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1 Department of Biomedical Sciences, Dental School, University of Maryland, Baltimore, 666 W. Baltimore Street, Baltimore, MD 21201, USA
2 Molecular and Cell Biology Program, University of Maryland, Baltimore, 108 N. Greene St, Baltimore, MD 21201, USA
Correspondence
Mark A. Strauch
mas002{at}dental.umaryland.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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; Strauch, 1993; Strauch & Hoch, 1993). The size and diversity of functions present in the AbrB regulon indicate the global role of AbrB in optimizing gene expression to suit a particular environment (Phillips & Strauch, 2002; Strauch, 1993). AbrB orthologues and paralogues have been found in all Bacillus, Clostridium and Listeria species sequenced to date. Others have been noted via BLAST (Altschul et al., 1990) searches in Oceanobacillus, Geobacillus, Ruminococcus, Thermoanaerobacter, Moorella, Desulfitobacterium, Exiguobacterium, Carboxydothermus, Pyrococcus, Sulfolobus and Ferroplasma databases.
The B. subtilis AbrB protein is a homotetramer of identical 10 500 Da subunits (Vaughn et al., 2000; Perego et al., 1988). The N-terminal domain (about 50 amino acids) of the protein is paradigmatic for a new class of DNA-binding motif (the looped-hinge helix fold) that achieves binding specificity through flexible interactions with DNA of varying sequences (Vaughn et al., 2000; Zuber, 2000). Based upon mutant analysis (Xu et al., 1996) and examination of purified N-terminal truncations of AbrB (Xu & Strauch, 2001), it has been proposed that most, if not all, of the DNA-binding specificity determinants reside in the N-terminal domain, with the function of the C-terminal being a multimerization domain. The AbrB protein from Bacillus anthracis is highly homologous to the B. subtilis protein (80/94 amino acid residue identities), with the N-terminal 62 residues being perfectly identical in the two species. Given this identity in DNA-binding domains, and our hypothesis that the C-terminal domains of AbrB homologues are primarily multimerization domains, we predicted that the proteins might be interchangeable in achieving AbrB-mediated regulatory effects in vivo, and that the sequence differences in the C-terminal domains of the proteins might have little, or no, effect on DNA-binding specificity. To test these predictions, we purified the B. anthracis AbrB protein (AbrBBA) and compared its in vitro binding specificity to that of the B. subtilis orthologue (AbrBBS). Additionally, we examined the ability of the two orthologues to substitute for each other in achieving in vivo regulatory effects.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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), with the following modifications: buffer B* (see below) replaced the buffer A of the original purification scheme, and the desalting step prior to fractionation on DEAE-trisacryl column was accomplished by dialysis rather than use of a desalting column. A 450 bp DNA fragment containing the B. anthracis abrB gene and its ribosome-binding site was amplified via PCR using pUTE460 (T. Koehler, University of Texas, Houston, TX, USA) as a template, and the oligonucleotide primers S95 (5'-CGGAATTCGTCCATAGGGTATTTTGTCG-3') and S96 (5'-AAACTGCAGATCGAGATTTGTCGAAACG-3'). After digestion with EcoRI and PstI (which cut at sites engineered at the flanks; see the underlining in the primers above), the fragment was ligated into the IPTG-inducible expression vector pKQV4 (Strauch et al., 1989b), creating plasmid pMAS3317, which was introduced into E. coli strain DH5
. The resultant expression strain (MAS3317) was grown at 37 °C in LB medium containing 50 µg ampicillin ml–1 to an OD600 of 0·6. IPTG was added to 1 mM and incubation continued for 2 h. The cells were harvested by centrifugation, washed and cracked open as has been described (Strauch et al., 1989b). Throughout the procedure the presence of the AbrBBA protein was monitored by SDS-polyacrylamide gel fractionation (4 % stacking gel/18 % separating gel; Laemmli buffer system). All the following steps were performed at 4 °C. The buffer (B*) used to prepare the crude extract and in the following steps was 10 mM Tris pH 8, 10 mM KCl, 0·1 mM MgCl2, 1 mM Na2EDTA, 10 mM 
-mercaptoethanol and 40 µg PMSF ml–1. The crude extract was subjected to streptomycin sulfate and ammonium sulfate precipitation steps as has been described for the B. subtilis protein, except that the ammonium sulphate cut used for subsequent steps was 30–75 %. After dialysis versus buffer B*, the (NH4)2SO4 cut was applied to a DEAE-trisacryl (BioSepra) column, washed with buffer B* and eluted with a 10–400 mM gradient of KCl in buffer B*. Fractions containing the AbrBBA protein were pooled, dialysed versus buffer C* (25 mM Tris pH 7·2, 5 mM KCl, 1 mM MgCl2, 1 mM Na2EDTA, 10 mM -mercaptoethanol and 40 µg PMSF ml–1) and applied to a heparin agarose (Sigma) column equilibrated with the same buffer. The column was washed with buffer C* and eluted with a 5–150 mM KCl gradient in C*. Fractions containing the AbrBBA protein were pooled and concentrated to approximately 2–3 mg ml–1 using Centriprep 3 devices (Amicon). Glycerol was added to approximately 25 % (v/v; final concentration) for storage at –80 °C. Protein concentrations were determined using the Bio-Rad (Bradford) assay reagent with BSA as the standard. Final protein purity was judged to be greater than 95 % by SDS-PAGE (data not shown).
