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Department of Microbiology and Molecular Genetics, University of Texas – Houston Health Science Center, 6431 Fannin St, Houston, TX 77030, USA
Correspondence
Theresa M. Koehler
Theresa.M.Koehler{at}uth.tmc.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Present address: Washington University School of Medicine, 660 South Euclid Avenue, Campus Box 8230, St Louis, MO 63110, USA. A supplementary table listing the primers used in this study and a supplementary figure showing in silico models of other AtxA-regulated promoters are available with the online version of this paper.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Anthrax toxin synthesis by Bacillus anthracis, the causative agent of anthrax, is dependent upon the presence of the regulator AtxA (Koehler et al., 1994
; Uchida et al., 1993). Steady-state levels of mRNA transcripts associated with the three toxin genes pagA, cya and lef, are dramatically reduced in atxA-null mutants (Dai et al., 1995). AtxA also affects transcription of the B. anthracis capsule biosynthetic operon capBCADE, in part via its positive control of the cap regulator acpA (Drysdale et al., 2004). The anthrax toxin proteins and the B. anthracis capsule are considered primary virulence factors of the bacterium. B. anthracis mutants devoid of the toxin and/or capsule genes are highly attenuated in some animal models. AtxA-mediated control of virulence during infection has also been established (Drysdale et al., 2005). atxA-null mutants are highly attenuated and the antibody response to all three toxin proteins is decreased significantly in mice infected with an atxA-null mutant compared with those infected with the parent strain (Dai et al., 1995).
Bioinformatic analysis of the predicted amino acid sequence of AtxA has revealed potential functional domains of the 56 kDa AtxA protein. AtxA is predicted to have an amino-terminal winged-helix DNA-binding domain. Nevertheless, specific nucleic acid-binding activity has not been reported. Recently, Tsvetanova et al. (2007) demonstrated that phosphorylation affects AtxA activity in vivo, suggesting that activation of AtxA-regulated genes is controlled by the phosphorylation state of AtxA. The predicted domains subject to phosphorylation bear the signature of phosphotransferase system-regulated domains (PRDs). Given that PRDs respond to carbohydrate availability (Stülke et al., 1998; Stülke & Hillen, 2000), the presence of functional PRDs in AtxA indicates a mechanistic link between environmental signalling and virulence gene expression.
In addition to AtxA, other regulators have been identified that affect toxin gene transcription. These include the transition state regulator and DNA-binding protein AbrB (Saile & Koehler, 2002), and the alternative sigma factor for RNA polymerase, SigH (Hadjifrangiskou et al., 2006). However, these regulators have been proposed to control expression of the atxA gene, rather than affect toxin gene expression directly. One regulatory protein, PagR, encoded by the second gene of the bicistronic pagAR operon, has been shown to bind to nucleic acid sequences in the promoter region of pagAR and function in weak autogenous control of the operon (Hoffmaster & Koehler, 1999; Mignot et al., 2003). PagR also binds to sequences in the promoter regions of two other genes, eag and sap, which encode S-layer proteins that have not been associated with virulence. Although specific regions of each PagR-regulated promoter were protected in DNase I footprint analyses, common DNA sequences resembling an operator motif could not be identified (Mignot et al., 2003).
Despite advances in knowledge of trans-acting regulators that affect transcription of B. anthracis virulence genes, a detailed analysis of the cis-acting features of AtxA-controlled genes is lacking. Microarray experiments employing parent and atxA-mutant strains have identified a number of atxA-regulated genes (Bourgogne et al., 2003), expanding the role of AtxA to that of a global regulator. Nevertheless, no sequence similarities have been noted in the promoter regions of genes within the AtxA regulon. The 5' ends of mRNA transcripts associated with the most stringently controlled atxA targets, pagAR, lef and cya, have been mapped (Dai et al., 1995; Koehler et al., 1994). While the lef and cya genes each possess a single apparent transcription start site, pagAR transcription appears to start at two distinct sites. The major start site, P1, is tightly controlled by atxA, while the P2 site, located closer to the translational start codon for pagA, appears to be expressed constitutively at a relatively low level (Koehler et al., 1994). AtxA-dependent transcription from the pagAR P1 site and the lef and cya start sites is induced during growth in elevated (5–20 %) atmospheric CO2 and/or in media containing dissolved bicarbonate (Bartkus & Leppla, 1989; Koehler et al., 1994). The CO2/bicarbonate signal may be relevant as a host signal during infection.
