A new Bacillus cereus DNA-binding protein, HlyIIR, negatively regulates expression of B. cereus haemolysin II

Zhanna I. Budarina1, Dmitri V. Nikitin1, Nikolay Zenkin2, Marina Zakharova1, Ekaterina Semenova2, Michael G. Shlyapnikov1, Ekaterina A. Rodikova3, Svetlana Masyukova1, Oleg Ogarkov2, Gleb E. Baida1, Alexander S. Solonin1 and Konstantin Severinov2

1 The Institute of Biochemistry and Physiology of Micro-organisms, Nauki Avenue, 5, Pushchino, 142292 Russia
2 Waksman Institute for Microbiology, Rutgers, The State University of New Jersey, 190 Frelinghuysen Road, Piscataway, NJ 08854, USA
3 Pushchino State University, Nauki Avenue, 5, Pushchino, 142292 Russia

Correspondence
Konstantin Severinov
severik{at}waksman.rutgers.edu
Alexander S. Solonin
solonin{at}ibpm.pushchino.ru


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Haemolysin II, HlyII, is one of several cytotoxic proteins produced by Bacillus cereus, an opportunistic human pathogen that causes food poisoning. The hlyII gene confers haemolytic activity to Escherichia coli cells. Here a new B. cereus gene, hlyIIR, which is located immediately downstream of hlyII and regulates hlyII expression, is reported. The deduced amino acid sequence of HlyIIR is similar to prokaryotic DNA-binding transcriptional regulators of the TetR/AcrA family. Measurements of haemolytic activity levels and of hlyII promoter activity levels using gene fusions and primer-extension assays demonstrated that, in E. coli, hlyII transcription decreased in the presence of hlyIIR. Recombinant HlyIIR binds to a 22 bp inverted DNA repeat centred 48 bp upstream of the hlyII promoter transcription initiation point. In vitro transcription studies showed that HlyIIR inhibits transcription from the hlyII promoter by binding to the 22 bp repeat and RNA polymerase, and by decreasing the formation of the catalytically competent open promoter complex.


Abbreviations: RNAP, RNA polymerase


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Bacteria of the Bacillus cereus group, B. cereus, Bacillus anthracis, Bacillus mucoides and Bacillus thuringiensis, are important human and animal pathogens. While the pathogenic properties of bacteria of the B. cereus group are clearly distinct, analyses of genomic sequences, particularly rRNA sequences, of member taxa reveal the very small amounts of sequence variation that would be expected for different strains of a single bacterial species (Ash et al., 1991). The differences in pathogenic properties of bacteria of the B. cereus group are due to the production of different and non-overlapping sets of cytotoxic proteins, some of which are encoded by plasmid-borne genes.

Haemolysin II, HlyII, is one of several cytotoxic proteins produced by B. cereus. B. cereus is a soil bacterium and an opportunistic human pathogen that causes sometimes-lethal food poisoning (Granum, 1997). Based on its deduced amino acid sequence, HlyII is a member of the family of oligomeric {beta}-barrel toxins (Baida et al., 1999; Gouaux, 1998). This family also includes a number of Staphylococcus aureus cytolysins ({alpha}-toxin, leukocidins, {gamma}-haemolysin), Clostridium perfringens {beta}-toxin (Gouaux, 1998) and B. cereus cytotoxin K (CytK) (Lund et al., 2000). These toxins are secreted in a soluble form and cause their cytotoxic effect by assembling into a transmembrane pore.

The synthesis of {beta}-barrel toxins is subject to genetic regulation. The synthesis of the prototypical member of the family, S. aureus {alpha}-toxin, occurs at the end of the exponential phase of growth and is regulated in a complex fashion by the agr (accessory gene regulator) (Recsei et al., 1986) and sar (Staphylococcus accessory regulator) loci. The products of agr genes are involved in positive regulation of {alpha}-toxin gene transcription. SarA, a pleiotropic regulator of S. aureus virulence genes, interacts directly with AT-rich sequences upstream of its target genes, which include the {alpha}-toxin gene; SarA also negatively controls expression of the agr genes (Chakrabarti & Misra, 2000). In the much less well-understood case of B. cereus CytK, putative binding sites for PlcR, a pleiotropic regulator of extracellular virulence factor gene expression in B. thuringiensis, have been identified (Lund et al., 2000), suggesting that cytK expression is subject to positive regulation.

HlyII is most closely related to CytK of B. cereus and to the {alpha}-toxin of S. aureus. Up to now, no regulatory system for the hlyII gene has been described. hlyII-like genes have been found in a number of B. cereus strains, in most of the B. thuringiensis strains tested (Budarina et al., 1994), and most recently in B. anthracis (Read et al., 2002). The extent of haemolytic activity in these bacilli varies both between these closely related species and between different strains of the same species; however, the reasons for such variation are not known.

We have previously reported the cloning of a fragment of B. cereus VKM-B771 genomic DNA containing the hlyII gene and adjacent sequences (Sinev et al., 1993). This fragment, when introduced on a plasmid into E. coli, led to a haemolytic phenotype (Sinev et al., 1993; Baida et al., 1999). A perfect 22 bp inverted repeat has been found 222 bp upstream of the hlyII ORF initiating ATG codon, and it was hypothesized that it could be required for transcription regulation of hlyII (Baida et al., 1999). However, no molecular mechanism for this regulation was proposed. In this paper, we describe a B. cereus ORF, hlyIIR, located downstream of hlyII. Sequence analysis revealed that HlyIIR (for HlyII Regulator) belongs to the Tet/AcrA family of DNA binding transcription regulators. When hlyIIR was introduced into E. coli cells harbouring hlyII plasmids, the haemolytic phenotype and hlyII transcription levels were decreased. In vitro, recombinant HlyIIR specifically interacted with the 22 bp inverted repeat which is centred 48 bp upstream from the hlyII promoter transcription initiation point. HlyIIR binding to promoter DNA did not affect promoter recognition by RNA polymerase (RNAP), but decreased the number of catalytically active open promoter complexes. Thus, our results indicate that HlyII synthesis is the subject of negative regulation by HlyIIR.


