Identification of the DNA-binding site of the Rgg-like regulator LasX within the lactocin S promoter region

Elizabeth L. Andersen Rawlinson{dagger}, Ingolf F. Nes and Morten Skaugen{ddagger}

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


   ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
LasX regulates the transcription of the divergent operons lasXY and lasAW, which specify the production of lactocin S in Lactobacillus sakei L45. Using histidine-tagged LasX, and a DNA fragment containing the complete intergenic lasAlasX region, electrophoresis mobility-shift (EMSA) analyses were employed to demonstrate that LasX binds to the lasA–lasX intergenic DNA. Two direct heptanucleotide motifs directly upstream of PlasA–W, and a third imperfect copy of this motif, overlapping the –10 element of PlasA–W, were identified as possible LasX-binding sites. To assess the role of the direct repeats in the binding of LasX to the intergenic lasA–lasX region, binding experiments were performed using DNA probes with different combinations of the repeats, and with arbitrarily chosen repeat substitutions. The result of these experiments demonstrated that only the middle repeat was required for the binding of LasX to the las-promoter region. This observation correlated with the results of subsequent reporter-gene analyses, thereby weakening the hypothesis of the involvement of the direct repeats in LasX-mediated transcription regulation. By analysing the ability of LasX to bind successively shortened derivatives of the original intergenic fragment, a tentative 19 bp minimum LasX-binding site was identified.


Abbreviations: EMSA, electrophoretic mobility-shift assay; UTR, untranslated region

{dagger}Present address: Norwegian Defence Research Establishment, PO Box 25, N-2027 Kjeller, Norway.

{ddagger}Present address: Department of Chemistry, Biotechnology and Food Science, Agricultural University of Norway, PO Box 5003, N-1432 Ås-NLH, Norway.


   INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Lactocin S (Mørtvedt & Nes, 1990) is a 37-residue lantibiotic produced by Lactobacillus sakei strains. A total of 11 genes, which are clustered together on a 50 kb plasmid termed pCIM1, specify the production of lactocin S in the original producer strain L. sakei L45 (Skaugen et al., 1997, 2002). The gene cluster is organized into two divergently oriented operons, lasAMNTUVPJW (lasAW) and lasXY; the former contains the biosynthetic, immunity and transport genes (Skaugen et al., 2002). It was recently established that the gene lasX encodes a protein involved in the transcriptional regulation of both the lasA–W and lasXY operons (Rawlinson et al., 2002). Using reporter-gene fusions, it was demonstrated that expression of lasX, supplied in trans, affected transcription from both of the promoters PlasA–W and PlasXY. LasX was shown to activate PlasA–W, while it repressed its own promoter PlasXY (Rawlinson et al., 2002). In light of these observations, and given that the predicted LasX protein has an amino-terminal helix–turn–helix motif (Skaugen et al., 2002), it was proposed that LasX might act by binding directly to the promoter region, as is the case for most transcriptional activators and repressors. Two directly repeated heptanucleotide motifs (TTATCCC) located directly upstream of the PlasA–W –35 region were identified as candidate DNA binding sites (Skaugen et al., 2002).

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 (Ajdic 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 helix–turn–helix 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.


   METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Bacterial strains and growth conditions.
Bacterial strains and plasmids used in this study are listed in Table 1. Escherichia coli Top10 (Invitrogen) was cultivated at 37 °C in Luria–Bertani (LB) broth with agitation, or on LB agar (1·5 %, w/v). When required, the medium was supplemented with 300 µg erythromycin ml–1, 25 µg chloramphenicol ml–1 or 100 µg ampicillin ml–1 (Sigma). E. coli BL21-SI (Invitrogen) was grown at 37 °C in LBON (LB without NaCl) broth with agitation, or on LBON agar (1·5 %, w/v); the medium was supplemented with 100 µg ampicillin ml–1, unless otherwise stated. L. sakei RV2002 strains were grown in Man–Rogosa–Sharpe (MRS) broth. When appropriate, the MRS medium was supplemented with 10 µg chloramphenicol ml–1 and/or 5 µg erythromycin ml–1. MRS agar (1·5 %, w/v) containing 1·5 µg erythromycin ml–1 and/or 5 µg chloramphenicol ml–1 was used for initial selection of L. sakei transformants.


