Bacterial Strain and maintenance: A non-toxigenic C. botulinum strain LNT01 subtype A1 (Johnson et al., 1997; Lin and Johnson, 1991) will be used in this study. The bacteria will be cultured anaerobically at 37oC and maintained as frozen stocks at -80oC in TPGY broth (50 g/liter trypticase peptone, 5 g/liter Bacto peptone, 4 g/liter D-glucose, 20 g/liter yeast extract, 1 g/liter cysteine - HCl, pH 7.4) containing 20% glycerol.
AIM I: To examine the role of the 5'-untranslated region of phosphoribosylaminoimidazole carboxylase.
Overall Rationale: Riboswitches are typically located at the 5' or 3' ends of the gene they regulate (Mandal et al., 2003; Breaker, 1997). The proximity of the putative purine riboswitch and phosphoribosylaminoimidazole carboxylase (PRAIC) suggests that this riboswitch may be playing a role in either transcription or translation of this gene. In view of the fact that PRAIC catalyzes the only carbon-carbon bond forming reaction in the inosine 5'-monophosphate pathway (IMP), I hypothesize that this putative riboswitch plays an indirect role in regulating the level of purine nucleotides by controlling the production of PRAIC. Establishing the involvement of this putative riboswitch in transcription and expression of PRAIC in the presence and absence of purine nucleotides will help understand how it influences the de novo biosynthesis of purine nucleotides. This section is divided into two sub-aims: sub-aim 1 focuses on the requirement of the 5'-UTR for transcription of PRAIC while sub-aim 2 investigates how the UTR affects expression of PRAIC in the presence and absence of purine nucleotides.
Sub-Aim 1: To investigate whether the 5'-untranslated region is required for the transcription of phosphoribosylaminoimidazole carboxylase gene.
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Rationale: Most riboswitches are typically transcribed together with the gene they regulate (Nechooshtan et al., 2009). This aim is intended to determine whether the 5'-untranslated region is required in order for phosphoribosylaminoimidazole carboxylase to be transcribed. Data from this aim will be important in understanding the nature of the role of this putative riboswitch.
Experimental Design: Genetic manipulations, cloning, and transformation procedures will be performed according to established techniques (Marshall et al., 2010; Cheah et al., 2007; Mandal et al., 2003; Sambrook et al., 1989). DNA polymerases, restriction enzymes, deoxynucleotides, T4 DNA ligase, plasmid isolation kits, and PCR-product purification kits will be obtained from New England BioLabs (Ipswich, MA) and used as specified by the supplier.
To investigate the requirement of UTR in the expression of PRAIC, transcriptional fusion will be used. DNA constructs carrying different sequence sizes from 100 bp upstream of 5'-UTR to 50 bp downstream of the start codon of PRAIC (-292 to + 50 from start codon of PRAIC) will be created as shown in Figure 3. This will be done by designing primers to amplify the regions A-D (as outlined in the Figure 3) of the PRAIC gene using template DNA from C. botulinum strain LNT01. These regions will be PCR amplified as BglII-Sph1 fragments and cloned into the amyE insertional vector pDR66 (Ireton et al., 1993) at a site directly upstream of a promoterless lacZ reporter gene. The integrity of each construct will be confirmed by sequencing. The plasmid constructs will be integrated into the amyE locus of LNT01 strain as single copies as described (Strauch et al., 2007; Ireton et al., 1993). Briefly, the pDR66 vector with the inserted constructs will be transformed into the wild type strains with selection for chloramphenicol resistance (5μg/ml). Disruption of the amyE locus will be determined by the absence of amylase activity by flooding TPGY agar plates containing 1% soluble starch with Lugol's iodine (Arcuri et al., 2000). Amy-positive colonies will be surrounded by a zone of clearance in the medium while Amy-negative colonies will have no zone of clearance.
Single colonies of the mutants will be grown overnight on TPGY broth containing 5 µg/mL chloramphenicol anaerobically at 37oC. The overnight culture will be diluted 1:100 in a fresh media and grown to OD600 of 0.4 or mid-exponential phase with and without different concentrations of adenine or guanine. The cells will be centrifuged and expression of the resulting lacZ transcriptional fusions monitored by β-galactosidase activity assay as described previously (Jaacks et al., 1989; Miller, 1972) using the supernatant.