In addition to sequence-specific control sites recognized by trans-acting regulators, promoter activity can be affected by local DNA structure. Specific structural features such as dynamic 3D geometry, thermostability and responsiveness to superhelicity, play significant roles in the ability of a DNA sequence to respond to or allow protein binding. In prokaryotes, small nucleoid-associated proteins, such as H-NS, IHF and HU, associate with the chromosomal DNA, affecting DNA topology, and ultimately gene expression on a global level (Garcia et al., 2007; Luijsterburg et al., 2006). In addition, there are multiple examples of the influence of intrinsic curvature on gene expression wherein local topology affects binding to specific regulatory proteins and subsequent protein-induced bending (Owen-Hughes et al., 1992; Rippe et al., 1995; Yamada et al., 1991; Zuber et al., 1994). A well-studied example is the Salmonella typhimurium put operon for proline utilization (Perez-Martin et al., 1994a, b), wherein operator sites for binding of a regulatory protein are separated by a region of curved DNA, and removal of the curved DNA results in lower levels of repression (Ostrovsky de Spicer et al., 1991). Also in S. typhimurium, osmoregulation of the proU promoter requires a curved sequence found 200 bp downstream of the transcription start site (Owen-Hughes et al., 1992). The LrpC protein of Bacillus subtilis is a global regulator that recognizes specific DNA structure. LvpC complexes with curved sequences containing phase-A tracts to wrap DNA and form nucleosome-like structures (Beloin et al., 2003).
To gain an understanding of the intrinsic properties of the anthrax toxin gene promoter DNA, we undertook a thorough characterization of the promoter regions. We hypothesized that the toxin gene promoters contain cis-acting regions with common sequence and/or structural features associated with atxA-dependent expression. In work reported here, we determined the minimal sequences of the toxin gene promoters that are required for atxA-dependent transcription, and employed in silico and in vitro analyses to demonstrate that the anthrax toxin promoters are characterized by intrinsic curvature.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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) and an isogenic atxA-null mutant, UT60 (Koehler et al., 1994), were used for this study. Escherichia coli strains TG1 (Zymoresearch) and GM2163 (New England Biolabs; NEB) were used as hosts for cloning. All strains were cultured in Luria–Bertani (LB) medium (Sambrook et al., 1989), unless indicated otherwise. B. anthracis cell extracts used for β-galactosidase assays were obtained from cells cultured with shaking at 37 °C in CaCO3 medium [CA medium (Thorne & Belton, 1957) buffered with 100 mM HEPES (pH 8.0) and 0.8 % (w/v) sodium bicarbonate], as described previously (Hadjifrangiskou et al., 2006). Antibiotics were purchased from Sigma–Aldrich or Fisher Scientific, and were added to media at the following concentrations when appropriate: ampicillin, 100 µg ml–1; kanamycin, 100 µg ml–1; and erythromycin, 150 µg ml–1 for E. coli and 5 µg ml–1 for B. anthracis.
Extraction of chromosomal DNA from B. anthracis cultures was performed using the Mo Bio Laboratories genomic isolation kit. The Wizard SV Plus Mini Prep kit (Promega) was used for extraction of plasmid DNA from E. coli. B. anthracis was electroporated with unmethylated plasmid DNA from E. coli GM2163, as described previously (Koehler et al., 1994). Recombinant DNA techniques were performed using standard procedures (Ausubel, 1993). Restriction enzymes and T4 DNA ligase were purchased from Promega and Fisher Scientific. Taq DNA polymerase was purchased from NEB and Pfu Turbo DNA polymerase from Stratagene. Plasmids created for deletion analyses of the pagA, cya and lef promoter regions are listed in Table 1. Supplementary Table S1 lists primers used in this study.
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. At least three independent cultures were evaluated for enzyme activity using duplicate samples from each culture. The figures show average promoter activity.
In silico promoter analysis.
