Different Requirements for σ Region 4 in BvgA Activation of the Bordetella pertussis promoters Pfim3 and PfhaB

doi:10.1016/j.jmb.2011.04.017

J Mol Biol. Author manuscript; available in PMC 2012 June 24.

Published in final edited form as:

J Mol Biol. 2011 June 24; 409(5): 692–709.

Published online 2011 April 22. doi: 10.1016/j.jmb.2011.04.017

PMCID: PMC3141349

NIHMSID: NIHMS298304

Different Requirements for σ Region 4 in BvgA Activation of the Bordetella pertussis promoters P_fim3 and P_fhaB

Kimberly B. Decker,^a Qing Chen,^b Meng-Lun Hsieh,^a Philip Boucher,^b^† Scott Stibitz,^b and Deborah M. Hinton^a^*

^aGene Expression and Regulation Section, Laboratory of Molecular and Cellular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, USA

^bDivision of Bacterial, Parasitic, and Allergenic Products, Center For Biologics Evaluation and Research, FDA, Bethesda, MD 20892, USA

^*For correspondence. Email: dhinton/at/helix.nih.gov; Tel. (+1) 301 496 9885; Fax (+1) 301 402 0053

^†Present address: Novartis Vaccines, Siena, Italy

The publisher's final edited version of this article is available at J Mol Biol

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Abstract

Bordetella pertussis BvgA is a global response regulator that activates virulence genes, including adhesin-encoding fim3 and fhaB. At the fhaB promoter, P_fhaB, a BvgA binding site lies immediately upstream of the −35 promoter element recognized by Region 4 of the σ subunit of RNA polymerase. We demonstrate that σ Region 4 is required for BvgA-activation of P_fhaB, a hallmark of Class II activation. In contrast, the promoter proximal BvgA binding site at P_fim3 includes the −35 region, which is composed of a tract of cytosines that lacks specific sequence information. We demonstrate that σ Region 4 is not required for BvgA activation at P_fim3. Nonetheless, Region 4 mutations that impair its typical interactions with core and with the −35 DNA affect P_fim3 transcription. Hydroxyl radical cleavage using RNA polymerase with σD581C-FeBABE positions Region 4 near the −35 region of P_fim3; cleavage using RNA polymerase with α276C–FeBABE or α302C–FeBABE also positions an αCTD within the −35 region, on a different helical face from the promoter proximal BvgA~P dimer. Our results suggest that the − 35 region of P_fim3 accommodates a BvgA~P dimer, an αCTD, and σ Region 4. Molecular modeling suggests how BvgA, σ Region 4, and α might coexist within this DNA in a conformation that suggests a novel mechanism of activation.

Keywords: BvgA, fim, σ, transcription, polymerase, promoter

Introduction

Pertussis is a reemerging disease in developed nations and a continued presence in the developing world. Although vaccines are available, they do not provide lifetime coverage; outbreaks of pertussis have occurred in populations that were vaccinated a few years earlier ^{1; 2; 3}. Bordetella pertussis, the causative agent of pertussis, employs a simple yet elegant system to regulate its virulence genes. In the environment of the human host, the BvgS-BvgA two-component system is active, resulting in the phosphorylation of BvgA (BvgA~P) (reviewed in ⁴). BvgA~P is a global activator, which binds to promoters of virulence genes with varied stoichiometry and affinity. Thus, upon entry into an inducing environment, the increasing intracellular concentration of BvgA~P orchestrates a temporal program of virulence gene expression ⁵. Genes that are activated early include those encoding adhesive proteins, such as fhaB (filamentous hemagglutinin) and fim2 and fim3 (fimbrial subunits). Late-activated genes include those encoding toxic virulence factors such as adenylate cyclase and pertussis toxin ⁶. The temporal program of gene expression is thought to be crucial for the virulence of the pathogen, as demonstrated in a murine model of B. pertussis respiratory infection ⁷. Thus, understanding the molecular mechanism of B. pertussis virulence gene expression and its regulation by BvgS-BvgA will help to develop better strategies to fight the disease.

As in all bacteria, a multisubunit RNA Polymerase (RNAP), consisting of a core of subunits (α₁ α₂, β, β’, ω) and a specificity factor, σ, transcribes the B. pertussis genome. Promoter recognition involves multiple interactions between regions of σ and the promoter DNA. For primary σ factors, such as σ^Bp in B. pertussis or σ⁷⁰ in Escherichia coli, these contacts can include Region 2 with the −10 element, Region 3 with the sequence ⁻¹⁵TG⁻¹⁴ (extended −10 element), and Region 4 with the −35 element (reviewed in ¹¹). As good matches to two out of three of these elements are typically sufficient for promoter activity, many promoters can be classified as belonging to the −35/−10, TGn/−10 (extended −10), or −35/TGn class. In addition, the C-terminal domains (CTDs) of the α subunits of RNAP contribute to promoter binding through their interaction with DNA sequences (UP elements) upstream of position −40 (reviewed in ¹²).

Regulated promoters require additional factors besides RNAP, usually to compensate for weak σ/DNA element interactions. In Class I activation, the activator may bind to several locations upstream of the −35 element and interact directly with the αCTDs; in Class II activation, the activator binds to a site adjacent to or overlapping the −35 element and contacts an αCTD and/or σ Region 4 (reviewed in ^{13; 14}). Class I and Class II activation may also function in combination with each other, but in each case, σ Region 4 is thought to be required in order to maintain contacts with the −35 region of the DNA.

σ appropriation is a different type of activation, used by bacteriophage T4, in which the binding site for the T4 activator MotA includes the −35 region of the DNA (reviewed in ¹⁵). Thus, MotA and σ Region 4 are essentially competing for the DNA. In this case, the T4-encoded co-activator AsiA binds to Region 4, remodeling its structure and preventing its interaction with the −35 portion of the promoter. However, σ Region 4 is still required because of its needed interactions with AsiA and MotA.