View this table:
[in this window]
[in a new window]
 
Table 1. Bacterial strains and plasmids

See text for plasmid construction details.

 
General DNA methodology.
Standard procedures were used for DNA manipulations (Sambrook et al., 1989). Enzymes used in restriction digestion and cloning were purchased from Promega, New England Biolabs and MBI Fermentas, and used as recommended by the manufacturers. Plasmid DNA was isolated from the E. coli strains and L. sakei RV2002 using Qiaprep Spin Miniprep Kit (Qiagen). Before the addition of the lysis solution during plasmid isolation, the L. sakei cells were incubated for 30 min at 37 °C in the presence of 5 mg lysozyme ml–1, 100 µg RNase ml–1 and 15 U mutanolysin ml–1 to aid cell lysis. QIAex PCR Purification Kit (Qiagen) was used for the purification of PCR products, and also in the extraction and purification of DNA from agarose gels. Initial cloning was carried out using chemically competent E. coli Top10. Purified plasmid was then introduced into chemically competent E. coli BL21-SI cells for protein expression. Plasmid transformation of L. sakei RV2002 was carried out by electroporation using the Bio-Rad Gene pulser, according to the protocol of Berthier et al. (1996).

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 NdeI–XhoI fragment of pER1 containing lasX was then subcloned into the NdeI–XhoI-digested pET22b(+) expression vector to produce the LasX-expression plasmid pER11.


View this table:
[in this window]
[in a new window]
 
Table 2. Oligonucleotide primers

Relevant restriction sites are in bold.

 
All plasmid constructs were sequenced to verify that no errors had been introduced during PCR and general handling. All plasmids are listed in Table 1.

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 ml–1. 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 P1–P8 (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 P9–P19 (see Fig. 5) to their complementary strands generated the double-stranded DNA probes P9–P19. The double-stranded DNA probes were end-labelled (Sambrook & Russell, 2001) with 32P by using T4 polynucleotide kinase (Fermentas) and [{gamma}-32P]ATP (Amersham Pharmacia Biosciences).



View larger version (34K):
[in this window]
[in a new window]
 
Fig. 1. Overview of the DNA probes AX and P1–P8 used in the EMSA experiments. (a) The upper diagram illustrates the lasAlasX intergenic region, and shows the organization and position of the promoters PlasA–W and PlasXY in relation to their respective genes. A schematic presentation of the AX probe used in DNA-binding experiments is shown underneath. The AX probe spans the entire 180 bp lasAlasX intergenic region, and part of the 5' end of both the lasX and lasA genes. The positions of the PlasA–W and PlasXY –35 and –10 elements, as well as the transcript initiation site of both lasXY and lasA–W, are indicated. (b) The sequences of the upper strands of the DNA probes P1–P8 used in EMSA experiments to determine the role of the direct repeats in the binding of LasX. The heptanucleotide repeats are in bold. The repeat substitutions are represented by underlined, lower-case letters.

 


View larger version (39K):
[in this window]
[in a new window]
 
Fig. 5. EMSA of probe P9 incubated with LasX-H6. Varying amounts (approx. 1·87, 3·75, 7·5, 15 and 30 ng) of partially purified LasX-H6 were incubated with DNA probe P9 (Fig. 5). Lanes: +, the binding reaction was conducted with LasX-H6; –, binding reactions were conducted with partially purified protein extract from cells not expressing LasX-H6 (approx. 30 ng); 0, no protein extract was added to the binding reaction.