DNA sequences were submitted to the DNA tools servers ‘bend.it’ and ‘model.it’ (http://hydra.icgeb.trieste.it/dna/) (Munteanu et al., 1998; Vlahovicek et al., 2003), and analysed for the presence of curvature using the Consensus (Trinucleotide) (Gabrielian & Pongor, 1996) and NMR (Dinucleotide) (Ulyanov & James, 1995) models. Figures show static 3D models generated using the model.it server based on the Consensus (Trinucleotide) model.
Circular permutation assays.
Promoter regions of the toxin genes were amplified using 7702 DNA as template, Pfu Turbo DNA polymerase (Stratagene), and the primer pairs MH197s/MH198as (PpagAR), MH201s/MH202as (Pcya) and MH199s/MH200as (Plef) (see Supplementary Table S1). The papR (BA5594, AE016879 : 5080849–5080995) promoter region was amplified using MH205s/MH206as to generate the 169 bp region from –170 to –2 in relation to the predicted translational start site. The resulting amplicons were cloned into the XbaI–SalI sites of pBend2 (Kim et al., 1989) to generate plasmids pUTE750 (PpagAR), pUTE755 (Pcya), pUTE749 (Plef) and pUTE757 (PpapR). Plasmids were digested with the following enzymes individually: BglII, NheI, SpeI, XhoI, EcoRV, PvuII and BamHI. Electrophoresis of the digested fragments was carried out in 5 and 8 % polyacrylamide gels, containing 10 % (v/v) glycerol in 1x TBE as described elsewhere (Kim et al., 1989; Sinden & Hagerman, 1984). DNA mobility was assessed at room temperature and at 4 °C. Experiments were performed four to six times for each promoter.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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, was cloned upstream of a promoterless lacZ gene in pHT304-18z (Agaisse & Lereclus, 1994). Each construct was introduced into the parent strain 7702 (Cataldi et al., 1992) and the isogenic atxA-null strain UT60 (Koehler et al., 1994). β-Galactosidase activity was assessed following growth under conditions that favour toxin gene expression (CaCO3 medium at 37 °C in an atmosphere containing 5 % CO2) (Dai et al., 1995). No significant promoter activity was detected in the UT60-derivatives, consistent with the requirement of atxA for toxin gene transcription.
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), corroborating and fine-tuning a previous report indicating that AtxA-dependent transcription of pagAR does not require DNA sequences more than 111 bp upstream of the P1 start site (Dai et al., 1995). Promoter activity was abolished in a reporter construct harbouring only 77 bp of sequence 5' of P1. Interestingly, the region implicated as being essential for pagAR transcription contains DNA sequences reported to be bound by the repressor PagR (Mignot et al., 2003). In the case of the cya promoter, the minimal functional control region extended 122 bp upstream of the transcription start (Fig. 1b). Shortening of the 5' end to 101 bp upstream of the start site reduced promoter activity approximately fivefold.
Analysis of the lef promoter region was less straightforward. AtxA-dependent expression of the lef promoter was evident in a construct harbouring only 70 bp upstream of the transcriptional start site for the gene (Fig. 1c). Yet constructs containing sequences from further upstream exhibited lower promoter activity. These results suggest that a repressor binding site may reside upstream of this region. Given that the transition state regulator AbrB has been shown to negatively regulate toxin gene expression (Saile & Koehler, 2002; Strauch et al., 2005), we measured the β-galactosidase activity of parental and abrB-null strains carrying reporters containing 70 bp of DNA upstream of the transcriptional start site (the minimal functional sequence) and reporters containing 195 bp of DNA upstream of the transcriptional start site. Although both constructs had higher overall activities in the abrB-null background, the fold-difference between them remained the same (data not shown), indicating that AbrB is not responsible for the low activity associated with the longer construct. To assess whether the shorter promoter fragment had lost its dependence on the presence of elevated CO2, we tested the β-galactosidase activity of parental strains carrying the two reporter constructs in the absence of elevated CO2. No significant activity was observed for either (data not shown).