Most of the virulence genes in B. pertussis have an organization that resembles that seen at ClassI/II activated promoters ^{16; 17}. For example, the fhaB gene is activated by three dimers of BvgA~P, which bind from −95 to −38 relative to the transcription start site ¹⁸ (Fig. 1a). In contrast, at the promoters for fim2 (P_fim2) and fim3 (P_fim3; Fig. 1a), the promoter proximal BvgA~P site fully includes the −35 region of the DNA. In addition, P_fim2 and P_fim3 both contain a tract of poly cytosines (C), which is located upstream of the −10 element and includes the −35 region ²⁰. The length of this C-tract regulates BvgA~P activation ²⁴. We have recently shown that the C-tract is needed to position BvgA~P correctly relative to the bound RNAP; however, there is little, if any, specific sequence information imparted by the tract ²⁰. The second unusual feature of P_fim3 is that it contains both the ⁻¹⁵TG⁻¹⁴ motif, which is recognized by σ Region 3, and a good match (⁻¹²TATTCT⁻⁷) to the −10 element, which is recognized by σ Region 2. Thus, P_fim3 appears to be a TGn/−10 (extended −10) promoter, and we would predict that it would exhibit some activity with RNAP alone ^{25; 26; 27}. However, P_fim3 displays very little activity in the absence of BvgA~P ²⁰.

Figure 1

The promoters P_fhaB, P_fim3−15C, P_RE#, P_RNAI and P_uvsX and Region 4 of wild type σ⁷⁰, σ^Bp and mutant σ proteins

In this study, we have investigated BvgA~P activation at P_fhaB and P_fim3. We show that σ Region 4 is required for BvgA~P activation at P_fhaB, providing further evidence that the promoter proximal BvgA~P functions as a Class II activator at this promoter. In contrast, BvgA~P activation at P_fim3 is different. Although we find that it does not require σ Region 4, we also find that Region 4 is still located close to the P_fim3 −35 DNA. Furthermore, using RNAP reconstituted with FeBABE-conjugated α subunits, we find that each α contacts the same region of P_fim3 as BvgA, but on a different helical face. Taken together, we speculate that within the activated complex at P_fim3, σ Region 4, BvgA~P, and an αCTD are all located within the −35 region C-tract of the DNA.

Results

Experimental design and description of σ mutants

The C-terminal portion of primary σ factors, such as E. coli σ⁷⁰, is composed of five alpha helices (H1–H5) with a turn (T) between H3 and H4 (Fig. 1b) ^{28; 29; 30}. Residues within σ⁷⁰ Region 4 (H1–4) and H5 perform several important functions, including interaction with the β-flap of core polymerase, recognition of −35 element promoter DNA, and interactions with regulators that are needed to activate promoters by Class II activation and by σ appropriation (Fig. 1b) (reviewed in ^{15; 31; 32}). The interaction with the β-flap positions Region 4 so that it is located close to the −35 DNA, which then allows residues within the second helix of the helix-turn-helix (H3-T-H4) to make specific contact with base determinants within the −35 DNA (Fig. 1b) ^{28; 33; 34; 35; 36; 37}.

To investigate the importance of various portions and functions of Region 4 in BvgA~P activation at P_fhaB and P_fim3, we employed several σ mutants, detailed below. The behavior of each mutant σ was evaluated using various promoters (Fig. 1a): the −35/−10 promoters P_RNAI ³⁸ and unactivated P_uvsX ³⁹, which require an interaction between Region 4 and the −35 DNA; the extended −10 promoter P_RE# ²⁵, which is much less dependent on an interaction with Region 4; and activated P_uvsX ³⁹, which uses contacts between Region 4 and the T4 activator MotA and the T4 co-activator AsiA (Fig. 1b). We used transcription conditions that recapitulate BvgA~P regulation of P_fhaB and P_fim3; specifically, for fim3 transcription a C-tract of 13 C’s is inactive while a tract of 14 or 15 C’s is active (Fig. 2a, lanes 13; Fig S1 ²⁰). We also limited transcription to a single round in order to observe how protein or DNA mutations affect the formation of transcriptionally competent complexes.

Figure 2

σ Region 4 is required for BvgA~P activation of P_fhaB, but not for P_fim3

We expected that two of the σ mutants, σ^{4.1+4.2 ala} and σ^580-3ala, might impair recognition of the −35 region of the promoter DNA. σ^{4.1+4.2 ala} contains alanine patches at residues 551–555 and 584–588, substitutions that replace residues that directly contact the −35 element or that interact with the β-flap (Fig. 1b). In addition, the Region 4.2 alanine mutations alone have previously been shown to eliminate σ appropriation at P_uvsX because they disrupt important interactions between the T4 co-activator AsiA and Region 4 (⁴⁰ (Fig. 1b)). As expected, transcription from the −35/−10 promoter P_RNAI (Fig. 2a, lanes 7–9) and unactivated and activated transcription from T4 P_uvsX (Fig. S2, lanes 1–4) is essentially abolished when using RNAP reconstituted with σ^{4.1+4.2 ala}. In addition, transcription from the extended −10 promoter P_RE# is also reduced (Fig. 2a, lanes 7–9), consistent with other work showing that non-specific contacts between the −35 region of an extended −10 promoter and Region 4 are beneficial ^{25; 41; 42}. The σ^580-3ala mutant contains an alanine patch within the turn of the DNA binding motif, H3-T-H4. As seen in Fig. 1b, these mutations might impart some impairment in the interaction of the binding motif with the −35 DNA or with the interaction with AsiA. Transcription from the −35/−10 promoters P_RNAI (Fig. 2a, lanes 10–12) and unactivated P_uvsX (Fig. S2, lane 9 vs. 13) is indeed impaired. However, MotA/AsiA activation of P_uvsX is similar to that seen with RNAP reconstituted with σ⁷⁰ (Fig. S2, lane 12 vs. 15).

The σ^Hyb4 mutant protein is a chimeric σ factor in which Region 4.2 and H5 of σ⁷⁰ (residues 565 to 613) have been replaced with the corresponding residues (280–330) of σ³⁸, the stationary phase σ factor of E. coli (Fig. 1b). Thus, the protein retains residues that contact the −35 element despite many amino acid changes throughout Region 4; in addition, it has been shown that σ³⁸ Region 4 interacts well with the β-flap ³⁷. However, some of the substitutions present in σ³⁸ include Region 4 residues involved in the MotA/σ⁷⁰ interaction (Fig. 1b), so they should render RNAP reconstituted with σ^Hyb4 significantly impaired at MotA/AsiA-mediated σ appropriation. Experiments with σ^Hyb4 thus sought to address whether BvgA activation resembles σ appropriation. As seen in Fig. 2a, lanes 4–6, and Fig. S2, lane 5, this mutant σ recognizes the P_RNAI, P_RE#, and unactivated P_uvsX promoters, although the overall activity of the σ^Hyb4 is lower than wt. In contrast, it is significantly impaired for MotA/AsiA activation (Fig. S2, lane 8).