 
Binding of LasX-H6 to DNA was carried out in a 20 µl reaction mixture containing 2·5 fmol [32P]DNA, 1 µg poly(dI-dC) (Amersham Pharmacia Biosciences), partially purified protein of concentrations ranging from 0 to 60 ng, and the appropriate amount of buffer (20 mM Tris/HCl, pH 8·4; 50 mM NaCl; 0·1 % Tween 20; 10 % glycerol; 1 mM EDTA). The DNA binding reactions were initiated by the addition of LasX-H6, and incubated at 30 °C for 30 min. Samples were then loaded on a pre-run 10 % non-denaturing polyacrylamide gel. The gels were run at 12 V cm–1 in 1x TBE buffer (89 mM Tris base, 89 mM boric acid, 2 mM EDTA) for 0·5–2·5 h at 4 °C. The gels were subsequently dried for 1 h at 80 °C, and the DNA–protein complexes were detected by direct autoradiography with Kodak Biomax MS film at –80 °C for 3–15 h.

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 XmaI–XbaI-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-X–221002-rP or F1-P–221002-rX. Following restriction enzyme digestion, the PCR products were ligated to PstI–XhoI-digested pER30, giving rise to pER301P and pER301X (see Table 1 and Fig. 7), respectively. The plasmids pER302P–pER308P were constructed in a similar manner, using RepAmpI as the initial template, and replacing P1 with one of the forward primers P2–P8.



View larger version (38K):
[in this window]
[in a new window]
 
Fig. 7. Analysis of the role of the three repeats in LasX-dependent regulation of PlasA–W. The figure shows a graphical representation of DNA probes P1–P8 fused to the reporter gene lacZ in the promoter-probe vector pER30. The repeats are shown as boxes. The transcription start sites of lasA–W and lasXY are shown, and they highlight the orientation of the promoter fragments with respect to lacZ. The plasmids were introduced into L. sakei RV2002 harbouring pELA50 or pMS6607C in order to assay the promoter activity in the presence and absence of LasX, respectively. {beta}-Galactosidase assays were performed as described in Methods. The data presented are the mean values of three {beta}-galactosidase assays (±SD). {beta}-Galactosidase actvity is given in arbitrary units (see Methods).

 
{beta}-Galactosidase assay.
lacZ expression in L. sakei RV2002 was assayed as follows. Bacterial cells from 10 ml culture were harvested in the late-exponential phase of growth (OD600 ~0·8), and resuspended in 500 µl Z buffer (100 mM sodium phosphate, pH 7; 10 mM KCl; 1 mM MgSO4; 50 mM {beta}-mercaptoethanol; 20 % glycerol). An 80 µl aliquot of the cell suspension was removed and transferred to a microtitre plate, and 30 µl acetone/toluene (9 : 1, v/v) was added to each aliquot, followed by incubation at 37 °C for 10 min to permeabilize the cells. Following this, 80 µl ONPG (4 mg ml–1) was added, and incubation continued at 30 °C. After 15 min, the reaction was stopped by adding 110 µl 1 M Na2CO3. Cellular debris was removed by centrifugation, and 200 µl supernatant was transferred to a new microtitre plate, and the A405 of the reaction mixture was measured using the Multiskan Ascent microtitre plate reader (Labsystems). These readings were converted to enzyme activity units (arbitrary units, AU) using the following formula (modified from Miller, 1972): (1000xA405)/(txvxOD600), where t is the assay incubation time in minutes, and v is the culture volume in millilitres.


   RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
LasX binds the intergenic lasAlasX region
LasX-H6 was cloned and expressed as described in Methods. Affinity chromatography purification of LasX-H6 was performed, leading to partial purification of the target protein. To obtain a negative control for the subsequent EMSA experiments, an extract from identically treated E. coli BL21 cells containing the expression vector pET22b(+) was prepared in parallel (results not shown).

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 PlasA–W 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.