The deletion analysis results indicated that the minimal functional promoter regions of the three toxin genes differ significantly in terms of overall sequence length and distance from the 5' ends of toxin gene mRNAs. To assess sequence similarities within the minimal functional promoter regions of the toxin genes, we generated alignments using CLUSTAL W (Chenna et al., 2003; http://www.ebi.ac.uk/clustalw/). Each sequence aligned was terminated at the nucleotide corresponding to the transcriptional start site of each promoter. Our results did not reveal significant sequence similarities between the three sequences, as depicted in Fig. 2. Overall, the pagAR, cya and lef promoter regions were highly A+T-rich (85, 79 and 80 % A+T, respectively) compared with the overall 64.6 % A+T content of the B. anthracis genome (Read et al., 2003). The minimal functional promoter regions also had prominent stretches of A- and T-tracts; however, a thorough comparison of the frequency of A- and T- tracts throughout the B. anthracis genome was not performed. Given that A- and T-tracts are implicated in the formation of kinks and/or bends in DNA, we wanted to examine the degree of curvature in the identified minimal functional promoters.
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; Pedersen et al., 2000) and methods available from the DNA tools web servers bend.it and model.it (http://hydra.icgeb.trieste.it/dna/) (Munteanu et al., 1998; Vlahovicek et al., 2003). Modelling of the promoter sequences was performed using the Consensus (Trinucleotide) and NMR (Dinucleotide) scales, as these have been shown experimentally to be the most reliable for predicting DNA curvature (Gabrielian & Pongor, 1996). Similar results were obtained using either property scale. Only the consensus bendability data are presented here.
3D models of the toxin gene promoters indicate common structural characteristics. For each promoter, a region of high curvature precedes the atxA-dependent transcription start. In the models shown in Fig. 3(b), the transcription start site of each promoter is in the same plane as an apparent kink. In the cases of the pagAR and lef promoters, the kink is located immediately downstream of the transcriptional start, while in the cya promoter the kink is located upstream.
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, 5'-deletion non-functional sequences).
In addition to the toxin genes, AtxA has been reported previously to influence expression of a number of other B. anthracis genes (Bourgogne et al., 2003). We modelled the promoter regions of two additional genes, pXO1-90 and pXO1-91, which are positively regulated 61-fold and 25-fold, respectively, by AtxA (Bourgogne et al., 2003). Models generated for these promoter regions shared the same characteristics as those identified for the toxin gene promoters (Supplementary Fig. S1), further supporting the hypothesis that local DNA structure is associated with regulation by AtxA.
In order to verify that the predicted structural features are unique to promoters of genes highly regulated by AtxA, we modelled a series of other DNA sequences of similar A+T content. Such sequences included promoter regions of non-AtxA-regulated genes on the B. anthracis plasmids and chromosome, as well as random intergenic regions. Our results revealed that the majority of modelled sequences were predicted to contain areas of high curvature; however, the identified structural features did not resemble those predicted for the toxin gene promoters (Fig. 3b).
Gel mobility assays indicate curvature of toxin gene promoters
Comparison of the minimal functional promoter regions using in silico analysis revealed that each promoter is characterized by regions of high curvature. DNA mobility in polyacrylamide gels is affected by DNA structure, in particular DNA curvature (Chastain et al., 1995; Hagerman, 1990). The rate of migration depends upon the angle and position of the DNA curves, as well as the physical properties of the gel (Chastain et al., 1995; Ostrovsky de Spicer et al., 1991; Ostrovsky de Spicer & Maloy, 1993). Curved DNA migrates aberrantly in non-denaturing polyacrylamide gels. Generally, bent DNA migrates more slowly with increasing concentration of polyacrylamide compared with non-curved sequences of the same length, but in some cases intrinsic DNA structure causes DNA to migrate faster in high-percentage polyacrylamide gels (Drak & Crothers, 1991; Matsugami et al., 2006; Ohyama et al., 1998; Schroth et al., 1992). To test for the presence of curvature, we examined the mobility of the minimal functional promoter regions in 5 and 8 % polyacrylamide gels. Fig. 4 is a schematic representation indicating the location and size of predicted bends (black bars) in each digestion fragment.