Subcloning of B. anthracis promoters for in vitro and in vivo assays.
A 372 bp fragment containing the atxA promoter region (–200 to +171, relative to the start of transcription) was PCR amplified using pUTE411 as the template and the following primers: 5'-GGGAATTCAG-TAACATTCATTAATCCTAAGCTAG-3' and 5'-TTGGATTCTTGTTCATTGATAAA-GTGTAGTAAA-3'. A 391 bp fragment containing the abrBBA promoter region (–236 to +155 relative to the start of transcription) and its intact Spo0A-binding region (‘Spo0A box’=TGNCGAA; Strauch et al., 1990) was amplified using pUTE415 as the template and the following primers: upstream, 5'-CGGAATTCGTTAATGCGTGATAAAAACGAG-3', and downstream, 5'-TAGGATCCAGTACGGCGTAATTCGATTGG-3'. To construct an abrBBA promoter with a disruption in the Spo0A box region, the upstream primer listed above was used with the downstream primer 5'-TAGGATCCTCGACAAAATACCCTATGGACA-3' to produce a 255 bp fragment (–236 to +17). This construct removed the second of the tandem Spo0A boxes in its entirety (the normal Spo0A box region is 5'-TGTCGAAAAATGACGAA-3'; the construct contains the 6 bp sequence TGTCGA of the Spo0A box at its 3'end). A 399 bp fragment containing the presumptive promoter region of the lef gene (–324 to +75 relative to the ATG start codon for the lef ORF) was amplified using pUTE2 as the template and the primers 5'-CGGAATTCGAAAAATGATAGAATCCCTACACT-3' and 5'-ATGGATCCCGGGACCACTCAAAGTAATTG-3'. A 400 bp fragment containing the presumptive cya promoter region (–317 to +83 relative to the ATG start codon of the cya gene) was amplified using pUTE5 as the template and the primers 5'-CGGAATTCCAGCTGAACTTTATCAACTTAGAATC-3' and 5'-ATGGATCCGCCTGTGAGGAGGATATAGCA-3'. A 326 bp fragment containing the presumptive promoter region of the pag gene (–230 to +96 relative to the ATG start codon of the pag gene) was amplified using pUTE4 as the template and the primers 5'-CGGATTCCGAACTGATACACGTATTTTAGCA-3' and 5'-AGGGATCCAACTTCTGCCTGAATCACCTCT-3'. The fragments were cloned into either pDH32, a lacZ transcriptional fusion vector integrative at the amyE gene of B. subtilis (Shimotsu & Henner, 1986), or pJM103 (Perego, 1993), or both. The appropriate B. subtilis strains were transformed with the pDH32 derivatives carrying the atxA and abrBBA promoters using standard procedures (Hoch, 1991), and were assayed for -galactosidase activity as described below. Derivatives of pJM103 were used to generate labelled fragments for use in footprinting experiments (see below).
Construction of reporter strains SQQ103 and SQQ118.