Finally, we tested mutants of E. coli σ⁷⁰ and the B. pertussis primary σ (σ^Bp) that lack all of Region 4, residues 530–613 in σ⁷⁰ and residues 675–760 in σ^Bp, respectively. Region 4 of E. coli and Region 4 of B. pertussis are 73% identical (Fig. 1b). Transcription from known promoters using RNA polymerase reconstituted with either E. coli σ⁷⁰ or σ^Bp has been shown to be similar ^{40; 43} and σ^Bp, like σ⁷⁰, is competent for MotA/AsiA mediated σ appropriation ⁴⁰. Thus, it is thought that Region 4 of each σ behaves similarly. As expected, RNA polymerase with σ^70ΔR4 had practically no activity at P_RNAI, and like σ^{4.1+4.2 ala}, its activity with P_RE# was significantly reduced although not as much as at P_RNAI (Fig. 2b, lanes 4–6 and 16–18). As expected, neither σ^70ΔR4 nor σ^BpΔR4 were active at P_uvsX with or without MotA/AsiA (data not shown). Surprisingly, σ^BpΔR4 retained some activity at P_RNAI (Fig. 2b, lanes 10–12 and 22–24), suggesting that there are differences in how the E. coli and the B. pertussis primary σ factors use this promoter. However, we have not examined this difference at P_RNAI further.

σ⁷⁰ Region 4 is required for BvgA~P activation at P_fhaB

The positions of the BvgA~P binding sites within P_fhaB are suggestive of a combination of Class I and Class II activation ^{16; 17}. In such an arrangement, σ Region 4 is typically required for activation, providing a direct contact with the promoter proximal activator (reviewed in ¹⁴). However, previous work has shown that at BvgA~P activated P_fhaB, the αCTDs are located on the same sequence but an alternate face from a BvgA~P dimer ¹⁸. This position differs from other Class I and Class II activators, in which the αCTDs are located adjacent to the activator ^{44; 45; 46}. Consequently, it is important to ask directly whether σ⁷⁰ Region 4 is needed for BvgA~P activation at P_fhaB.

We find that Region 4 is indeed needed for BvgA~P activation at P_fhaB. RNAP reconstituted with σ^{4.1+4.2 ala}, σ^70ΔR4, or σ^BpΔR4 is not functional at this promoter (Fig. 2a, lanes 7–9 and 2B, lanes 4–6 and 10–12). In contrast, transcription with RNAP containing σ^Hyb4 or σ^{580-3 ala}, which retain contacts with the −35 DNA and the β-flap, is much less affected (Fig. 2a, lanes 4–6 and 10–12). Taken together, these results are consistent with the idea that BvgA~P activation at P_fhaB relies on Region 4 contacts with the −35 region of the DNA, a hallmark of Class II activation.

σ Region 4 is not required for BvgA~P activation at P_fim3

Unlike at P_fhaB, the promoter proximal BvgA~P binding site at P_fim3 completely overlaps the −35 portion of the promoter (Fig. 1a). This positioning suggests several possibilities for the importance of Region 4 at P_fim3. First, this BvgA~P site at P_fim3 is in a similar location to the PhoB site at the promoter P_pstS ²³. PhoB is known to contact a portion of Region 4 that is not typically used by Class II activators (^{47; 48} (Fig. 1b)), and as expected, deletion of σ⁷⁰ Region 4 eliminates PhoB activation at P_pstS ⁴¹. However, another activator whose binding site overlaps the −35 region is bacteriophage T4 MotA, which uses σ appropriation. Thus, if similar to MotA, BvgA~P activation at P_fim3 might require a repositioning of Region 4 to allow the downstream BvgA~P dimer to interact with its binding site. Finally, BvgA~P might need an unrecognized reconfiguration or accommodation of Region 4 in order to activate at P_fim3. Besides these considerations, the fim3 promoter is also unusual in that its −35 region contains a C-tract, whose length determines whether the promoter is inactive (<13 or >15 C’s) or active (14 or 15 C’s) (²⁰ (Fig. S1)). We have previously shown that the C-tract appears to function simply as a spatial unit that does not impart significant sequence specificity ²⁰. In fact, although both BvgA and σ Region 4 have a “C” base within their respective consensus binding sites, mutation of each C throughout the C-tract is inconsequential ²⁰. This finding would be consistent with either a model in which Region 4 is removed from its normal position on the DNA by the presence of BvgA~P or a model in which Region 4 stays in its typical position through protein-protein contacts, despite the absence of specific contacts with the DNA.

As a first step in determining which of these models are possible at P_fim3, we assayed the effect of the σ mutants on BvgA~P activation at this promoter. In contrast to the results with P_fhaB, our transcription analyses with P_fim3 indicated that none of the tested σ mutants eliminated BvgA~P activation (Fig. 2a and b). In particular, RNAP reconstituted with σ^{4.1+4.2 ala}, which was essentially nonfunctional with every other tested promoter, yielded 60% of the BvgA~P activated transcription seen with σ⁷⁰, (Fig. 2a, lanes 7–9) and RNAP containing either σ^70Δ4 or σ^BpΔ4 still retained some activity for BvgA~P activation at P_fim3 (Fig. 2b, lanes 16–18 and 22–24).

To analyze the role of σ Region 4 by a separate and independent method, we asked how the dramatic remodeling of Region 4 imposed by AsiA (²⁹; reviewed in ^{15; 49}) affects BvgA~P activation at P_fim3. The binding of AsiA to Region 4 changes the structure of Region 4, eliminating its ability to interact with either the −35 DNA or the β-flap of core polymerase. In these transcriptions, we added NTPs and heparin to the transcription complexes after only 1 min. Thus, the formation of stable complexes was performed under stringent conditions. Consistent with our previous results, we found that AsiA-modified RNAP transcribes from P_fim3 in a BvgA~P-dependent manner but is inactive at P_fhaB (Fig. 3, lanes 4–6). Although it is formally possible that BvgA~P displaces AsiA from σ⁷⁰, this seems highly unlikely. Other work has shown that the interaction between AsiA and σ Region 4 is extremely tight; urea is used to disrupt the association ⁵⁰.