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) and analysed the migration of the circularly permuted fragments on 5 and 8 % polyacrylamide gels (Fig. 5). The PpapR DNA fragment was used as a negative control because in silico modelling revealed no predicted curvature. When electrophoresed in 8 % polyacrylamide gels, the toxin gene promoters exhibited an overall retarded migration that was not observed in the migration pattern of the papR promoter (Fig. 5a). While the 301 bp papR promoter fragment migrated consistently with the 300 bp marker, the 301 bp pagAR promoter fragment migrated to a position well above the 300 bp marker. The slowest-migrating pagAR promoter fragments were those generated using BglII and NheI, which place the pagAR promoter insert at the end of the digested fragment (Fig. 4). The fastest-migrating fragment, and the only one with an apparent size below 400 bp, was the fragment resulting from SpeI digestion. In this fragment the pagAR promoter insert is close to the centre. Surprisingly, the fragment generated with XhoI, which positions the pagAR promoter 6 bp closer to the centre compared with the SpeI digest, migrates much more slowly. These results were reproduced in multiple independent experiments, including a trial in which the enzyme concentration was reduced 10-fold. The digests yielded a similar migration pattern following electrophoresis on 5 % polyacrylamide gels at 4 °C (Fig. 5b). To verify that the SpeI-digested fragment was not shorter due to star activity of the SpeI enzyme, digestions were repeated, and the SpeI fragment was electrophoresed on a 2 % agarose gel, purified and subjected to PCR with pBend2-specific primers. A PCR product was generated, indicating the integrity of the digested fragment (data not shown).
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, the pBend2 constructs containing the lef and cya promoters were digested with BglII, EcoRV and BamHI, placing the promoter inserts at the far right, centre, and far left of the fragments depicted in Fig. 4, respectively.
The cya promoter fragment (270 bp) migrated at rates similar to or slower than that of the 300 bp size marker (Fig. 5a), indicating the presence of curvature. According to our modelling results, the bends preceding the cya transcription start site are found between –90 and –28 bp, a region found roughly in the centre of the 137 bp promoter fragment cloned in pBend2. As expected, when this fragment was digested with BglII or EcoRV, both of which place the predicted bend close to the centre of the digested fragment (Fig. 4), the fragments migrated with an apparent size of 300 bp (Fig. 5b). Digestion with BamHI, which places the predicted area of curvature at the extreme left of the digested fragment, resulted in faster migration and the digested sequence showed an apparent size of 275 bp (Fig. 5b).
The lef promoter fragment (284 bp) had an overall faster migration on 5 and 8 % polyacrylamide gels, running with an apparent size closer to 250 bp (Fig. 5), but differences in mobility of the digested lef pBend constructs were observed on the high-percentage polyacrylamide gels. Based on our in silico models, the area of high curvature within the lef promoter region is predicted to lie 
20 bp upstream of the lef transcription start and occupies 30 bp (Fig. 4). On 5 % gels (Fig. 5b), significant differences in the mobility of the digested lef pBend constructs were not readily apparent. Yet 8 % gels (Fig. 5a) showed that while the lef pBend2 construct digested with BglII, which places the predicted bend close to the centre, ran with an apparent size of 250 bp, digestion with EcoRV and BamHI, which shifts the location of the predicted bend away from the centre and to the extreme left, increased the rate of migration of the digested lef fragment to an apparent size close to 225 bp. It is important to note that for all promoters tested, the migration patterns of all digested fragments corresponded to actual fragment size, when electrophoresed on 2 % agarose gels in 1x TBE. Similar results have been reported for other curved promoters, including the put control region of S. typhimurium (Ostrovsky de Spicer et al., 1991).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Hirota & Ohyama, 1995). The minimal functional pagAR, cya and lef promoter regions determined in this study are 85, 79 and 80 % A+T, respectively: percentages significantly higher than the overall 64.6 % A+T content of the B. anthracis genome (Read et al., 2003). The retarded migration patterns obtained in our electrophoretic mobility analyses indicate significant curvature, exceeding that predicted based on A+T content alone (Calladine et al., 1988; Hirota & Ohyama, 1995).