Expected Results: It is anticipated that β-galactosidase activity will be significantly higher in cells carrying the constructs with an active promoter likely located at the UTR. No β-galactosidase activity is expected to be observed in cells carrying constructs without an active promoter (construct D in Figure 3). Transcription of PRAIC is likely to occur when the 5'-UTR is present. Construct A is expected to produce the highest activity with little to no activity in constructs B and C. This expected increase in transcription of PRAIC is a measure of promoter activity, which is proposed to be located at 5' UTR. Lack of activity from constructs carrying the whole UTR or truncated UTR (constructs A-C) will indicate that the 5'-UTR is most likely not required for transcription of PRAIC.
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Limitations and Alternative Approaches: C. botulinum is very difficult to manipulate genetically and may result in failure in transforming the cells with the constructs. As an alternative, conjugative transfer of the constructs will be done by first transforming conjugative E. coli strain CA434 with the constructs. The E. coli transformants will then be mated with C. botulinum for the constructs to be transferred by conjugation. It is probable that data from β-galactosidase assay may not be consistent to enable reliable comparison of activities between the samples. Messenger RNA transcript analysis by quantitative real time PCR (qRT-PCR) will be used as an alternative. Briefly, total RNA or mRNA will be isolated from wild type LNT01 strain and reverse transcribed to obtain cDNA. This cDNA will be used as template in a PCR with primers with fluorescent-labeled taqman designed to amplify -292 to +50 regions from PRAIC start codon. Amplification of a long PCR product with a size spanning the UTR and PRAIC will indicate that their transcription is linked. This will be confirmed by cloning and sequencing. Primers specific for the triose phosphate isomerase gene will be used to amplify this gene as a control and normalization.
Figure 3: UTR-PRAIC constructs from -292 to +50 regions from PRAIC start codon that will be used for LacZ transcriptional fusions. R= 5' end of the putative riboswitch, ATG = start codon of the PRAIC gene. A-D= Constructs that will be created.
Sub-Aim 2: To examine the effect of the 5'-UTR on expression of phosphoribosylaminoimidazole carboxylase under different purine nucleotides conditions.
Rationale: The presence of cognate metabolites of riboswitches repress the genes involved in their synthesis (Cheah et al., 2007; André et al., 2008; Corbino et al., 2005). Availability of a metabolite at sufficiently high concentrations induces a structural alteration in the 5'-untranslated region that influences the expression of the target gene. This sub-aim seeks to evaluate how the 5'-UTR affects the expression of PRAIC in the presence and absence of purine nucleotides.
Experimental Design: A translational fusion with lacZ reporter gene inserted in frame within the PRAIC gene will be used. A pDR66-based plasmid construct will be created carrying the entire region of the 5'-UTR and PRAIC with lacZ gene inserted in frame within the PRAIC gene at +140 from the start codon at the BglII restriction site. A second construct without the 5' UTR will also be created. These constructs will be integrated into the amyE locus of C. botulinum LNT01 wild type strain, selected, and confirmed as described in sub-aim 1.
Single colonies of the translational fusion mutants will be grown overnight on TPGY broth containing 5 µg/mL chloramphenicol anaerobically at 37oC. The overnight culture will be diluted 1:100 in a fresh media and incubated with and without adenine or guanine at different concentrations. Samples will be taken at different time intervals for analysis. The cells will be centrifuged and β-galactosidase activity performed using the supernatant.
To further evaluate the relative amounts of β-galactosidase and PRAIC hybrid protein expressed, Western blot analysis will also be performed. This technique will be performed according to established procedure (Burnette, 1981). Briefly, the culture will be sonicated and total proteins separated by sodium dodecyl sulfate electrophoresis and transferred unto a nitrocellulose membrane. The membrane containing the transferred proteins will be incubated with antibodies specific for PRAIC, β-galactosidase, and triose phosphate isomerase (for normalization or control). SuperSignal West Dura Chemiluminescent Substrate (Thermo Scientific, Waltham, MA) will be used to probe the membrane for the presence of the proteins following blocking and washing. The membrane will be incubated with HRP-conjugated secondary antibody and following washing, incubated with the chemiluminescent substrate prepared according to the manufacturer's instructions. Imaging and analysis of the blotted membrane will be done using ChemiImager 4000 (Alpha Innotech Cell Biosciences, Santa Clara, CA). Comparison of the relative amounts of each protein will be made to evaluate how the 5'-UTR affects the expression of PRAIC under different purine nucleotides conditions.