   METHODS
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INTRODUCTION
METHODS
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Bacterial strains and growth conditions.
The Escherichia coli strain Z85 [thi, {Delta}(lac-proAB), {Delta}(srl-recA), hsdR, supE, Tn10(Tcr), (F', –traD, proAB, lacI, {Delta}M15)] was used in haemolytic phenotype detection experiments and as a host for plasmid DNA. The hlyIIR protein was expressed in E. coli strain M15pRep4 (Kmr, lac, ara, gal, mtl, F). Both strains were grown in LB liquid medium (10 g bactotryptone, 5 g yeast extract, 5 g sodium chloride l–1) at 37 °C. For growth on plates, the LB medium was supplemented with 15 g agar l–1.

Haemolytic phenotype detection.
The haemolytic phenotype of recombinant cells was tested by the appearance of haemolytic zones (clearance zones) around colonies grown on LB agar containing a 1 % suspension of human erythrocytes. Cells were grown at 37 °C overnight and were then incubated for an additional 8–12 hours at 20 °C.

To measure the haemolytic phenotype quantitatively, cells were grown in LB medium at 20 °C with vigorous aeration until the onset of stationary phase. Haemolytic activity in cultured medium was measured as described by Baida et al. (1999).

Plasmids and DNA manipulations.
Molecular cloning was performed using standard protocols. Sequencing reactions were carried out using the FemtoMol sequencing kit (Promega).

Previously described plasmids pUJ1 and pUJ2 (Sinev et al., 1993) contain the 2·9 kb EcoRI fragment of B. cereus VKM-B771 genomic DNA containing the hlyII gene cloned in opposing orientation in a pUC19 vector. For construction of the pH2 plasmid, the hlyII gene was PCR amplified using 5' primer ACGTTGTAAAACGACGGCCAGTG (anneals to the pUC19 vector part of pUJ1, upstream of the EcoRI site used for the cloning of the 2·9 kb fragment of B. cereus DNA) and 3' primer AATAAAGCTTTTATACTCATATTTG complementary to DNA 38–63 nt downstream of the hlyII termination codon and carrying an engineered HindIII site (underlined). The amplified product was treated with EcoRI and HindIII and cloned into the pUC19 vector. For construction of the pRH2 plasmid, the hlyIIR gene was amplified with the same 5' primer as for the hlyII amplification together with 3' primer GATGGATCCTTATTTCATCAAAAT, which anneals to B. cereus DNA 24–44 nt downstream of the hlyII termination codon and carries an engineered BamHI site (underlined), using the pUJ2 plasmid as a template. The amplified product was treated with EcoRI and BamHI and cloned into the p15KS vector. For construction of the pFH1Lac plasmid, the 2420 bp BamHI–HindIII fragment of pUJ1 was replaced with the 3070 bp BamHI–HindIII lacZ fragment from plasmid pCL471 (Zabeau & Stanley, 1982). Plasmid pHR, expressing hexahistidine-tagged HlyIIR, was obtained by cloning the PCR-amplified hlyIIR ORF between the BamHI and HindIII sites of the pQE30 expression vector.

{beta}-Galactosidase assays.
The amounts of {beta}-galactosidase in E. coli cell extracts were determined with a quantitative colorimetric assay using ONPG as a substrate, as described elsewhere (Nikolaev et al., 1992).

Primer extension reactions.
Total RNA was extracted either from E. coli Z85 cells transformed with appropriate hlyII plasmids and grown in LB medium overnight or from cells scraped from the surface of LB plates containing 1 % human erythrocytes. RNA was purified with the RNeasy RNA isolation mini kit (Qiagen), following the manufacturer's instructions. RNA samples were then treated with RNase-free DNase and repurified using the Qiagen RNeasy protocol. Total RNA (5 µg) was analysed by electrophoresis in 1 % agarose gels to ascertain that the rRNA was intact. For primer extension experiments, 10 µg of total RNA was reverse transcribed with 5 U AMV Reverse Transcriptase (Roche) for 40 minutes at 42 °C in 1x AMV Reverse Transcriptase buffer, supplied by the manufacturer, in the presence of 1 pmol of the 32P-end-labelled primer CGGACGCTACGGCAACACTTTTAGCTATTCCC, which is complementary to hlyII ORF positions 15–46. A side-by-side sequencing reaction was performed with the same end-labelled primer and the pUJ1 plasmid as a template, using the Cycle Sequencing system (Promega) according to manufacturer's protocol. Primer extension reactions were terminated by the addition of 50 µl RNase solution (100 U) (Qiagen), incubated for 15 minutes at room temperature and phenol/chloroform extracted, after which nucleic acids were precipitated with ethanol. The pellet was dissolved in formamide-containing loading buffer, and products were resolved on a 6 % sequencing gel and visualised using a PhosphoImager (Molecular Dynamics).