Our modelling and circular permutation assays revealed that the most highly expressed toxin gene promoter, the pagAR promoter (Cataldi et al., 1992), is characterized by the most pronounced curvature. In silico models predict that the pagAR promoter contains two bends oriented perpendicularly. The first bend is predicted to begin 58 bp upstream of the AtxA-mediated transcription start and occupy a 48 bp region, terminating 10 bp upstream of the start. The second bend is predicted to be 30 bp downstream of the transcription start, terminating at the end of the sequence. The presence and organization of the two bends in the pagAR promoter is further supported by aberrant migration of the different pagAR circular permutations in electrophoretic mobility analyses. In silico models generated for the cya and lef promoters suggest that these DNA sequences have a similar 3D structure (see Fig. 3, third row). In the case of the cya promoter DNA, the area with pronounced curvature is found the furthest upstream with respect to the atxA-regulated transcriptional start site, at approximately –100 bp. Interestingly, a 5'-deletion fragment spanning 101 bp upstream of the cya transcriptional start site was not functional. The area of high curvature predicted for the lef promoter region lies 40–50 bp upstream of the transcriptional start, at a distance similar to that found in the pagAR promoter. Neither the lef or cya promoter regions are characterized by additional areas of high curvature. This was also reflected in the migration patterns observed in our circular permutation analyses.
In addition to at least one area of high curvature present in the three toxin promoter regions, our modelling analyses predict the presence of a distinct kink in close proximity to the atxA-regulated transcription start sites. In the case of the pagAR and lef promoter regions, the predicted kink in the DNA is found in very close proximity to the start sites, indicating a significant similarity between the two regions, in addition to the location of the area of high curvature. Notably, in limited 3'-deletion analyses not shown here, we observed that 80 bp of sequence downstream of the transcriptional start sites for pagAR and lef was required for wild-type promoter activity. On the other hand, in the cya promoter region, in which the kink is predicted to be farther upstream of the transcriptional start site, only 15 bp of sequence downstream of the transcriptional start site, in addition to 122 bp upstream, was enough to produce wild-type levels of activity. These observations suggest that the kinked region may be of importance to the activity of these promoters. The kinked regions identified in the toxin gene promoters have no apparent sequence similarities. Future work will include further characterization of the positioning and sequence composition of these regions.
It is notable that B. anthracis toxin gene expression is enhanced during growth of the bacterium in elevated atmospheric CO2 or dissolved bicarbonate (Sirard et al., 1994). It is possible that alterations in the inherent DNA topology of the B. anthracis toxin gene promoters occur in response to growth under these conditions. Local DNA structure can be affected by environmental conditions. DNA topology changes due to environmental cues, including osmolarity (Jordi et al., 1995), temperature (Goldstein & Drlica, 1984) and oxygen availability (Dorman et al., 1988), have been reported. For example, a DNA topological change required for transcription from the proU promoter in S. typhimurium is induced under conditions of high osmolarity (Jordi et al., 1995; Owen-Hughes et al., 1992; Yamada et al., 1991; Zuber et al., 1994). In the same organism, DNA topology of the aniG promoter is affected by changes in pH (Karem & Foster, 1993).
Overall, our analysis of the promoter regions of the anthrax toxin promoters has revealed the minimal regions required for promoter activity, and areas of high curvature within these sequences. The lack of any known sigma factor consensus sequences may indicate that in the absence of AtxA these plasmid-borne promoter regions are not favoured for expression by the conventional transcription machinery. The presence of high intrinsic curvature may play a role in recognition of the promoters by transcription machinery and/or trans-acting regulators such as AtxA. Structural models of our non-functional deleted promoters predict alterations in the intrinsic curvature, suggesting that the architecture of the DNA within these regions plays a significant part in toxin gene expression. We postulate that in an organism with a high A+T content, such robust intrinsic curvature may preclude the need for nucleosome architects such as H-NS and HU. Our study is, to our knowledge, the first comprehensive analysis of the toxin gene promoter regions and cis-acting sequences important for expression. These findings will complement ongoing investigations of AtxA and other trans-acting factors that affect promoter expression.
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ACKNOWLEDGEMENTS |
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Edited by: T. Abee
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Received 19 December 2007;
revised 1 May 2008;
accepted 8 May 2008.
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