Figure 3

The native structure of σ Region 4 is not required for BvgA~P activation of P_fim3 but is required for activation of P_fhaB

Taken together, our results demonstrate that BvgA~P activation at P_fim3 does not require σ Region 4 and thus, it differs from Class II activation, PhoB activation, or MotA/AsiA mediated σ appropriation. Two simple possibilities then emerge: 1) BvgA~P somehow replaces σ Region 4, making its presence superfluous or 2) Region 4 retains its typical position within RNAP, which would position it close to the −35 region of the DNA, but this position neither interferes with BvgA~P nor is required for BvgA~P activation.

The extended −10 motif of P_fim3 contributes to BvgA~P activation

The ⁻¹⁵TG⁻¹⁴ sequence (extended −10 motif) provides an important contact for RNAP through σ⁷⁰ Region 3 and has been identified in nearly one-quarter of E. coli promoters (reviewed in ¹¹). The presence of this motif together with a good −10 element has been shown to be sufficient for transcription with or without Region 4 ²⁵. However, even though P_fim3 contains the ⁻¹⁵TG⁻¹⁴ sequence, P_fim3 is dependent on BvgA~P for a high level of activity in vitro and in vivo (Fig. 2a; ²⁰). To investigate in vitro whether the Region 3/⁻¹⁵TG⁻¹⁴ contact was required for this activation, we tested P_fim3 mutant promoters in which the ⁻¹⁵TG⁻¹⁴ was changed to either TA (P_fim3−TA) or AC (P_fim3−AC). In the presence of BvgA~P, both mutant promoters yielded a modest (~2 to 3-fold) decrease in the level of transcription, indicating a preference for the ⁻¹⁵TG⁻¹⁴ sequence over TA or AC (Fig 4a, lanes 3–6; Fig. 4b, lanes 4–9).

Figure 4

The extended −10 element of P_fim3 is essential for activity when σ Region 4 is mutated

To test the importance of the ⁻¹⁵TG⁻¹⁴ sequence in vivo, we took advantage of transcriptional gene fusions with the luxCDABE operon of Photorhabdus luminescens, encoding a bacterial luciferase enzyme and other enzymes required for luciferase substrate synthesis. B. pertussis strains were constructed in which the P_fim3 promoter, harboring specific substitutions within ⁻¹⁵TG⁻¹⁴, was fused with the lux operon. The resulting strains were tested for light output (Fig. 5a left panel, with quantitation in Fig. 5b). As previously shown ^{20; 24}, P_fim3−13C, containing a shortened (C)-tract of 13 Cs, had zero promoter activity, while the presence of the longer (C)-tract in P_fim3−15C rendered the promoter active. As in vitro, substitution of the ⁻¹⁵TG⁻¹⁴ sequence of P_fim3−15C with TA yielded only a modest (~2-fold) decrease in promoter activity. However, other substitutions were significantly more deleterious, although a low level of activity (~10%) was still observed with the CG and AC mutations. The discrepancy between the in vitro and in vivo effects of the AC mutation may be attributed to the conditions of the in vitro system, in which there is excess RNAP holoenzyme and no competition with endogenous promoters. As expected, all of the promoters were inactive in the presence of the virulence modulator MgSO₄ (Fig. 5a right panel; Fig. 5b). This compound is known to down regulate the BvgS-BvgA two-component system and, in doing so, inhibit virulence gene activation by BvgA~P ^{51; 52}. This confirms that the active P_fim3 derivatives are still regulated by BvgA~P. Taken with our in vitro data, we conclude that the extended −10 motif of P_fim3 contributes to BvgA~P activation. Thus, even though P_fim3 does not behave like a “classic” extended −10 promoter, which is active in the presence of RNAP alone, it does appear to use the extended −10 element for optimal activity with BvgA~P.

Figure 5

Specific substitutions within the ⁻¹⁵TG⁻¹⁴ sequence of Pfim3 are deleterious in vivo

Region 4 affects transcription from P_fim3

We next sought to understand how BvgA~P activation functions when both Region 3 contacts with the DNA and Region 4 contacts with the −35 DNA and core polymerase are missing or weakened. To this end, we used P_fim3, P_fim3−TA, and P_fim3−AC and RNAP reconstituted with σ⁷⁰, σ^{4.1+4.2 ala}, or σ^ΔR4. With the RNAPs reconstituted with the mutant σ proteins, we observed a strong dependence on the extended −10 motif within P_fim3 (Fig. 4a, lanes 7–12; Fig. 4b, lanes 10–18).

Because the 4.1+4.2 ala mutations impair the ability of Region 4 to correctly interact with the −35 DNA and with the β-flap, our results above suggested that during BvgA~P activated transcription from P_fim3−TA and P_fim3−AC, σ Region 4 retains its typical position relative to the −35 DNA and core polymerase, and that an interaction of Region 4 with the −35 DNA might compensate for the loss of the Region 3/⁻¹⁵TG⁻¹⁴ contact. If this were also true at the wt P_fim3 promoter, then our results would imply that the position of Region 4 within the P_fim3/BvgA~P/RNAP complex might resemble its position at a typical −35/ −10 promoter. To investigate this possibility, we examined the abortive initiation products from P_fim3 using RNAP reconstituted with either σ⁷⁰ or σ^{4.1+4.2 ala}. The short RNA abortive transcripts are synthesized and released from RNAP before the RNAP/promoter complex transitions from an initiating to elongating complex (reviewed in ⁵³). Previous work has indicated that mutations within σ⁷⁰ Region 4 that reduce its interaction with the −35 DNA element and the β-flap facilitate promoter clearance, thus decreasing the level of abortive products relative to full length RNA ^{42; 53; 54; 55}. When using P_fim3 DNA, we observed that the level of abortive products made by RNAP reconstituted with wt σ⁷⁰ is much greater than the level observed using RNAP reconstituted with σ^{4.1+4.2 ala} (Fig. 6). These results are inconsistent with a model in which the presence of promoter proximal BvgA~P simply releases Region 4 from its typical place within RNAP. Instead these results suggest that σ Region 4 either retains its typical contacts with core polymerase and thus, is positioned to potentially interact with the −35 C-tract DNA or that the presence of BvgA~P has moved Region 4 to a new location where it can now make other contacts with polymerase and/or a portion of the DNA.