Proteins.
E. coli M15pREP4 transformed with the pHR plasmid was inoculated into 200 ml LB medium containing 60 mg ampicillin l–1 and 10 mg kanamycin l–1. When the OD600 of the culture reached 0·5–0·7, cells were induced by the addition of 4 mM IPTG. After 3 h induction, cells were harvested and resuspended in 5 ml Buffer A (40 mM Tris/HCl, pH 7·5, 50 mM NaCl). Cells were lysed by sonication, cell debris was removed by centrifugation and the cell extract was loaded onto a Ni-NTA agarose column equilibrated with Buffer A. The column was washed and HlyIIR was eluted with 250 mM imidazole in buffer A. HlyIIR-containing fractions were pooled and dialysed into buffer B (40 mM Tris/HCl, pH 7·5, 50 mM NaCl, 1 mM EDTA, 1 mM {beta}-mercaptoethanol). The protein was then loaded onto a 1 ml MonoQ column equilibrated in the same buffer. The column was developed with a linear gradient of NaCl concentration (50–1000 mM) in buffer B. Fractions containing homogeneous HlyIIR were pooled, concentrated and stored under 50 % (v/v) glycerol at –20 °C.

E. coli RNAP core and {sigma}70 holoenzymes, and recombinant {sigma}70 and {sigma}565 were purified as described by Minakhin et al. (2003). B. cereus RNAP {sigma}A holoenzyme was partially purified from B. cereus VKM-B771 by performing polyethyleneimine P fractionation and heparin agarose affinity chromatography, using the standard procedure for E. coli RNAP purification. The resultant enzyme was ~75 % pure.

DNA binding assays, footprinting and in vitro transcription.
The standard HlyIIR–DNA binding reaction contained, in 20 µl buffer (10 mM Tris/HCl, pH 7·5, 10 mM NaCl, 1 mM MgCl2), various amounts of HlyIIR, 200 ng radioactively labelled DNA or 1 µg unlabelled DNA, and 1 µg BSA. Complexes were allowed to form for 20 min at 37 °C. Reactions were then loaded on 4 % or 12 % polyacrylamide gels, or on 0·8 % agarose gels. DNA complexes separated on polyacrylamide gels were revealed by autoradiography; complexes resolved by agarose gel electrophoresis were stained by ethidium bromide and visualized with UV irradiation. Where indicated, 0·5, 1 and 2 µg of pUC19 competitor DNA was added to samples prior to electrophoresis.

The hlyII promoter-containing DNA fragments used in in vitro transcription and footprinting reactions were created by PCR, using pUJ1 as a template. The standard, ‘wild-type’ fragment corresponded to positions –94 to +214 relative to the hlyII transcription initiation start point. ‘Mutant’ fragments, lacking B. cereus sequences 27 and 47 bases upstream of the hlyII promoter initiation start point, were created by PCR.

To form open promoter complexes, 3 pmol E. coli RNAP {sigma}70 holoenzyme or B. cereus RNAP {sigma}A holoenzyme (purified as described by Kashlev et al., 1996) was preincubated in 10 µl transcription buffer (20 mM Tris/HCl, pH 7·9, 40 mM KCl, 10 mM MgCl2) with 1 pmol of hlyII promoter DNA fragment for 10 min at 37 °C. HlyIIR (50 pmol) was added to the reaction either before or after RNAP addition and open complex formation. After the HlyIIR addition, the reaction mixture was incubated for 10 min at 37 °C. Transcription complexes were next subjected to native gel analysis or footprinting, or were supplemented with 500 µM GTP, 50 µM ATP and CTP, and [{alpha}-32P]UTP (30 Ci mmol–1) to analyse in vitro transcription products. Transcription reactions were allowed to proceed for 10 min at 37 °C, and were terminated by the addition of an equal volume of formamide-containing gel loading buffer. Reaction products were separated on a 20 % polyacrylamide, 6 M urea gel and revealed by PhosphoImager analysis. For native gel and footprinting analysis, the DNA fragment containing the hlyII promoter was 3'-end-labelled at the non-template strand with Klenow enzyme, using standard procedures. Samples containing hlyII promoter complexes were loaded directly on a 4 % (37·5 : 1) polyacrylamide native gel, footprinted with DNase I or probed with KMnO4, as described by Severinova et al. (1998). The samples were then analysed on a 6 % sequencing gel and revealed on a PhosphoImager.

For primer extension analysis of in vitro transcribed RNA, promoter complexes were formed during a 10 min 37 °C incubation of 10 pmol E. coli or B. cereus RNAP and 5 pmol hlyII promoter-containing fragment in 100 µl transcription buffer. NTP (500 µM) was added and the reaction continued for 30 min at 37 °C. Reactions were extracted with chloroform, and nucleic acids were precipitated with ethanol, dissolved in water and subjected to the primer extension assay. In vitro transcribed RNA was reverse-transcribed with 100 U Super Script III enzyme (Invitrogen) in the presence of 1 pmol 32P-end-labelled primer complementary to nucleotide positions 158–182 of the hlyII gene. Primer extension reactions were carried out for 50 min at 50 °C and terminated by a 5 min incubation at 85 °C. RNA was removed by RNase H treatment. Reaction products were extracted with chloroform, precipitated with ethanol and dissolved in formamide-containing loading buffer. As a reference, sequencing reactions were performed on a hlyII promoter-containing DNA fragment using the same end-labelled primer as the one used in the primer extension reaction. The fmol DNA Cycle Sequencing System (Promega) was used for sequencing, according to the manufacturer's protocol. The extension products were resolved on an 8 % sequencing gel, together with the sequencing reactions, and visualised using a PhosphorImager.


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The hlyIIR gene negatively regulates B. cereus hlyII expression in E. coli
Previously, we reported that E. coli plasmid pUJ1, containing a ~2·9 1kb EcoRI fragment of B. cereus VKM-B771 DNA cloned in pUC19, allowed E. coli cells to lyse erythrocytes (Sinev et al., 1993; Baida et al., 1999). The haemolytic activity was attributed to the presence of the hlyII (haemolysin II) gene, which was located in the left-hand side of the 2·9 kb EcoRI fragment (Baida et al., 1999).