Expected Results: In the presence of purine nucleotides, the β-galactosidase activity is expected to be reduced significantly in mutants that carry the 5'-UTR. This will probably be a consequence of allosteric control of PRAIC by the putative riboswitch to regulate the cellular levels of purine nucleotides. The reduction in activity is expected to decrease further with an increasing amount of purines. Conversely, β-galactosidase expression will increase in the absence of purine nucleotides in mutants with the 5'-UTR. Translational mutants without the 5'-UTR are anticipated to be un-responsive (in terms of activity) to the presence or absence of purine nucleotides. Accordingly, the Western blot data is expected to show an increase in intensity of the PRAIC and β-galactosidase protein bands in UTR-containing translational fusion mutants incubated without purine nucleotides compared to purine-treated cells.
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Limitations and Alternative Approaches: It is probable that the adenine or guanine nucleotides may not have any effect on the expression of PRAIC. Other closely related purine nucleotides including xanthine, hypoxanthine, 2-aminopurine, 3-methylxanthine, 2-amino-6-bromopurine, and pterin will be evaluated as an alternative. Quantitative real-time PCR will also be used to determine the level of PRAIC mRNA produced in the presence and absence of the purine nucleotides as an alternative approach, in case of inconsistent β-galactosidase activity.
AIM II: To investigate whether purine nucleotides selectively bind to the 5'-UTR of phosphoribosylaminoimidazole carboxylase and to evaluate if this metabolite binding alters its secondary structural characteristics.
Rationale: For cellular metabolic homeostasis to be achieved, a bacterium must be able to sense the amount of its target metabolite and also stop any potential regulatory crosstalk with other molecules that might activate genetic modulation. Certainly, a hallmark of riboswitches is their capability to discriminate between closely related metabolites (Barrick and Breaker, 2007; Mandal et al; 2003). An important feature of riboswitch function is communication of metabolite binding to an expression platform, which holds the regulatory effect (Grundy and Henkin, 1998; Miranda-Rios et al., 2001; Nahvi et al., 2002). As a result, metabolite binding is an important event that initiates processes that underlie the regulatory function of riboswitches. In the absence of a cognate metabolite, riboswitches generally fold into a structure that does not interfere with the expression of their target gene. I hypothesize that specific binding of purine nucleotides to the 5'-untranslated region of phosphoribosylaminoimidazole carboxylase alters its structure, which may repress the expression of this enzyme during purine nucleotide-rich conditions. The rationale for this aim is to demonstrate that binding occurs between purine nucleotides (either adenine or guanine) and the 5'-UTR of PRAIC and to probe the potential structural modulations that may occur due to this binding. This will ultimately lead to identification of the specific metabolite for this putative riboswitch as well as mapping of the aptamer domain and expression platform.
Experimental Design: To establish binding of the purine nucleotides to the 5'-UTR of PRAIC, in-line probing assay (Regulski and Breaker, 2008; Nahvi et al., 2002; Mandal et al., 2003; Soukup and Breaker, 1999b) will be used. This assay, also a tool to study RNA secondary structure characteristics, is based on the natural instability and structure dependent spontaneous cleavage of RNA under normal conditions. Regions of RNA that are single stranded are more flexible and degrade over time. However, binding of a ligand stabilizes and modulates the structure resulting in cleavage patterns that can be interpreted to assess ligand binding and structural characteristics.