Figure 6

Disrupting the σ Region 4 contacts with the −35 DNA and with the β-flap of core dramatically reduces the amount of P_fim3 abortive transcripts

Residue 581 of σ Region 4 is located near the −35 region of P_fim3 DNA

To ask directly if σ Region 4 is located near its typical position, we used RNA polymerase reconstituted with a mutant σ⁷⁰ (σ C132S/C291S/ C295S/ D581C), which has a single cysteine residue at position 581. Conjugation with the reactive moiety FeBABE generated σD581C-FeBABE. Since residue 581 of σ⁷⁰ lies at the C-terminal end of H3 of Region 4 (Fig. 1b), it should be located near the −35 region of promoter DNA. Multiple studies ^{56; 57; 58; 59; 60; 61} have demonstrated that upon generation of the cleavage reaction, a bacterial σ conjugated at this location cleaves promoter DNA in the vicinity of the −35 region. Specifically, for the template strand of galP1, major cleavage sites are observed between −38 to −41, while weaker sites are seen at −28/−29⁵⁹; our control reactions with galP1 produced similar results (Fig S3). When using P_fim3−15C end-labeled on the template strand, we found that RNAP containing σD581C-FeBABE resulted in cleavage around positions−41 and −30 (Fig. 7a). End-labeled P_fim3−14C DNA also was cleaved around position −41 but the cleavage bands were less intense (Fig. S4). These cleavage sites are consistent with Region 4 being near the −35 DNA. Taken with the previous results that indicate that Region 4 is indeed needed for BvgA~P activation of P_fim3 that lacks the ⁻¹⁵TG⁻¹⁴ element, we speculate that Region 4 retains its typical position in the presence of BvgA~P. However, we cannot eliminate the formal possibility that the conjugated RNAP is cleaving a subset of the complexes that differ from the transcriptionally competent species.

Figure 7

σ-FeBABE and αCTD-FeBABE cleavage of P_fim3

The RNAP αCTDs bind to the same region of P_fim3 as BvgA~P

At P_fhaB, the αCTDs and BvgA proteins have been mapped to the same region, but different helical faces, of the DNA ¹⁸. Given the differences in the requirement for σ⁷⁰ Region 4 at P_fhaB and P_fim3, we wondered if the αCTDs are also located in a similar position relative to BvgA~P at P_fim3−14C. (A C-tract of either 14 or 15 is an active configuration for this promoter (Fig. S1, ^{20; 24}).) To investigate this question, we used an RNAP that had been reconstituted with α subunits in which an FeBABE moiety had been conjugated to residue 276 or 302, the same conjugates previously used to map the position of the αCTDs at P_fhaB ¹⁸. Hydroxyl radical cleavage of P_fim3−14C DNA, after initiation of the Fenton reaction, generated two major sets of cuts for each conjugate. α302-FeBABE yielded cleavage at −61 and −39 on the template strand and −56 and −33 on the non-template strand; α276-FeBABE yielded cleavage at −53 and −31 on the template strand and −49 and −25 on the non-template strand (Fig. 7b). These cleavage sites are consistent with each αCTD contacting the DNA in the minor groove on a DNA face different from that occupied by the corresponding BvgA~P dimer, i. e. with the same relative orientation as that at P_fhaB ¹⁸. However, when compared to P_fhaB, the cleavage sites for the promoter proximal αCTD at P_fim3 are shifted slightly downstream (approximately 4–5 bp) relative to BvgA~P.

Discussion

Within RNAP, both the α and σ subunits play crucial roles in promoter binding and regulation of transcription. Residues within the αCTD can interact with promoter DNA upstream of −40 and with various transcriptional activators (reviewed in ^{12; 45}). σ Regions 2, 3, and 4 interact with promoter elements within the −10, the ⁻¹⁵TG⁻¹⁴, and the −35 element, respectively (reviewed in ¹¹). In addition, σ Region 4 residues can also interact with various binding partners, including the β-flap of core polymerase ^{37; 62; 63}, and activators needed for Class II activation and σ appropriation (discussed in ^{14; 31; 64}; Fig. 1b).

BvgA~P, the master regulator of B. pertussis virulence genes, binds to multiple sites upstream of each of its targeted promoters. At most promoters that have been analyzed, weaker BvgA~P binding sites are located downstream of a strong binding site and rely on cooperativity of BvgA~P binding to activate transcription (^{65; 66}, reviewed in ⁶⁷). Because the affinity of each site for BvgA~P varies, there is a hierarchy of promoter activity as the concentration of BvgA~P increases, which in turn leads to a temporal pattern of regulation. Adhesin and adhesin-related genes such as fhaB, fim2, and fim3 are expressed early as virulence is being established. It is crucial to understand the mechanism of activation of these adhesin genes, both because the gene products are involved in early steps of pathogenesis and because they are components of the acellular vaccine. However, in order to understand mechanism, one must first determine how the various players (BvgA~P and subunits of RNAP) are positioned within the transcriptionally competent complexes of P_fhaB and P_fim3. For P_fhaB, a strong BvgA~P site is located at positions −95 to −82, while two weaker sites lie at regular helical turns downstream (−73 to −60 and −51 to −38; Fig. 1a). Previous work ¹⁸ has shown that activation of P_fhaB is dependent on the αCTDs. We show here that this activation is also dependent on σ Region 4. This dependence and the P_fhaB organization imply that BvgA~P uses a combination Class I/II mechanism of activation at this promoter, as has been seen for GcvA activation at P_gcvT ⁶⁸, UhpA activation at P_uhpT ^{69; 70}, and synthetic promoters activated by tandem CRP molecules ^{44; 71}. In such a mechanism, contacts between the promoter proximal activator and/or αCTD and σ⁷⁰ Region 4 facilitate the interaction of RNAP with non-ideal promoter elements (Fig. 8a and b), aiding both in RNAP recruitment and in the formation of the stable, transcriptionally competent open complex (reviewed in ¹³). However, unlike these classic systems, the positions of the αCTD binding sites within P_fhaB ¹⁸ are located on a different helical face from the BvgA~P binding site rather than immediately upstream or downstream as is seen for CRP ⁷² (Fig. 8a and b). Thus, BvgA~P at P_fhaB represents a different flavor of this type of activation.