The hlyII gene (412 codons) is much shorter than the B. cereus DNA fragment of pUJ1. We determined the sequence of the entire 2·9 kb EcoRI fragment of pUJ1 and identified an additional, 201-codon-long ORF downstream of hlyII. The new ORF has an appropriately positioned Shine–Dalgarno sequence. The new ORF is transcribed in the same direction as hlyII; it is preceded by a putative promoter and is followed by a putative {rho}-independent transcription terminator, suggesting that it is expressed independently of hlyII.

To determine whether the product of the additional ORF is involved in haemolysis or not, E. coli expression plasmids containing either hlyII or the new gene were created (Fig. 1a), and their ability to impart haemolytic activity to E. coli was tested. As expected, the pUC19-based hlyII plasmid pH2 imparted haemolytic activity to E. coli (data not shown; see also Fig. 1b). In contrast, plasmid pRH2, which carried the downstream ORF with its putative regulatory elements cloned in the p15KS vector, did not impart haemolytic activity to E. coli, thus excluding the possibility that the product of the second gene is involved in haemolysis directly (data not shown; see also Fig. 1b).



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Fig. 1. The hlyIIR gene decreases haemolytic activity of E. coli cells carrying the hlyII haemolysin gene. (a) Schematic representation of plasmids used. (b) E. coli cells transformed with the indicated plasmids were plated on LB agar plates containing 1 % human erythrocytes. The results of overnight growth are presented. Clearing around cells carrying the pRH2 hlyII plasmid and p15KS vector plasmid indicate haemolysis. The bar graph on the right indicates the results of quantitative haemolytic activity measurements using extracts of cells carrying the indicated plasmids.

 
Since replicons of the pUC19 and p15KS plasmids are compatible, we tested the haemolytic activity of E. coli cells harbouring both hlyII and the downstream ORF plasmids. The haemolytic activity of cells harbouring the pH2 (hlyII) plasmid and the p15KS vector plasmid was about 10 times higher than the haemolytic activity of cells carrying pH2 and pRH2 (Fig. 1b). As expected, no haemolytic activity was observed in cells harbouring pRH2 and the pUC19 vector plasmid (Fig. 1b). The result thus suggests that the product of the new ORF, while not involved in haemolysis directly, negatively regulates hlyII expression or HlyII activity. Accordingly, we named the new gene hlyIIR (for hlyII Regulator).

Comparisons of the deduced HlyIIR sequence with protein sequences in public databases revealed that HlyIIR is similar through much of its length to a number of oligomeric DNA-binding bacterial transcriptional regulators. An NCBI Conserved Domain Search revealed that HlyIIR amino acids 1–166 aligned with the AcrR conserved domain, while amino acids 12–58 also gave significant alignment with the TetR conserved domain, which contains a helix–turn–helix DNA-binding element. HlyIIR has the most similarity to other proteins in its N-terminal TetR domain. The C-terminal portion of HlyIIR is more divergent. A similar trend has been observed with other proteins of this class (see, for example, Namwat et al., 2001). This likely reflects the fact that the C-terminal portions of AcrR/TetR family proteins are involved in homomultimerization and interactions with cofactors, which are highly diverse, while the architecture of the compact N-terminal domain is constrained by its essential DNA-binding function.

HlyIIR inhibits transcription from the hlyII promoter
The similarity between HlyIIR and the AcrR/TetR family of DNA-binding transcriptional regulators strongly suggests that HlyIIR affects HlyII haemolysis by decreasing hlyII expression at the level of transcription. To prove this conjecture, two types of experiments were performed. First, we constructed plasmid pFH1Lac, harbouring a fusion of the upstream regulatory region of hlyII with the lacZ gene (Fig. 2a), and we determined the effect of a compatible plasmid carrying hlyIIR on the amount of {beta}-galactosidase activity in cells carrying pFH1Lac. The results, which are presented in Fig. 2b, show that in the presence of vector plasmid p15KS substantial levels of {beta}-galactosidase activity were obtained in cells harbouring pFH1Lac. However, when hlyIIR plasmid pRH2 was substituted for p15KS, a 10-fold decrease in {beta}-galactosidase activity was observed. As expected, no {beta}-galactosidase activity was found in cells harbouring pRH2 and the pUC19 vector. The results thus support the idea that HlyIIR inhibits hlyII transcription.



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Fig. 2. HlyIIR decreases transcription from the hlyII promoter. (a) Schematic representation of the pFH1lac fusion plasmid. The hlyII sequences are shown in grey. (b) E. coli cells were transformed with the indicated plasmids, and {beta}-galactosidase activity in cell extracts was determined. (c) Total RNA was purified from cells carrying wild-type hlyII in the presence or absence of hlyIIR, or from cells carrying p{Delta}Ssp, a pRH2 derivative lacking sequences between the EcoRI and SspI sites, in the absence of the hlyIIR plasmid, and primer extension using an oligonucleotide annealing at the beginning of hlyII was performed. The products were resolved on a 6 % denaturing gel alongside the products of a sequencing reaction obtained using the same primer and the pRH2 plasmid as a template. An autoradiograph is presented. (d) Total RNA was purified from B. cereus VKM-B771 and primer extension using an oligonucleotide annealing at the beginning of hlyII was performed. The products were resolved on a 6 % denaturing gel alongside the products of a sequencing reaction obtained using the same primer and the pRH2 plasmid as a template. An autoradiograph is presented. (e) DNA sequence at and around the transcription initiation start point of the hlyII promoter. The 22 bp inverted repeat is indicated by arrows. The likely extended –10 motif is underlined. The transcription initiation start point (determined by primer extension, Fig. 2c) is shown in lower case. The SspI site is also indicated (see text).