View larger version (36K):
[in this window]
[in a new window]
 
Fig. 2. Electrophoretic mobility shift assay (EMSA) of DNA probes AX and M incubated with LasX-H6. AX represents a DNA fragment that contains the lasAlasX intergenic region (Fig. 1), whereas M represents a DNA fragment containing an arbitrarily chosen sequence from within the lasM gene (Skaugen et al., 1997). The AX probe was incubated with increasing amounts (approx. 1·87, 3·75, 15, 30, 45 and 60 ng) of partially purified LasX-H6, whereas the M probe was incubated with approximately 60 ng partially purified LasX-H6 (lanes marked +). Lanes –, partially purified protein extract from cells not expressing LasX-H6 (approx. 60 ng) was added to the binding reaction; lanes 0, no protein was added to the binding reaction.

 
Identification of the specific LasX DNA-binding site(s)
Transcription activators usually bind to specific DNA sequence motifs immediately upstream of their target promoters, and a candidate regulatory site was recently identified in the lasA–X intergenic region (Skaugen et al., 2002). This proposed cis-acting regulatory element consists of two heptanucleotide direct repeats (TTATCCC) situated directly upstream of the PlasA–W –35 element (Fig. 1), in the –40 to –70 region relative to the lasAW transcript initiation site. A third, imperfect repeat (TTtaCCC), overlapping the –10 element of PlasA–W (Fig. 1b), was also identified (Skaugen et al., 2002). In order to determine whether or not these repeats are targets for LasX, an EMSA was carried out using a DNA probe (P1) that spans the promoter elements as well as the three repeats, and a DNA probe (P2) of the same length in which all three repeats were replaced by an arbitrarily chosen sequence (Fig. 1). The DNA probes were incubated with partially purified LasX-H6, and, as can be seen in Fig. 3, the presence of LasX-H6 created a shift in the mobility of probe P1. No shift in the mobility of the DNA probe P2 was observed, supporting the notion that the repeats may be involved in the binding of LasX to the lasAlasX promoter region.



View larger version (45K):
[in this window]
[in a new window]
 
Fig. 3. EMSA of DNA probes P1 and P2 incubated with LasX-H6. The DNA-binding experiment was conducted with an 89 bp DNA probe, which contained the lactocin S promoters, as well as the three heptanucleotide repeats (P1), and the DNA probe of the same length in which the repeat had been replaced by the randomly chosen sequence (P2). The DNA probes P1 and P2 (Fig. 1) were incubated with increasing amounts (approx. 1·87, 3·75, 15 and 30 ng) of partially purified LasX-H6 (lanes marked +). Lanes –, DNA probes were incubated with partially purified protein extract from cells not expressing LasX-H6 (approx. 30 ng); lanes 0, no protein was added to the binding reaction.

 
In order to evaluate the significance of the individual repeat units in the binding of LasX, additional binding experiments were carried out. The DNA probes P3, which contains the first two repeats, P4, which contains the first and third repeat, and P5, which contains the middle and third repeat (Fig. 1), were incubated with partially purified LasX-H6, and analysed by EMSA. As can be seen from the results in Fig. 4(a), a mobility shift was observed with probes P3 and P5, but not with probe P4. When using DNA fragments containing just a single repeat (P6–P8), mobility shift was observed only for P7, which retains the middle repeat unit. These results strongly suggest that binding of LasX is independent of the flanking repeats.



View larger version (80K):
[in this window]
[in a new window]
 
Fig. 4. EMSA of DNA probes P1–P8 incubated with LasX-H6. Partially purified LasX-H6 (3·75 ng) was incubated with 32P-labelled DNA probes P1–P8 (Fig. 1). Lanes: +, the binding reaction was performed with partially purified LasX-H6; 0, no protein was added to the binding reaction.

 
LasX binds to nucleotides within a 19 bp fragment upstream of the PlasA–W –35 element
In light of the results reported above, where the sequence containing the middle repeat unit was shown to be sufficient for LasX binding, an analysis using a 38 bp DNA fragment (P9) spanning the middle repeat, but excluding the first and third repeats (nucleotides 23–60 of P1, Fig. 1b), was performed. The results of this experiment (Fig. 5) show that the mobility of the DNA probe P9 was altered after incubation with partially purified LasX-H6, indicating DNA–protein complex formation.