Figure 8

Mechanisms of gene activation and P_fim3 molecular model

In contrast to P_fhaB, the promoters for the fimbrial genes fim2 and fim3 display architectures in which the promoter proximal BvgA~P binding site fully overlaps the −35 region of the promoter (Fig. 1a). While such an arrangement is dissimilar from many well-characterized Class II activators, including CRP, it is similar to the architecture of λcII at λP_RE ^{21; 57; 73; 74}, E. coli PhoB at P_pstS ^{2,3; 41; 47; 75}, Enterococcus faecium VanR_B, a regulator of vancomycin resistance, at P_RB ²²; and bacteriophage T4 MotA at T4 middle promoters (reviewed in ¹⁵). Based on the cII/DNA structure ²¹, it is likely that in these cases except MotA, the activator dimer uses each H-T-H motif to interact with the major groove of the DNA on either side of the σ Region 4 interaction with the major groove of the −35 DNA (Fig. 8c). Additional contacts at P_pstS between PhoB and σ Region 4, such as D570 and E575 ^{47; 48} (Fig. 1b), or between the promoter proximal αCTD and σ Region 4 at cII-activated λ P_RE ²¹ are thought to help to “lock” Region 4 onto the DNA. Indeed, the presence of Region 4 is required for PhoB activity ⁴¹ and the interaction of Region 4 with the −35 element of P_RE is not changed by the presence of cII ⁵⁷. Furthermore, like P_fim3, λP_RE and E. faecium P_RB both contain the ⁻¹⁵TG⁻¹⁴ motif that is recognized by σ Region 3. These similarities suggest that the architecture of BvgA~P at P_fim3 might share features with these activators.

Despite its similarities with the cII, VanR_B, and PhoB systems, BvgA~P at P_fim3 has significant differences. First, the −35 region of P_fim3 (and P_fim2) includes the C-tract, whose length varies stochastically during replication. The length of the C-tract determines promoter activity ²⁴, by positioning BvgA~P relative to the promoter ²⁰. The C-tract appears to function simply as a spatial unit that does not impart significant sequence specificity. In fact, although both BvgA and σ Region 4 have a “C” base within their respective consensus binding sites, mutations of each C throughout the C-tract are inconsequential ²⁰. Consequently, it appears that neither BvgA~P nor σ Region 4 have any specific recognition determinants within this portion of the DNA. Thus, it is perhaps not particularly surprising that BvgA~P activation at P_fim3 does not require σ Region 4. However, it is surprising that the presence of Region 4 contributes to maximal P_fim3 activity, that disrupting the typical contacts between Region 4 and core polymerase and the −35 DNA significantly decreases the synthesis of P_fim3 abortive transcripts, and that FeBABE cleavage analysis suggests that Region 4 is located close to the −35 DNA. We suggest then that Region 4 retains its typical position within the BvgA~P activated P_fim3 complex and thus, is positioned to potentially interact with the −35 C-tract DNA. Furthermore, the promoter proximal αCTD at P_fim3 is also positioned to interact with a portion of the C-tract, even though its typical binding site involves A/T rich sequences ¹². A major question then is what is the “glue” that holds these various protein/nonspecific DNA interactions together?

Our results suggest the BvgA~P/RNAP/P_fim3 complex must accommodate BvgA~P, an αCTD, and σ Region 4, all within the −35 region of the promoter (Fig. 8c). To ask whether such a simultaneous engagement is possible, we have generated a speculative molecular model, which combines the structures of T. aquaticus σ Region 4 bound to −35 element DNA ²⁸ (PDB 1KU7) and αCTD bound to UP element DNA ⁷² (PDB 1LB2) with a structure-based model of the BvgA~P DNA binding domain (BvgA residues 148–209) bound to its optimal binding site ¹⁸. (The BvgA~P/DNA interaction is based on the structure of the NarL/DNA complex ⁷⁶ (PDB 1JE8), since BvgA is a member of the NarL family of response regulators.) In the model shown in Fig. 8d, the downstream BvgA~P dimer binds within the major groove along one face of the DNA, from −61 to −26 relative to the transcription start site, while σ Region 4 binds in the major groove on the opposite face of the DNA double helix, positioned between the two BvgA~P monomers. The αCTDs are centered at −55 (promoter distal) and at −32 (promoter proximal), based on the FeBABE analyses of Fig. 7. Such a position is approximately 4 to 5 bp farther downstream than that predicted from the α302-FeBABE analysis of the cII/RNAP/P_RE complex ⁷³. In the generated model at P_fim3, there is little, if any, predicted interaction between BvgA~P and σ Region 4. This model is consistent with our inability to demonstrate an interaction between BvgA or BvgA~P and σ using native gel shifts or bacterial two-hybrid assays (data not shown). A prediction of the model is that αCTD is required for activation, either through an interaction with BvgA~P or σ Region 4. Interactions between the upstream and downstream BvgA dimers may also help stabilize the various protein/nonspecific DNA interactions within the C-tract DNA. Additional experiments are underway to test this model and assemble a detailed map of the transcription activation complex at P_fim3.

Materials and Methods

Bacteria strains and cultures

The strains used in this study are listed in Table 1. Luria-Bertani (LB) agar or broth ⁷⁷ and Bordet Gengou (BG) plate ²⁰ were used for the growth of E. coli strains and B. pertussis strains, respectively. To obtain the modulating conditions repressing the bvgAS locus activities, MgSO₄ (50mM) was added to the BG plates. Unless indicated elsewhere, the antibiotics used in LB agar were ampicillin (200µg/ml), gentamicin sulfate (5µg/ml) and kanamycin sulfate (10 µg/ml). The antibiotics used in BG agar were gentamicin sulfate (5µg/ml) and streptomycin sulfate (100µg/ml).

Table 1

Bacteria strains and plasmids used in this study

DNA

Plasmids in this study are listed in Table 1. Plasmids were constructed using standard cloning procedures. Transcription template plasmids pfha ¹⁹, pP_RE# ²⁰, and pDKT90 (for P_uvsX; ³⁹) have been described. Plasmid pSS3447 is a derivative of pTE103 with the multi-cloning site (MCS) replaced with one of our own design and a 100-bp deletion between the MCS and the terminator. Template plasmids pP_fim3−15C, P_fim3−TA and P_fim3−AC and pP_fim3−14C were constructed by ligating the EcoRI/SalI fragments containing derivatives of the fim3−15C promoter constructed in pSS4159 ²⁰ with plasmid pSS3447 that had been cut with the same restriction enzymes. The P_fhaB, P_fim3, and P_fim3 extended −10 mutant templates were transcribed as supercoiled plasmids; a transcription termination site located downstream of the promoters yields RNAs of 401 (for P_fhaB) and 261 nt (for P_fim3 and P_fim3 mutants). pP_RE# was digested with NaeI to yield a linear template that produces a run-off RNA of 296 nt; pDKT90 was digested with BsaAI to yield a linear template that produces a run-off P_uvsX RNA of 490 nt. P_RNAI was present on each of these pBR-based plasmids and yields a transcript of 108 nt because of an internal transcription terminator ³⁸.