 
We next performed primer extension analysis using RNA prepared from E. coli cells transformed with hlyII plasmids in the presence or absence of hlyIIR. In RNA prepared from cells harbouring the hlyII gene alone, a single primer extension product corresponding to a transcription start point 174 bases upstream of the hlyII-initiating ATG was observed (Fig. 2c, lane 2). The hlyII transcription start point determined by primer extension is preceded by an appropriately located extended –10 promoter element, TGTGTTTTAAT (Fig. 2e, consensus sequence TGTGXTATAAT, where X denotes any nucleotide; Gaal et al., 2001). Sequence analysis reveals no –35 consensus promoter element in the hlyII promoter, in agreement with the fact that extended –10 class promoters do not require this element for activity (Barne et al., 1997). As expected, the amount of primer extension product was about 10 times lower in cells harbouring both hlyII and hlyIIR (Fig. 2c, compare lanes 1 and 2). The result thus confirms that HlyIIR negatively regulates hlyII expression at the level of transcription. No hlyII primer extension product was detected with RNA prepared from cells harbouring the p{Delta}Ssp plasmid, a derivative of pH2 in which the hlyII upstream sequences have been removed as far as an SspI site located 180 bp upstream of the hlyII-initiating ATG (Fig. 2c, lane 3). This is an expected result, since the deletion removed the extended –10 promoter element (Fig. 2e). Cells harbouring p{Delta}Ssp did not exhibit any haemolytic activity (data not shown).

The transcription start site identified in E. coli corresponds to the natural transcription start site in B. cereus, since a primer extension experiment performed with RNA prepared from B. cereus VKM-B771 revealed the same primer extension product as that determined in E. coli (Fig. 2d).

HlyIIR interacts with a palindromic DNA sequence upstream of the hlyII promoter
The hlyIIR gene was cloned into the E. coli expression plasmid and the recombinant HlyIIR protein was overproduced and purified from cell extracts to homogeneity as an N-terminal hexahistidine fusion. The DNA-binding activity of HlyIIR was tested in gel retardation assay using EcoRI- and BamHI-digested pH2. The results, which are presented in Fig. 3, show that only the 480 bp EcoRI–BamHI fragment of pH2, which includes the entire upstream sequence of hlyII, was shifted in the presence of HlyIIR (Fig. 3a, indicated by an arrow). In contrast, the electrophoretic mobilities of the two larger pH2 fragments remained unchanged in the presence of HlyIIR (Fig. 3a, compare lanes 1 and 2). To further narrow the HlyIIR binding site, the 480 bp EcoRI–BamHI fragment of pH2 was purified, treated with AluI (cleaves at positions 200 and 270 with respect to the left-hand end of the fragment) and the gel retardation experiment with HlyIIR was repeated. As expected, in the absence of HlyIIR, three major DNA fragments were observed (Fig. 3a, lane 3). In the presence of recombinant HlyIIR, retardation of the largest, 200 bp, fragment but not of the smaller, 180 and 70 bp, fragments was observed (Fig. 3a, lane 4). The interaction of HlyIIR with the 200 bp fragment was specific, since the addition of a 10-fold excess of linearized pUC19 plasmid DNA did not change the retardation pattern (Fig. 3b, lane 5).



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Fig. 3. Recombinant HlyIIR interacts with an inverted repeat located upstream of the hlyII promoter. (a) Lanes 1 and 2: BamHI and EcoRI-digested plasmid pRH2 was incubated in the presence or absence of HlyIIR, and reaction products were resolved by electrophoresis in 0·8 % agarose and revealed by ethidium bromide staining. Lanes 3–5: a 480 bp BamHI–EcoRI fragment of pRH2 was digested with AluI, HlyIIR was added where indicated and reaction products were resolved by native 10 % PAGE and revealed by ethidium bromide staining. In lane 5, reactions were challenged with excess pUC19 DNA before loading on the gel. In lane 3, undigested/partially AluI-digested DNA fragments are visible. (b) DNase I footprinting of HlyIIR–hlyII promoter complexes. A 200 bp long EcoRI–AluI fragment of pRH2 containing the hlyII promoter and upstream sequences (Fig. 3a) was radioactively labelled at the template strand, combined with increasing concentrations of HlyIIR and treated with DNase I. Reaction products were resolved by 6 % denaturing gel electrophoresis and revealed by autoradiography. The two vertical arrows at the left indicate the position of the 22 bp inverted repeat upstream of the hlyII promoter. (c) Binding of HlyIIR to a double-stranded 46 bp oligonucleotide containing the 22 bp inverted repeat. A 46 nt oligonucleotide containing the 22 nt inverted repeat was radioactively labelled, self-annealed, gel-purified and combined with increasing amounts of HlyIIR. Reaction products were resolved by native PAGE and revealed by autoradiography. In lane 5, reactions were challenged with excess pUC19 DNA before loading on the gel.

 
To locate the HlyIIR-binding site within the 200 bp fragment, we footprinted HlyIIR DNA complexes with DNase I. The results, presented in Fig. 3b, indicated that HlyIIR specifically protected ~50 bp of DNA, between positions –75 to –25 relative to the hlyII transcription initiation start point, from DNase I digestion. The protected region corresponds to the 22 bp inverted repeat centred 48 bp upstream from the hlyII transcription initiation start point (Fig. 2d).

We next proceeded to establish that the 22 bp inverted repeat is sufficient for interaction with HlyIIR. We used a synthetic double-stranded DNA probe that contained the entire 22 bp inverted repeat. Gel-retardation experiments revealed that HlyIIR readily shifted this oligonucleotide (Fig. 3c, compare lanes 1 and 3) and that the HlyIIR–DNA complex was specific, as it withstood competition with a large excess of pUC19 DNA (Fig. 3c, compare lanes 4 and 5). We conclude that the 22 bp inverted repeat contains the HlyIIR binding site.