In order to define a ‘minimum’ binding site for LasX binding, DNA probes P10–P19 (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 PlasA–W –35 element. The reason for the weak complex formation observed for P14 is unclear.



View larger version (37K):
[in this window]
[in a new window]
 
Fig. 6. (a) Overview of the DNA probes P10–P19 used in EMSAs to identify the binding site of LasX. The nucleotide sequences of the upper strands of the DNA probes P10–P19 are shown in relation to the DNA probes P1 and P9 used in earlier EMSAs (Figs 3, 4 and 5). The repeats are highlighted in bold. (b) EMSA of DNA probes P10–P19 incubated with LasX-H6. Lanes: +, the binding reaction was conducted with partially purified LasX-H6 (approx. 30 ng); 0, no protein was added to the binding reaction.

 
Reporter-gene studies
Although the results reported above suggest that LasX does not bind the first and last repeat, it cannot be excluded that these constitute low(er) affinity binding site(s) which escape detection in the EMSA experiments. Therefore, in order to assess the in vivo significance of the three repeats in the LasX-dependent regulation of PlasA–W, a reporter assay was set up. A reporter vector (pER30) was constructed using lacZ from E. coli as a reporter gene. Initially, the P1–P8 (Fig. 1b) fragments were amplified using the primer pair F1-X–R1-P or F1-P–R1-X (Table 2). The resulting amplicons were digested with XhoI and PstI, and inserted in front of the lacZ reporter gene of pER30. The resulting constructs were introduced into L. sakei RV2002 harbouring the lasX-expression plasmid pELA50 or the control vector pMS6607c (Table 1), allowing the promoter to be analysed in the presence and absence of LasX, respectively. However, no {beta}-galactosidase activity was detected in cells harbouring the PlasA–WlacZ fusion construct in the presence of LasX (results not shown). This was in contradiction to the results of a previous promoter–reporter-gene study (Rawlinson et al., 2002), where LasX was shown to have a clear stimulatory effect on PlasA–W. Although the DNA probe P1 (Fig. 1b) contains both PlasA–W and PlasXY, and encompasses the transcription start sites of both lasXY and lasA–W, it lacks a 110 bp region of the lasA–W 5' untranslated region (UTR), which was included in the previously reported reporter constructs (Rawlinson et al., 2002). The DNA probe P1 was consequently modified to include the 110 bp 5' UTR (see Methods), before being cloned into the reporter vector pER30. The resulting constructs pER301P and pER301X (Table 1) were subsequently introduced into the expression host L. sakei RV2002 carrying pELA50 and pMS6607c (Table 1). Cell extracts of these two-plasmid L. sakei RV2002 clones were prepared, and promoter activity was analysed by measuring {beta}-galactosidase activity. The results are presented in Fig. 7.

{beta}-Galactosidase expression from the PlasA–WlacZ fusion construct (pER301P), in the absence of LasX (pMS6607c), was close to background expression levels (pER30/pMS6607c). In the presence of LasX (pELA50), {beta}-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 PlasA–W, and also indicates that the 5' UTR is important for the LasX-stimulated transcription of lasA–W.

For the PlasXYlacZ fusion (pER301X) in the absence of LasX (pMS6607c), the {beta}-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 P2–P8 fragments were modified as described above for P1, cloned into pER30 in the reverse PlasA–WlacZ orientation, and the clones assayed for {beta}-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.