To assess the transcriptional activity of promoters in vivo, we used vector pSS3967 that contains the luxCDABE operon of Photorhabdus luminescens, oriT and 1.8 kb sequence of B. pertussis. Plasmids pSS4159 (P_fim3−13C) and pQC1157 (P_fim3−15C) contain the fim3 promoter region from −130 to +33, relative to the transcription start site. Fragments were cloned into the EcoRI and SalI sites upstream of luxCDABE in pSS3967. The mutagenesis of the extended −10 motif was performed in pQC1157 with the methods described previously ²⁰ to generate plasmids pQC1245 - pQC1250 and pQC1352. E. coli DH5α was used as a host strain for plasmid construction. After sequencing confirmation, the P_fim3 variants in pSS3967 were conjugated via E. coli strain SM10 into B. pertussis strain BP536 as described previously ²⁰.

Wild type σ⁷⁰ and σ^Bp were produced using pLHN12 (described in ⁷⁸) and pETsigmaBp ²⁰, respectively. pETσ^4.1+4.2ala was constructed by digesting both pETσ551–552, 554–555A and pETσ584–588A ⁴⁰ with ClaI and EcoRI. The ClaI restriction site is located between the sequence coding for σ⁷⁰ Regions 4.1 and 4.2. Complementary fragments were then ligated together to yield pETσ^4.1+4.2ala, which produces His₆-tagged σ^{4.1+4.2 ala}. To construct pETσ^580-3A, we used the Quikchange procedure (Stratagene) and primers that introduced codons for alanine at σ⁷⁰ residues 580–583. pETσ^ΔR4 was derived from a variant of pETσ^fl, pETσ^flCF (gift of L. Knipling), in which the XhoI site downstream of the σ⁷⁰ gene (at bp 2087) had been removed. Digestion of pETσ^flCF with XhoI and AvrII removed the fragment encoding the C-terminal end of σ⁷⁰ (residues 528–613). The cut plasmid was then ligated to a XhoI/AvaII linker that recreated the codons for residues 528 and 529 followed by a stop codon. The resulting plasmid produced His₆-tagged σ^ΔR4. pETσ^BpΔR4 was constructed by PCR amplification of the region encoding residues 1–674 from pETsigmaBp (Chen et al., 2010), using primers, which included an NdeI site (upstream) and EcoRI site (downstream). The resulting fragment was ligated to the vector pET28a(+) (Novagen), which had been digested with NdeI and EcoRI. The resulting plasmid expressed His₆-tagged σ^BpΔR4. To construct pETσ^{Hyb 4}, we first digested pBRα-σHyb4 (gift of S. Dove and A. Hochschild) with XhoI and SalI to isolate the fragment encoding σ⁷⁰ residues 528–564 followed in frame with σ³⁸ residues 280–330. This fragment was then ligated into pETσ^flCF that had been previously digested with XhoI/SalI to yield pETσ^{Hyb 4}, which produced His₆-tagged σ^{Hyb 4}. Sequences of the primers are available from the authors upon request. DNA sequencing (Center for Biologics Evaluation and Research core facility, FDA) throughout the cloned regions confirmed the presence of only the desired sequences.

The Cys(−)rpoD plasmid (gift of C. Meares; described in ⁷⁹ encodes a cysteine-less σ, in which the three native cysteine residues at positions 132, 291, and 295 were converted to serines. To create pETσD581C, this plasmid was first digested with BfuAI. The fragment containing the cysteine to serine mutations was ligated to BfuAI-digested pETσ^flCF (described above), resulting in a plasmid encoding His₆-tagged σ-Cys(−). We then used the Quikchange procedure (Stratagene) and primers that introduced a codon for cysteine at residue D581. Plasmid pDL934, containing the galP1 promoter region, was the kind gift of D. Lewis and S. Adhya.

P_fim3−15C, P_fim3−14C, and galP1 DNA, 5’-³²P-end labeled on the template (bottom) or nontemplate (top) strand, were prepared as described ²⁰ using PCR with appropriate primers and the plasmids pP_fim3−15C, pP_fim3−14C, and pDL934.

Proteins

E. coli RNAP core was purchased from Epicentre Technologies. The purification of wild type σ⁷⁰ ⁸⁰, His₆-tagged AsiA ^{81; 82}, MotA ⁶⁴, and BvgA and σ^Bp ²⁰ have been described. The His₆-tagged proteins σ^{Hyb 4}, σ^{4.1+4.2 ala}, σ^580-3ala, and σ C132S/C291S/C295S/D581C (referred to as σD581C) were purified from BL21(DE3)/pLysE ⁸³ cultures containing the desired pETσ plasmid as described ⁸⁴, by denaturation of inclusion bodies containing the protein, Ni²⁺ resin affinity chromatography under denaturing conditions, and then a slow renaturation of the protein. The His₆-tagged σ^ΔR4 and σ^BpΔR4 were purified similarly, except that after sonication of the cells, these proteins were found in the 17,400 × g supernatant rather than in inclusion bodies; consequently, they were subjected to Ni²⁺ resin chromatography under native conditions. Generation of α276C and α302C and isolation of RNAP containing each of these mutants was performed as described ¹⁸.