Transcription inhibition by HlyIIR in vitro
We next studied the effect of recombinant HlyIIR on transcription from the hlyII promoter in vitro. Both E. coli RNAP {sigma}70 holoenzyme and B. cereus {sigma}A holoenzyme produced a single transcript from a DNA fragment containing the hlyII promoter (Fig. 4a, lanes 1 and 3, respectively). In the presence of HlyIIR, no transcript was produced by either enzyme (Fig. 4a, lanes 2 and 4). Primer extension revealed that both enzymes initiated transcription from the same point (Fig. 4b), which corresponded to the in vivo start point utilized in E. coli (Fig. 2c).



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Fig. 4. Inhibition of hlyII transcription by HlyIIR in vitro. (a) E. coli RNAP {sigma}70 holoenzyme or B. cereus {sigma}A holoenzyme were used to transcribe from a hlyII promoter-containing DNA fragment in vitro in the presence or absence of HlyIIR. Products were resolved by denaturing PAGE and revealed by autoradiography. (b) E. coli RNAP {sigma}70 holoenzyme or B. cereus {sigma}A holoenzyme were used to transcribe from a hlyII promoter-containing DNA fragment in vitro. The RNA product was purified and primer extension using an oligonucleotide annealing at the beginning of hlyII was performed. The products were resolved on an 8 % denaturing gel alongside the products of a sequencing reaction obtained using the same primer and the hlyII DNA fragment as a template. An autoradiograph is presented. An approximately 10-fold excess of RNAP and hlyII promoter fragment was used in an in vitro transcription reaction by B. cereus RNAP to obtain the same amount of primer extension product as that obtained with the E. coli enzyme.

 
Because under our in vitro conditions, which were optimized for E. coli RNAP transcription, B. cereus RNAP was only about 10 % active compared to the E. coli enzyme (Fig. 4a, compare lanes 1 and 3), the mechanistic experiments presented below were conducted with the E. coli {sigma}70 holoenzyme. A steady-state multiple-round transcription assay demonstrated that the addition of HlyIIR strongly inhibited transcription from the hlyII promoter regardless of whether HlyIIR was added before or after {sigma}70 holoenzyme (Fig. 5a, compare lane 1 with lanes 2 and 3). When the transcription experiment was repeated with a modified hlyII promoter template that lacked the promoter-distal copy of the 22 bp inverted repeat, HlyIIR had little effect on transcription (Fig. 5a, compare lane 4 with lanes 5 and 6). HlyIIR also inhibited transcription by an RNAP holoenzyme ({sigma}565) reconstituted from E. coli RNAP core enzyme and mutant {sigma}70 which lacks the C-terminal conserved region 4.2 (Minakhin et al., 2003) (data not shown). The result thus confirms that the hlyII promoter belongs to the extended –10 class (since {sigma}70 region 4.2 interactions with the –35 promoter consensus element are not required for transcription from the extended –10 class promoters) and suggests that {sigma}70 region 4.2 is not the target of transcription inhibition by HlyIIR. We attempted to determine the effect of HlyIIR on in vitro transcription from a hlyII promoter derivative that lacked both copies of the inverted repeat. However, such a DNA fragment had no promoter activity in vitro, despite the fact that the extended –10 promoter element was retained, suggesting that upstream elements of the hlyII promoter contribute to efficient promoter utilization. Be that as it may, we conclude that HlyIIR acts as a transcription inhibitor in vitro, and that transcription inhibition is dependent on the presence of an intact HlyIIR binding site, the 22 bp inverted repeat.



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Fig. 5. The mechanism of transcription inhibition by HlyIIR. (a) The indicated proteins were combined with a DNA fragment containing the wild-type hlyII promoter (hlyII, lanes 1–3), or with a mutant fragment that had the promoter-distal copy of the 22 bp inverted repeat removed by site-directed mutagenesis (hlyII{Delta}, lanes 4–6) in the presence of GTP, ATP, CTP and [{alpha}-32P]UTP. In lanes containing both E. coli RNAP and HlyIIR, asterisks indicate which of the two proteins was added first. Transcription reactions were allowed to proceed for 10 min at 37 °C; products were resolved by denaturing PAGE and revealed by autoradiography. (b) The indicated proteins were combined with a radioactively labelled DNA fragment containing the hlyII promoter. Complexes were allowed to form for 10 min at 37 °C, reactions were challenged with 100 µg heparin ml–1 and immediately loaded in native polyacrylamide gel. After electrophoresis, protein–DNA complexes were revealed by autoradiography. (c) Promoter complexes were formed as above and probed with DNase I. Reaction products were resolved by denaturing PAGE and revealed by autoradiography. (d) Promoter complexes were formed as above and probed by KMnO4. Reaction products were resolved by denaturing PAGE and revealed by autoradiography.

