, imperfect copy (TTtaCCC) of this motif overlaps the 10 element of PlasAW (Skaugen et al., 2002
).
The main purpose of the present study was to establish whether or not LasX binds specifically to the promoter region, and, if so, to analyse the role of the direct repeats in this putative proteinDNA interaction. The results of the EMSA experiments demonstrated conclusively that only the middle repeat in the promoter region is required for LasX binding, and, by analysing the ability of LasX to bind DNA fragments shortened from either side of the heptanucleotide motif, a tentative 19 bp minimum binding site was identified (Fig. 6).
Although the in vitro studies suggested that LasX does not bind to the flanking repeats, the possibility that they constitute low(er) affinity binding sites escaping detection under the experimental conditions used cannot be excluded. A reporter-gene assay was therefore set up to further investigate the role of the repeat unit in LasX-mediated regulation of gene expression in vivo. The results of these assays confirmed the results of the previous promoterreporter-gene study (Rawlinson et al., 2002), and demonstrated that LasX has a stimulatory effect on PlasAW (pER301P, Fig. 7
) activity, and that PlasXY is a weak constitutive promoter, which is downregulated in the presence of LasX (pER301X, Fig. 7
). Interestingly, the reporter system used in this study seems to be more sensitive than the one used previously (Rawlinson et al., 2002
), leading to an improved correlation between the observed mRNA levels (Skaugen et al., 2002
) and the promoter activity determined by the reporter-gene assay.
The results reported above indicate that the presence of the middle repeat is required for LasX-dependent activation of PlasAW (pER304P and pER307P, Fig. 7). The data also show that while the flanking repeats were not essential for induction of PlasAW, their substitution did cause a moderate reduction in maximal expression levels. It still cannot be excluded, therefore, that the flanking repeats may be involved in modulating the interaction of LasX with PlasAW.
The position of the proposed binding site suggests that LasX-mediated transcription stimulation may involve proteinprotein contact between LasX and RNA polymerase (Hochschild & Dove, 1998; Rhodius & Busby, 1998
). Given that the other members of the Rgg protein family have the same mechanism of transcription activation, it follows that the distance between the transcription initiation point and the transcription factor binding site should be approximately equal for all promoters activated by an Rgg-like protein. One implication of this assumption is that once the transcription start point of an Rgg-activated promoter has been determined, the activator-binding site may subsequently be identified simply by aligning the sequences upstream of the start point.
Following this line of reasoning, a segment of the lasXlasA intergenic region was aligned with the corresponding regions from six other promoters reported or likely to be recognized by Rgg-like proteins. The alignment (Fig. 8a) shows a low overall similarity between the different promoters, but a short consensus motif can be identified. This motif is situated within the proposed LasX-binding site, as well as the 36 bp region recently shown to be required for Rgg-dependent gtfG transcription in S. gordonii (Vickerman & Minick, 2002
). Interestingly, the close relationship between LasX and RopB is reflected in an alignment of the respective target promoter regions. This alignment also highlights the presence in the proposed LasX binding site similarity of a short palindrome, which could be involved in the binding of the regulator.
In the present study, we have shown that LasX is a DNA-binding protein and, through in vitro DNA-binding studies complemented with in vivo reporter-gene analysis, a 19 bp region immediately upstream of the PlasAW 35 region was identified as the LasX-binding site. These results provide a starting point for future, more detailed analyses of the molecular interaction between an Rgg-like regulator and its DNA target. They also provide an experimental basis for addressing unresolved issues associated with the regulation of lactocin S production, such as the influence of pH, temperature and oxygen on the lantibiotic production phenotype.
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ACKNOWLEDGEMENTS |
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Received 28 May 2004;
revised 11 November 2004;
accepted 30 November 2004.
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