An approximately 450 bp fragment containing the yxbB promoter (Yoshida et al., 1995, 1999; Molle et al., 2003; Hamon et al., 2004) was inserted into the lacZ fusion vector pDG1729 (Guerout-Fleury et al., 1996; pDG1729 is an integrative plasmid that inserts in the thrC locus of B. subtilis) to create pQQ23. The spo0A abrB strain 1S11 (Xu et al., 1996) was transformed with pQQ23, with selection for spectinomycin resistance (75 µg ml–1) and screening for threonine auxotrophy, to produce strain SQQ107. DNA fragments (about 450 bp) containing the abrB genes from B. subtilis and B. anthracis, and their respective ribosome-binding sequences, were inserted into the amyE-integrative vector pDR67 (Ireton et al., 1993) in order to place the genes under control of the IPTG-inducible pSpac promoter present on pDR67. These latter plasmids, pQQ43 and pQQ47, were propagated in a pcn mutant E. coli strain (TP611) as it had been determined empirically that pSpac derivative plasmids expressing wild-type alleles of abrB were deleterious to E. coli cells. Plasmids pQQ43 and pQQ47 were introduced into SQQ107, with selection for chloramphenicol resistance (5 µg ml–1) and screening for the absence of amylase activity (on nutrient agar plates containing cornstarch), to create SQQ103 and SQQ118, respectively.
DNase I footprinting assays.
Target DNA fragments containing promoter regions were obtained from the appropriate clones (see above, Table 1 and figures), and end-labelled using [-32P]dATP (Amersham) and the Klenow enzyme (New England Biolabs). DNase I protection assays were performed essentially as has been previously described (Strauch et al., 1989b; Xu & Strauch, 2001), but with the following modifications: AbrB binding was carried out in a 15 µl reaction volume at room temperature for 10 min followed by Dnase I (6·3 µg ml–1 final concentration) treatment for 10 s at room temperature.
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-Galactosidase assays.-galactosidase activity were grown in Schaeffer's medium and the assays were performed as has been described previously (Ferrari et al., 1986). All assays were repeated at least twice with no significant differences in either the temporal pattern or the relative levels of expression (between strains) being observed. |
RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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), and have hypothesized that the C-terminal residues are required primarily for stable multimerization of the native homotetramer and provide no direct DNA-binding specificity determinants (Xu et al., 1996; Xu & Strauch, 2001). Given the identical nature of the N-domains and the significantly different C-domains of AbrBBS and AbrBBA, comparison of the DNA-binding specificity of these two orthologues provided a strong test for our hypotheses.
We first performed DNase I footprinting assays of AbrBBA and AbrBBS binding to the promoter region of the abrBBS gene. As shown in Fig. 1, the extent of binding by the two proteins is indistinguishable at this target. Reasoning that expression of the B. anthracis AbrB is probably subject to autoregulation as is the B. subtilis protein (also see below), we next examined binding of the proteins to a DNA fragment containing the abrBBA promoter region. Fig. 2 shows the result of a DNase I footprinting experiment demonstrating that the extent of binding by the two proteins is indistinguishable at this target also. Fig. 3 provides a comparison of the sequences of the two promoters and shows the extent of AbrB binding to each. Previous work had divided the AbrB-binding region on the abrBBS promoter into a higher-affinity interaction region (–14 to –43) and a contiguous upstream region (–44 to about –120) that displayed an apparently lower affinity for binding (Strauch et al., 1989b; Xu et al., 1996; Strauch, 1995a). The position (relative to RNA polymerase recognition elements) of AbrB binding on the abrBBA promoter coincides with the higher-affinity region on the abrBBS target (although the former extends about one turn of the helix farther downstream than the latter). In contrast, the B. anthracis abrB promoter lacks the upstream lower-affinity region, possibly due to the upstream presence of the sequence 5'-TTTTTAAAAA-3' (from –52 to –61), which is not found in the abrBBS sequence. The physiological or regulatory relevance of the upstream TTTTTAAAAA sequence, and the lack of upstream AbrB binding, is not clearly understood but in vitro binding of the two AbrB orthologues was identical, supporting the hypothesis that sequence specificity results from interaction of the identical N-domains of the proteins with the DNA targets.
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, which depicts binding to DNA fragments containing two synthetic sites selected for high-affinity binding by AbrBBS (Xu & Strauch, 1996). Identical specificity (data not shown) was also seen at the AbrBBS targets from the B. subtilis yxbB and spo0E (Strauch, 1995a) genes.