In vitro transcription reactions

BvgA was phosphorylated by incubation for 30 min at room temperature in a solution containing 20 mM Tris-Cl (pH 8) and 20 mM acetyl phosphate. Transcription reactions (10 µl) containing 0.1 pmol DNA, the indicated amount of BvgA~P, 0.75 pmol reconstituted RNAP (sigma:core of 2.5:1), 60 mM Tris-Cl (pH 8), 25 mM acetyl phosphate, 15 mM NaCl, 10 mM KCl, 4 mM Hepes, 2 mM MgCl₂, 3 mM magnesium acetate, 0.4 mM dithiothreitol, 0.3 mM ethylenediaminetetraacetic acid (EDTA), 25 µg/ml BSA, 20% glycerol (v/v), and 0.002% Triton X-100 were incubated at 37o C for 15 min before the addition of heparin to a final concentration of 200 µg/ml and NTPs to a final concentration of 250 µM ATP, GTP, and CTP and 25 µM [α-³²P]UTP (4.4×10³ dpm/pmol) (Fig. 2, ,3,3, ,4)4) or 250 µM ATP, GTP, and UTP and 50 µM [γ-³²P]GTP (2.5×10⁴ dpm/pmol) (Fig. 6). For transcriptions using AsiA-associated RNAP (Fig. 3), σ was incubated with 6-fold excess of AsiA for 10 min at 37° C before RNAP reconstitution. When using P_uvsX (Fig. S2), reactions were performed as described ⁶⁴. Reactions were stopped by the addition of 25 µl gel loading solution (1X TBE, 7 M urea, 0.1% (w/v) bromophenol blue, 0.1% (w/v) xylene cyanol FF), and the resulting solution was heated at 95° C for two minutes before electrophoresis on 7 M urea, 4% or 23% polyacrylamide denaturing gels run in 0.5X TBE. Gels were exposed to either film or a phosphorimaging screen, which was subsequently scanned using a Fujfilm FLA-5100 phosphorimager. Autoradiograms or phosphorimages were then analyzed on a Powerlook 2100XL densitometer and band intensities quantified using Quantity One software from Bio-Rad, Inc.

Luciferase activity assay

B. pertussis strains integrated with P_fim3 variants were streaked in sectors on BG plates supplied with gentamicin. After incubation for 48 hours at 37° C, luciferase activity was visualized by imaging with an IVIS−50 instrument (Caliper Life Sciences) and quantified using Living Image software v2.6.1 (Caliper Life Sciences) running in Igor Pro Carbon (WaveMetrics), as described previously ²⁰.

Cleavage of P_fim3 DNA with RNAP reconstituted with σ-FeBABE or αCTD-FeBABE

Conjugation of σ C132S/C291S/C295S/D581C (referred to as σD581C) was performed essentially as described ⁸⁵: σD581C (2 nmol) was dialyzed into storage buffer without DTT (50 mM Tris-Cl (pH 7.9), 50 mM NaCl, 0.1 mM EDTA (pH 8.0), 0.01% Triton X-100, and 50% glycerol) for 1 hr at 4°C to remove DTT. FeBABE ((iron (S)-1-(p-bromoacetamidobenzyl)ethylenediaminetetraacetate, 10 nmol, Dojindo Laboratories) was added and the mixture incubated for 1 hr at 37°C, then dialyzed against storage buffer containing 0.1 mM DTT for 1 hr at 4°C to remove free FeBABE. Conjugation efficiency (50%) was determined by mass spec analysis performed by the NIDDK Mass Spec Core Facility (population of protein with mass equal to σD581C-FeBABE divided by total population of σD581C and σD581C-FeBABE protein). Protein activity of the conjugated σD581C-FeBABE was estimated to be 35% based on its activity in in vitro transcription.

FeBABE cleavage reactions with RNAP that was reconstituted with σD581C-FeBABE were performed using a protocol similar to that of Colland et al ⁵⁶. Equimolar σD581C-FeBABE and RNAP core were incubated for 10 min at 37° C to reconstitute holoenzyme. Binding reactions consisted of 1.7 pmol reconstituted holoenzyme, 3.2 pmol BvgA~P, and 0.2 pmol ³²P-labeled fim3 DNA or 0.3 pmol ³²P-labeled galP1 DNA in 150 mM potassium glutamate, 40 mM Tris-acetate (pH 7.9), 34 mM NaCl, 9 mM Tris-Cl (pH 8), 5 mM KCl, 4 mM magnesium acetate, 2 mM acetyl phosphate, 2 mM Hepes-OH (pH 7.4), 1 mM MgCl₂, 0.3 mM DTT, 0.2 mM EDTA, 100 µg/ml BSA, 14% glycerol, 0.0005% TritonX-100. Reactions (10 µl total volume) were incubated for 10 min at 37° C. Heparin (0.5 µg in 0.5 µl) was added 1 min prior to the addition of 2 µl 100 mM sodium ascorbate and 2 µl 0.6% H₂O₂. The cleavage reaction was allowed to proceed for 10 min at 37° C at which point 110 µl quench buffer (72 mM thiourea, 4.5 µg/ml calf thymus DNA, 2.7 mM Tris-Cl (pH 7.9), 0.27 mM EDTA) were added. The end-labeled DNA was also used in G+A sequencing reactions ⁸⁶ to map cleavage coordinates. DNA was phenol extracted and ethanol precipitated, then separated by gel electrophoresis on an 8% acrylamide denaturing gel in 0.5 X TBE. Gels were exposed to film and analyzed as described above.

Cleavage of ³²P end-labeled P_fim3-14C DNA using RNAP with α276C and α302C that had been conjugated with FeBABE was performed essentially as described for P_fhaB ¹⁸.

Molecular modeling

Using MolSoft ICM software, the −33 nucleotide from the structure of T. aquaticus σ Region 4 bound to DNA (PDB 1 KU7) ²⁸ was superimposed with the −33 nucleotide from the model of BvgA~P bound to DNA ¹⁸ so that the major grooves aligned. A monomer of RNAP αCTD was added to the model by superimposing the −35 region minor groove from the structure of the CAP-αCTD-DNA complex (PDB 1LB2) ⁷² with the −35 region minor groove of the aforementioned σ Region 4/BvgA model, yielding a molecular model of the ternary complex of σ Region 4, BvgA~P, αCTD, and −35 region DNA.

Supplementary Material

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Acknowledgements

We are grateful to C. Meares for the Cys(−)rpoD plasmid, to D. Lewis and S. Adhya for pDL934, to S. Dove and A. Hochschild for pBRα-σHyb4, to T. James for the molecular modeling, and to L. Knipling for the construction of pETσ^flCF and pETσ^Hyb4 and purification of σ^Hyb4. We also thank R. Bonocora, L. Knipling, T. James, A. Boulanger-Castaing, S. Jha, and C. Jones for helpful discussions. K. Decker is a graduate student in the Graduate Partnership Program, Johns Hopkins University-National Institutes of Health. This research was supported in part by the Intramural Research Program of the NIH, National Institute of Diabetes and Digestive and Kidney Diseases.

Footnotes

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