View larger version (33K):
[in this window]
[in a new window]
 
Fig. 8. Alignment of the promoter region of lasA with the target promoter of other Rgg-like regulators. (a) Alignment with the gtfG promoter of S. gordonii (Sulavik et al., 1992; Vickerman & Minick, 2002), the gtfR promoter of S. oralis (Fujiwara et al., 2000), the promoters of mutA of the mutacin II (Qi et al., 1999) and mutacin 1140 (Hillman et al., 1998) systems in S. mutans, the promoter of gadCB of L. lactis (Sanders et al., 1998), and the promoter of speB from S. pyogenes (Lyon et al., 1998; Neely et al., 2003). The –19 nucleotide LasX-binding site that lies within the –38 to –56 region of the lasAlasX intergenic region is indicated. The 36 bp section of the gtfG promoter region that is essential in Rgg regulation of gtfG (Vickerman & Minick, 2002) and the –10 elements of the promoter regions are underlined. The identified transcription initiation sites are in bold. The distance of the transcript initiation site from the coding sequence of the target gene is indicated in parentheses. Conserved residues are shaded, and invariant residues are indicated by asterisks. (b) Alignment of the lasA promoter region with the corresponding region of the ropBspeB intergenic region. Identical nucleotides are shaded.

 
An alignment of the lasA promoter segment with the corresponding promoter segment of S. pyogenes speB (Fig. 8b) alone showed only a 27 % overall identity, but almost 53 % identity (10 out of 19) in the –38 to –56 region. This alignment also points to the presence of a perfect inverted repeat, 5'-TAATATATTA-3', which is included in the region required for LasX binding in vitro, and overlaps the directly repeated heptanucleotide.


   DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The pCIM1 lactocin S locus is composed of the tightly clustered, oppositely oriented lasXY and lasA–W operons, both of which are required for the expression of the lactocin S production phenotype (Skaugen et al., 1997, 2002). While the nine-ORF lasA–W operon encodes the pre-lactocin S peptide (LasA), as well as putative biosynthetic, immunity, processing and transport proteins, it has been shown that lasX, which is the proximal of the two genes in the divergently transcribed lasXY operon, is involved in the regulation of lasA–W gene expression. The two las operons are separated by a 180 bp non-coding region, and through reporter-gene experiments, it was recently demonstrated that the expression of lasX in trans affected the transcription from the lasXY promoter (PlasXY), as well as from the PlasA–W promoter (Rawlinson et al., 2002). It was shown that LasX activated PlasA–W, whereas it repressed PlasXY. The lasX gene product LasX belongs to the weakly interrelated group of regulatory proteins often referred to as Rgg-like regulators.

The inactivation of lasX leads to a strong reduction in the levels of lasA (and lasA–W) mRNA (Skaugen et al., 2002), suggesting that the primary function of LasX is the stimulation of transcription from PlasA–W. 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 helix–turn–helix 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 PlasA–W (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 protein–DNA 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 promoter–reporter-gene study (Rawlinson et al., 2002), and demonstrated that LasX has a stimulatory effect on PlasA–W (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 PlasA–W (pER304P and pER307P, Fig. 7). The data also show that while the flanking repeats were not essential for induction of PlasA–W, 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 PlasA–W.

The position of the proposed binding site suggests that LasX-mediated transcription stimulation may involve protein–protein 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 lasX–lasA 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 PlasA–W –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.


   ACKNOWLEDGEMENTS
 
This work was funded by the Norwegian Research Council, grant 8110729/130. Thanks to Catherine K. Halvorsen for her kind assistance in performing the {beta}-galactosidase assays.


   REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Ajdic, D., McShan, W. M., McLaughlin, R. E. & 16 other authors (2002). Genome sequence of Streptococcus mutans UA159, a cariogenic dental pathogen. Proc Natl Acad Sci U S A 99, 14434–14439.[Abstract/Free Full Text]

Berthier, F., Zagorec, M., Champomier-Vergès, M., Ehrlich, S. D. & Morel-Deville, F. (1996). Efficient transformation of Lactobacillus sakei by electroporation. Microbiology 142, 1273–1279.