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; Baillie et al., 1998). We examined the ability of the AbrB proteins to interact with the putative promoter regions of the B. anthracis toxin component genes (cya, lef and pag) and to the promoter for the toxin regulatory gene, atxA (Dai et al., 1995). No binding was observed to a fragment containing positions –230 to +96 (relative to the ATG start codon) of the pag gene or to a fragment containing positions –317 to +83 (relative to the ATG start codon) of the cya gene (data not shown). A rather weak AbrB binding interaction (data not shown) was observed upstream of the lef gene at positions –260 to –232 relative to the mapped transcription start site (Dai et al., 1995). As this site is well upstream of the lef promoter, its direct regulatory significance for lethal factor expression seems doubtful. A much stronger AbrB–DNA interaction (Fig. 5) was observed in the vicinity of the promoter for atxA expression (Dai et al., 1995). Relative to the start point of transcription, AbrB protected a region extending from –67 to about –25 on the template strand (–67 to –18 on the non-template strand; see Fig. 6). Given this location that overlaps with crucial promoter elements, and a previous report indicating that AbrB negatively regulates atxA expression (Saile & Koehler, 2002), it seems logical to conclude that AbrB binding here has a direct regulatory effect in vivo.
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, the atxA promoter is expressed in B. subtilis and subject to AbrB-dependent regulation. Expression was significantly increased in an abrB mutant strain (relative to wild-type) during both vegetative growth and the early stages of stationary phase/sporulation. The decreased expression seen in spo0A or spo0H backgrounds was abrogated if an abrB mutation was also present in the strain, and the pattern and level of expression seen in the double mutants were indistinguishable from those seen in the abrB mutant. Thus the spo0A and spo0H effects appear to be solely due to their causing an increase in intracellular levels of AbrB (Strauch et al., 1990).
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). The negligible expression of the abrBBA promoter in wild-type and spo0A contrasts with the pattern seen for the B. subtilis abrB promoter in that expression of the latter is detectable in both these backgrounds, with expression in spo0A actually being slightly higher than wild-type (Strauch et al., 1989a). We suspected that our observed results with the abrBBA promoter might have been due to it being more sensitive to AbrB-mediated negative regulation than the abrBBS promoter.
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), we next examined the expression of an abrBBA variant promoter that did not possess an intact Spo0A-box region (see Methods) in order to eliminate the direct Spo0A-binding component from our analysis. Expression of the truncated construct was significant in a wild-type background, negligible in a spo0A background and greatly overexpressed (relative to the wild-type) in both abrB and spo0A abrB backgrounds (Fig. 9). The simplest explanation of these patterns is that overexpression of AbrBBS (resulting from lack of Spo0A transcriptional repression of the native abrB gene, in a spo0A background) completely blocks transcription from the abrBBA promoter, presumably due, at least in part, to a strong AbrBBS–abrBBA DNA-binding interaction that effectively prevents RNA polymerase access. Since overexpression of AbrBBS (due to a spo0A mutation) does not completely shut off expression of the abrBBS promoter, with (Strauch et al., 1989a) or without intact Spo0A-boxes (data not shown), it is possible that the abrBBA recognition site provides a stronger binding target for AbrBBS interaction than does the abrBBS recognition site, at least under the B. subtilis intracellular conditions. However, we have not been able to detect any significant difference in the strength of binding of AbrBBS to the abrBBA or abrBBS promoters in vitro, nor have we detected any significant difference in the binding of AbrBBA to these regions (data not shown). Assuming AbrB binding strength is also approximately the same at both the abrBBS and abrBBA promoters in vivo, perhaps the RNA polymerase interaction at the B. anthracis abrB promoter is weaker than at the homologous promoter in B. subtilis. Thus AbrB binding at the former might be more effective at blocking transcription than at the latter. If this were true, the nucleotide sequence variations between the promoters would no doubt be a major contributing factor. In this regard it is interesting to note the differing –10 sequences and the stretch of upstream A and T residues in the B. anthracis promoter, which might be a region of bending or curvature (Crothers et al., 1990). Nevertheless, our observations show that the abrBBA promoter is an in vivo binding and regulatory target for AbrBBS. Although work has been published that demonstrates Spo0A regulation of abrB in B. anthracis (Saile & Koehler, 2002), we are unaware of any published data directly demonstrating autoregulation of abrB expression in B. anthracis. Combined with our other observations, we predict that the B. anthracis abrB gene is autoregulated in a manner similar, or identical, to the situation in B. subtilis.