 
The inhibitory effect of HlyIIR could be due to simple exclusion of RNAP from the promoter, or inactivation of RNAP–hlyII promoter complexes. A gel-retardation experiment performed in the presence of the DNA competitor heparin revealed that, in the presence of HlyIIR and RNAP {sigma}70 holoenzyme, a supershifted complex with electrophoretic mobility higher than that of either HlyIIR complex (Fig. 5b, lane 1) or RNAP open complex (Fig. 5b, lane 2) was formed (Fig. 5b, lanes 3 and 4). The formation of the supershifted complex was independent of the order of addition of RNAP or HlyIIR to the hlyII promoter fragment. The result thus indicated the presence of heparin-resistant, transcriptionally inactive ternary complexes containing promoter DNA, HlyIIR and RNAP. Therefore, transcription inhibition by HlyIIR is not a simple consequence of steric exclusion of RNAP from the promoter by bound HlyIIR, since in this case no ternary complexes are expected. Closer inspection of the band-shift gel of Fig. 5b revealed that in the presence of HlyIIR or RNAP alone, all of the promoter DNA fragment was complexed with proteins (Fig. 5b, lanes 1 and 2, respectively). However, when RNAP was added to HlyIIR–DNA complexes, a mixture of supershifted complexes and HlyIIR-only complexes was observed (Fig. 5b, lane 3), suggesting that HlyIIR decreases the affinity of RNAP for promoter DNA or, alternatively, that HlyIIR destabilizes promoter complexes. We favour the first possibility, since when HlyIIR was added to preformed RNAP promoter complexes, only the supershifted band corresponding to the ternary complex was observed (Fig. 5b, lane 4).

The hlyII promoter complexes formed by RNAP in the presence or absence of HlyIIR were studied by DNase I footprinting. The results are presented in Fig. 5c. As can be seen, HlyIIR and RNAP alone produced distinct DNase I footprints (Fig. 5c, compare lane 2 with lanes 3 and 4). HlyIIR alone protected DNA from –17 to –60 (Fig. 5c, lane 3), while RNAP alone protected the DNA from positions +20 to –13 (Fig. 5c, lane 4). In addition, RNAP changed the pattern of promoter DNA digestion by DNase I further upstream. DNA at around positions –24, –34, –44, –54 and, most strikingly, –64 became hypersensitive to DNase in the presence of RNAP. The extended protection of promoter upstream sequences by RNAP is probably due to wrapping of the very AT-rich upstream promoter DNA around RNAP (Burns et al., 1999). In the presence of HlyIIR and RNAP, promoter DNA between positions +20 and –13 remained fully protected (Fig. 5c, lanes 5 and 6). The pattern of upstream promoter DNA protection was changed and became distinct from those seen in the presence of either HlyIIR or RNAP alone. The extent of hypersensitivity in the upstream promoter DNA decreased in the presence of HlyIIR. However, in contrast to the situation seen in the presence of HlyIIR alone, no full protection of upstream promoter DNA was seen in the presence of both HlyIIR and RNAP. Several quantitative differences between the HlyIIR/RNAP footprint and the RNAP-alone footprint suggested that HlyIIR was present in these complexes (Fig. 5c, arrows). First, the striking hypersensitive band at –64 was either missing (when RNAP was added to the HlyIIR–DNA complex, Fig. 5c, lane 5) or significantly reduced (when HlyIIR was added to RNAP–promoter complexes, Fig. 5c, lane 6) in the presence of both HlyIIR and RNAP. Second, hypersensitive bands at positions –44 and –33 were also missing when both proteins were present in footprinting reactions. We take these data, together with the band shift results of Fig. 5b, as an indication that promoter complexes containing both HlyIIR and RNAP can form on the hlyII promoter. It appears that, in the ternary complex, contacts made by either HlyIIR or RNAP are different from contacts made when either of these proteins is present alone.

The hlyII promoter complexes were also probed by KMnO4, a chemical agent specific for single-stranded thymines (Fig. 5d). As can be seen, and as expected, RNAP, but not HlyIIR, caused the appearance of KMnO4-sensitive bands at positions +3, –1, –2, –5 and –12 (Fig. 5d, compare lanes 2 and 3). The degree of KMnO4 sensitivity decreased ~3·5-fold in the presence of HlyIIR, but the pattern of sensitive thymines remained the same (Fig. 5d, compare lane 3 with lanes 4 and 5). The result thus suggests that HlyIIR negatively regulates hlyII transcription by interfering with the formation of catalytically active RNAP open promoter complexes.

The footprinting results presented above suggest that HlyIIR may interact with the RNAP holoenzyme, at least in the context of the hlyII promoter complex. To determine whether HlyIIR can interact with free RNAP, a native gel analysis of reactions containing pure HlyIIR, E. coli RNAP core or {sigma}70 holoenzyme, and reactions containing HlyIIR with either core or {sigma}70 holoenzyme was performed (Fig. 6a). As can be seen, HlyIIR caused a change in the electrophoretic mobility of both the core and the holoenzyme (compare lanes 2 and 4 with lanes 2 and 5, respectively), suggesting an interaction. Analysis of the polypeptide composition of bands from the native gels revealed that bands shifted in the presence of HlyIIR contained, in addition to the expected RNAP subunits, the HlyIIR protein (Fig. 6b, lanes 4 and 6). We therefore conclude that HlyIIR is able to bind RNAP core and holoenzyme in solution, in the absence of the hlyII promoter DNA.



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Fig. 6. HlyIIR binds RNAP core and holoenzymes. The indicated proteins were combined and resolved by native 4–15 % Phastgel. The gel was stained (a), bands were excised and polypeptides were resolved by SDS-PAGE and revealed by Coomassie staining (b).

 

   DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The principal result of this work is the demonstration that heterologous expression of B. cereus haemolysin II in E. coli is negatively regulated by the product of the hlyIIR gene at the level of transcription. We demonstrate that HlyIIR interacts with a palindromic sequence centred 48 bp upstream of the hlyII transcription start point. The location of the HlyIIR binding site made it likely that HlyIIR would exert its negative effects on transcription by steric hindrance; however, this turned out not to be the case, since HlyIIR is able to interact with RNAP, and the resultant binary complex forms a ternary complex with hlyII promoter DNA. In vitro footprinting data indicate that the DNA contacts made by RNAP and HlyIIR in the ternary complex are different from the contacts made when either protein binds hlyII promoter DNA on its own. Although ternary complexes are heparin resistant, the extent of promoter opening is significantly decreased compared to binary RNAP–hlyII promoter complexes. Thus, it appears that HlyIIR interferes with the process of isomerization of the RNAP closed promoter complex to the catalytically active open promoter complex. This interference must be due to specific contacts between HlyIIR and the RNAP core, since HlyIIR binds with the same affinity to both core and holoenzymes, suggesting that the {sigma} subunit is not involved in the interaction.