Chaussee, M. S., Ajdic, D. & Ferretti, J. J. (1999). The rgg gene of Streptococcus pyogenes NZ131 positively influences extracellular SPE B production. Infect Immun 67, 1715–1722.[Abstract/Free Full Text]

Chaussee, M. S., Watson, R. O., Smoot, J. C. & Musser, J. M. (2001). Identification of Rgg-regulated exoproteins of Streptococcus pyogenes. Infect Immun 69, 822–831.[Abstract/Free Full Text]

Chaussee, M. S., Sylva, G. L., Sturdevant, D. E., Smoot, L. M., Graham, M. R., Watson, R. O. & Musser, J. M. (2002). Rgg influences the expression of multiple regulatory loci to coregulate virulence factor expression in Streptococcus pyogenes. Infect Immun 70, 762–770.[Abstract/Free Full Text]

Chaussee, M. S., Somerville, G. A., Reitzer, L. & Musser, J. M. (2003). Rgg coordinates virulence factor synthesis and metabolism in Streptococcus pyogenes. J Bacteriol 185, 6016–6024.[Abstract/Free Full Text]

Ferretti, J. J., McShan, W. M., Ajdic, D. & 20 other authors (2001). Complete genome sequence of an M1 strain of Streptococcus pyogenes. Proc Natl Acad Sci U S A 98, 4658–4663.[Abstract/Free Full Text]

Fried, M. G. (1989). Measurement of protein–DNA interaction parameters by electrophoresis mobility shift assay. Electrophoresis 10, 366–376.[Medline]

Fujiwara, T., Hoshino, T., Ooshima, T., Sobue, S. & Hamada, S. (2000). Purification, characterization, and molecular analysis of the gene encoding glucosyltransferase from Streptococcus oralis. Infect Immun 68, 2475–2483.[Abstract/Free Full Text]

Glaser, P., Frangeul, L., Buchrieser, C. & 52 other authors (2001). Comparative genomics of Listeria species. Science 294, 849–852.[Abstract/Free Full Text]

Glaser, P., Rusniok, C., Buchrieser, C. & 9 other authors (2002). Genome sequence of Streptococcus agalactiae, a pathogen causing invasive neonatal disease. Mol Microbiol 45, 1499–1513.[CrossRef][Medline]

Hillman, J. D., Novak, J., Sagura, E. & 8 other authors (1998). Genetic and biochemical analysis of mutacin 1140, a lantibiotic from Streptococcus mutans. Infect Immun 66, 2743–2749.[Abstract/Free Full Text]

Hochschild, A. & Dove, S. L. (1998). Protein–protein contacts that activate and repress prokaryotic transcription. Cell 92, 597–600.[Medline]

Huffman, J. L. & Brennan, R. G. (2002). Prokaryotic transcription regulators: more than just the helix–turn–helix motif. Curr Opin Struct Biol 12, 98–106.[CrossRef][Medline]

Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685.[Medline]

Lyon, W. R., Gibson, C. M. & Caparon, M. G. (1998). A role for trigger factor and an rgg-like regulator in the transcription, secretion and processing of the cysteine proteinase of Streptococcus pyogenes. EMBO J 17, 6263–6275.[Abstract/Free Full Text]

Miller, J. H. (1972). Experiments in Molecular Genetics. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.

Mørtvedt, C. I. & Nes, I. F. (1990). Plasmid-associated bacteriocin production by a Lactobacillus sakei strain. J Gen Microbiol 136, 1601–1607.

Neely, M. N., Lyon, W. R., Runft, D. L. & Caparon, M. (2003). Role of RopB in growth phase expression of the SpeB cysteine protease of Streptococcus pyogenes. J Bacteriol 185, 5166–5174.[Abstract/Free Full Text]

Qi, F., Chen, P. & Caufield, P. W. (1999). Functional analyses of the promoters in the lantibiotic mutacin II biosynthetic locus in Streptococcus mutans. Appl Environ Microbiol 65, 652–658.[Abstract/Free Full Text]

Rawlinson, E. L., Nes, I. F. & Skaugen, M. (2002). LasX, a transcriptional regulator of the lactocin S biosynthetic genes in Lactobacillus sakei L45, acts both as an activator and a repressor. Biochimie 84, 559–567.[CrossRef][Medline]

Rhodius, V. A. & Busby, S. J. (1998). Positive activation of gene expression. Curr Opin Microbiol 1, 152–159.[CrossRef][Medline]

Sambrook, J. & Russell, D. W. (2001). Molecular Cloning: a Laboratory Manual, 3rd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.

Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.

Sanders, J. W., Leenhouts, K., Burghoorn, J., Brands, J. R., Venema, G. & Kok, J. (1998). A chloride-inducible acid resistance mechanism in Lactococcus lactis and its regulation. Mol Microbiol 27, 299–310.[CrossRef][Medline]

Skaugen, M., Abildgaard, C. I. M. & Nes, I. F. (1997). Organization and expression of a gene cluster involved in the biosynthesis of the lantibiotic lactocin S. Mol Gen Genet 253, 674–686.[CrossRef][Medline]

Skaugen, M., Andersen, E. L., Christie, V. H. & Nes, I. F. (2002). Identification, characterization, and expression of a second, bicistronic, operon involved in the production of lactocin S in Lactobacillus sakei L45. Appl Environ Microbiol 68, 720–727.[Abstract/Free Full Text]

Stentz, R., Loizel, C., Malleret, C. & Zagorec, M. (2000). Development of genetic tools for Lactobacillus sakei: disruption of the {beta}-galactosidase gene and use of lacZ as a reporter gene to study regulation of the putative copper ATPase, AtkB. Appl Environ Microbiol 66, 4272–4278.[Abstract/Free Full Text]

Sulavik, M. S. & Clewell, D. B. (1996). Rgg is a positive transcriptional regulator of the Streptococcus gordonii gtfG gene. J Bacteriol 178, 5826–5830.[Abstract/Free Full Text]

Sulavik, M. C., Tardif, G. & Clewell, D. B. (1992). Identification of a gene, rgg, which regulates expression of glucosyltransferase and influences the Spp phenotype of Streptococcus gordonii Challis. J Bacteriol 174, 3577–3586.[Abstract]

Tettelin, H., Nelson, K. E., Paulsen, I. T. & 36 other authors (2001). Complete genome sequence of a virulent isolate of Streptococcus pneumoniae. Science 293, 498–506.[Abstract/Free Full Text]

Vickerman, M. M. & Minick, P. E. (2002). Genetic analysis of the rgggtfG junctional region and its role in Streptococcus gordonii glucosyltransferase activity. Infect Immun 70, 1703–1714.[Abstract/Free Full Text]

Vickerman, M. M., Wang, M. & Baker, L. J. (2003). An amino acid change near the carboxyl terminus of the Streptococcus gordonii regulatory protein Rgg affects its abilities to bind DNA and influence expression of the glucosyltransferase gene gtfG. Microbiology 149, 399–406.[CrossRef][Medline]

Wintjens, R. T., Rooman, M. J. & Wodak, S. J. (1996). Automatic classification and analysis of {alpha}{alpha}-turn motifs in proteins. J Mol Biol 255, 235–253.[CrossRef][Medline]

Received 28 May 2004; revised 11 November 2004; accepted 30 November 2004.



This Article
Abstract
Full Text (PDF)
Alert me when this article is cited
Alert me if a correction is posted
Citation Map
Services
Email this article to a friend
Similar articles in this journal
Similar articles in PubMed
Alert me to new issues of the journal
Download to citation manager
Google Scholar
Articles by Rawlinson, E. L. A.
Articles by Skaugen, M.
Articles citing this Article
PubMed
PubMed Citation
Articles by Rawlinson, E. L. A.
Articles by Skaugen, M.
Agricola
Articles by Rawlinson, E. L. A.
Articles by Skaugen, M.


HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
INT J SYST EVOL MICROBIOL MICROBIOLOGY J GEN VIROL
J MED MICROBIOL ALL SGM JOURNALS
Copyright © 2005 Society for General Microbiology.