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illustrates the expression of the yxbB–lacZ reporter in these strains with and without IPTG induction (i.e. with and without expression of the abrB genes). In the absence of IPTG induction, yxbB–lacZ expression patterns were identical for the two strains. In the presence of IPTG, induction of either AbrBBS or AbrBBA led to repression of the yxbB–lacZ reporter. The slight difference in repression level seen for the two induced strains may be attributable to a post-transcriptional event affecting AbrB protein levels. We believe the most likely cause is a difference in translational efficiency of the abrB genes since the two pSpac constructs have slightly different ribosome-binding sites upstream of the abrB genes (each being preceded by its native RBS). Nevertheless, the results clearly show that the B. anthracis AbrB protein can complement the deletion of the abrBBS gene in a B. subtilis cellular environment.
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; Xu et al., 1996; Strauch, 1996). NMR structural studies revealed a new class of DNA-binding motif (the looped-hinge helix fold) that is formed via the association of AbrB N-domains (Vaughn et al., 2000). We further hypothesized that the C-terminal domain of the protein was primarily, or solely, a higher-order multimerization domain with no function in direct contact or recognition of target binding sites. The AbrB protein from B. anthracis has perfect amino acid sequence identity to the B. subtilis protein over the first 62 residues, but differs significantly (14 non-identities) from it in the last 32 residues. This sequence difference results in the B. anthracis protein having a significantly more acidic pI than its orthologue. To test our domain hypothesis, we examined the in vitro binding specificity and in vivo regulatory interchangeability of these two AbrB variants.
Binding specificity of the two AbrB proteins was indistinguishable at each of the target DNA sequences examined (Figs 1–6
). Based on in vivo assays in an identical intracellular environment (B. subtilis), the proteins appeared essentially interchangeable in their ability to regulate each of the target promoters we examined (Figs 7–10), even when the target was a promoter not found in the protein's normal milieu (e.g. atxA regulation by AbrBBS and yxbB regulation by AbrBBA). Thus, we conclude that the C-domains of the B. subtilis and B. anthracis AbrB proteins, despite their differing sequences, do not contribute to the DNA-binding specificity of the proteins. This strongly supports a notion that the N-terminal and C-terminal domains of AbrB homologues are separable modules: the N-terminal domain providing the structure that directly recognizes and interacts with DNA target sequences, and the C-domain involved solely in a multimerization interaction necessary for stable association of the active multimeric form of the protein. Finally, our results have potential implications regarding regulatory phenomena controlling toxin production in B. anthracis. Previous observations (Saile & Koehler, 2002) indicated that anthrax toxin production is regulated by AbrB and that atxA expression is elevated in abrB mutants. Our results suggest that direct AbrB binding to the atxA promoter, but not direct AbrB binding to the pag, lef or cya promoters, is responsible (at least in part) for the observed AbrB effects on toxin gene expression.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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Baillie, L., Moir, A. & Manchee, R. (1998). The expression of the protective antigen of Bacillus anthracis in Bacillus subtilis. J Appl Microbiol 84, 741–746.[CrossRef][Medline]
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Vaughn, J. L., Feher, V., Naylor, S., Strauch, M. A. & Cavanagh, J. (2000). Novel DNA binding domain and genetic regulation model of Bacillus subtilis transition state regulator AbrB. Nat Struct Biol 7, 1139–1146.[CrossRef][Medline]
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Xu, K. & Strauch, M. A. (2001). DNA-binding activity of amino-terminal domains of the Bacillus subtilis AbrB protein. J Bacteriol 183, 4094–4098.
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Received 2 December 2004;
revised 22 February 2005;
accepted 23 February 2005.
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), spo0A (
), abrB (
), spo0H (
), spo0A abrB (
) and spo0H abrB (
). Zero on the abscissa denotes the approximate end of exponential growth and entry into stationary phase.
: B. anthracis AbrB under pSpac control. Zero on the abscissa denotes the approximate end of exponential growth and entry into stationary phase. IPTG was added to the indicated cultures at approximately 1 h prior to the end of exponential growth.