Although our data on hlyII promoter regulation were obtained in a heterologous E. coli system, we believe that the hlyII promoter identified in this study is also active in B. cereus, since, in vitro, B. cereus RNAP initiates hlyII transcription at the same site as E. coli RNAP, indicating that the relative positions of HlyIIR and RNAP bound to the hlyII promoter are the same in E. coli and B. cereus. Sinev et al. (1993) and Budarina et al. (1994) reported that in both B. cereus and E. coli cells carrying the pUJ1 plasmid (contains both hlyII and hlyIIR), haemolysin II production was activated at the late logarithmic stage of growth. While the reason(s) for this is not known, it may have to do either with negative regulation of hlyIIR expression or with the regulation of HlyIIR binding to DNA by low-molecular-mass ligands whose nature is yet to be determined.

Analyses of genomic sequences of B. anthracis demonstrate that sequences homologous to both hlyII and hlyIIR are present, as expected from the very close similarity between B. cereus and B. anthracis. However, unlike B. cereus, the anthrax bacterium is not generally known to produce haemolysins. Sequence analysis of B. anthracis DNA gives some clues to this apparent paradox. While the B. anthracis A2012 strain hlyII sequence is identical to the B. cereus VKM-B771 sequence (Read et al., 2002), an A corresponding to B. cereus hlyII position 232 is missing from the B. anthracis hlyII gene. In addition, a G at hlyII position 1116 is substituted for an A. The frame shift resulting from the absence of an A at position 232 likely renders B. anthracis haemolysin II non-functional. In contrast, while the hlyII genes from B. cereus strains ATCC 14579 (Ivanova et al., 2003) and BGSC 6A5 (Miles et al., 2002) have accumulated a significant number of point substitutions compared to the VKM-B771 sequence, they have maintained an intact reading frame which, at least in the case of BGSC 6A5, is known to code for a functional haemolysin (Miles et al., 2002).

The relative position of the the hlyII and hlyIIR genes, and the palindromic HlyIIR binding sites in front of hlyII is conserved. This allows some of the errors in annotation which arise due to sequence similarities between the protein sequences of HlyII and CytK haemolysins to be corrected. Thus, in the recently published genome of B. cereus ATCC 10987 (Rasko et al., 2004) a gene annotated as CytK is followed by a gene encoding a HlyIIR protein. The haemolysin gene is preceded by a palindromic sequence similar to the HlyIIR binding site. Since CytK genes are not followed by genes encoding TetR-like regulators and are not preceded by HlyIIR binding sites, we suggest that the B. cereus ATCC 10987 cytotoxin K gene in fact encodes HlyII. As is also the case with other B. cereus hlyII genes, the ATCC 10987 gene encodes a full-sized protein.

Interestingly, the ORF of the hlyII gene (annotated as an {alpha}-haemolysin precursor) is also interrupted in B. cereus G9241 (Hoffmaster et al., 2004), the only sequenced B. cereus strain which is associated with an illness resembling inhalation anthrax and which carries a circular plasmid very similar to B. anthracis toxin-encoding plasmid pXO1. The result may indicate that some aspects of HlyII function may be incompatible with anthrax toxin function. In this regard, the presence of frame-shift mutations in hlyII genes from B. anthracis, but not in those from B. cereus, is reminiscent of the previously described situation with the plcR gene, which encodes the major positive regulator of pathogenicity factors, including haemolysins, in B. cereus. The ORF of the plcR gene is interrupted in B. anthracis, which explains why B. anthracis does not produce pathogenicity proteins characteristic of B. cereus. Inspection of the DNA sequence upstream of hlyII and hlyIIR reveals no PlcR-binding sites, suggesting that haemolysin II production is not subject to direct regulation by PlcR.

The sequence of the B. anthracis A2012 hlyIIR gene is identical to that of B. cereus hlyIIR, except for a single G to A substitution that changes Gly2 in B. cereus HlyIIR to Glu in the B. anthracis protein. Further, the 22 bp palindromic HlyIIR operator is also present in front of B. anthracis hlyII. However, a single G to A change at the tenth position of the promoter-distal copy of the inverted repeat has occurred. A corresponding C to T change has occurred in the promoter-proximal copy of the B. anthracis repeat, thus preserving the perfect 22 bp palindrome, and suggesting that this DNA site continues to play a regulatory role. It is formally possible that a single Gly to Glu amino acid substitution changes the operator specificity of B. anthracis HlyIIR. It is not clear why B. anthracis would keep a non-functional haemolysin II gene as well as a gene encoding an apparently functional regulator protein. Reports on the isolation of haemolytic B. anthracis strains from several natural sources (Mikshis et al., 1999) indicate that haemolysis in B. anthracis may be more widespread than previously anticipated. A systematic comparison of hlyII sequences from various anthracoid bacilli strains and correlation with the haemolytic activity of these strains may thus be warranted.


   ACKNOWLEDGEMENTS
 
This work was supported by NIH RO1 grant GM59295 and an NRC/DTRA exchange fellowship (K. S.) and the Russian Foundation for Basic Research (Project 03-04-48623) (A. S.). The work of D. V. N. was supported by a Russian Science Support Foundation grant.


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DISCUSSION
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Received 5 March 2004; revised 23 June 2004; accepted 4 August 2004.



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