A double-stranded DNA template corresponding to the entire 5'-UTR of PRAIC will be generated by PCR with primers that will introduce T7 RNA polymerase promoter sequence to enable subsequent transcription. A truncated form of the construct that encompasses 5'-half of the UTR will also be amplified. To enable identification of the aptamer domain and the expression platform, mutations in the UTR will be generated by site directed mutagenesis by PCR method (Higuchi et al., 1988). Briefly, this will be done using a plasmid carrying the UTR-PRAIC region as template DNA and two tail-to-tail divergent primers of which one will carry the desired mutation. Mutations will be made in the stems (P1-P3 in Figure 2), loops, and the loop junction. The amplified DNA will be in vitro transcribed using T7 RNA polymerase to generate the RNA of the UTR and subsequently, labeled with 32P at the 5' end. The in vitro transcription will be performed using MAXIscript In Vitro Transcription Kit (Ambion, Austin, TX) according to the manufacturer's protocol. Briefly, the DNA will be incubated for 60 minutes at 37oC with nucleotide triphosphates (ATP, CTP, GTP, UTP, and [α-32P] UTP), and T7 RNA polymerase in an appropriate buffer. Following the incubation period, the transcripts will be purified by gel filtration using Ambion NucAway spin columns (Ambion). The purified RNA will be incubated for different time periods at 25oC in a buffer containing 50 mM Tris (pH 8.5), 20 mM MgCl2, and 100 mM KCl with and without 1 nM to 300 µM purine nucleotides (adenine or guanine). As a control, the RNA constructs will also be incubated with RNase T1 (which cleaves specifically at G residues) and under alkaline conditions. Denaturing polyacrylamide gel (10%) will be used to separate the spontaneous RNA cleavage products and analyzed using a Typhoon PhosphorImager (GE Healthcare, Piscataway, NJ).
Equilibrium dialysis assay will be used to further demonstrate and characterize the type of purine this riboswitch preferentially bind. This will be performed according to established protocol (Cheah et al., 2007; Mandal et al., 2003). Briefly, the purine nucleotides (adenine and guanine) will be labeled with tritium. A substantial shift in tritiated purine is expected to occur in a two-chamber dialysis apparatus when an excess of functional RNA is added to one chamber. This shifted equilibrium should return to unity upon addition of an excess of unlabeled competitor ligand. This assay will be conducted using a 5-Cell Equilibrium Dialyzer (Spectrum Laboratories, Rancho Dominguez, CA), in which chambers A and B are separated by a 5000 MWCO membrane. The composition of buffer will be 50 mM Tris-HCl (pH 8.5), 20 mM MgCl2, and 100 mM KCl. For the experiment, chamber A will contain 3H-adenine or 3H-guanine while the in vitro transcribed 5' UTR RNAs will be placed in chamber B. The samples will be equilibrated for 10 hrs at 25oC and monitored by liquid scintillation counter. The apparent dissociation constant (KD) will also be determined to evaluate metabolite binding specificity. This will be done by plotting the normalized fraction of RNA cleaved against the logarithm of the concentration of metabolite used.
Expected Results: The spontaneous cleavage patterns of the RNA are expected to undergo a significant alteration in the presence of purine nucleotides. Based on analysis of the cleavage patterns and mutagenesis studies, regions on the UTR that become highly structured in the presence of purines will be mapped to establish the aptamer and expression platform domains. Mutation of residues important for metabolite binding such as residues C-74 or U-74 in the aptamer domain is expected to abolish such binding. The specific purine (adenine or guanine) that is recognized by this riboswitch will significantly alter the cleavage pattern of the RNA. Increasing the concentration of the cognate metabolite (adenine or guanine) will progressively decrease the extent of spontaneous cleavage. This will be due to the binding of the metabolite to the RNA thus, stabilizing it from spontaneous cleavage. A shift in 3H-adenine or 3H-guanine from chambers A to B is expected when the UTR RNA is added to chamber B. However, this shift is anticipated to return to equilibrium if sufficient amount of unlabeled adenine or guanine is added to chamber B to compete for UTR binding.
Limitations and Alternative Approaches: It is possible that the rate of spontaneous RNA cleavage may vary between sample treatments. This will alter the observed cleavage patterns as some samples may undergo incomplete cleavage. As an alternative approach, RNA footprinting structure probing with dimethyl sulfate (DMS) will be used (Tijerina et al., 2007). DMS methylates adenosine at N1 and cytosine at N3 and this prevents hydrogen bonding between the bases modified by DMS. The RNA will be incubated with DMS under controlled conditions that do not allow formation of the folded structure and also at conditions that allow folding in the presence or absence of purine nucleotides. Following incubation, samples will be analyzed by primer extension, polyacrylamide gel electrophoresis, and quantitative analysis of the cleavage patterns.
AIM III: To investigate the effect of the 5'-UTR of Phosphoribosylaminoimidazole carboxylase on the production of the inosine 5'-monophosphate biosynthetic pathway intermediate proteins and their substrates in C. botulinum.
Rationale: Phosphoribosylaminoimidazole carboxylase (PRAIC) plays a significant role in de novo biosynthesis of inosine 5'-monophosphate (IMP), where it catalyzes the only carbon-carbon bond formation reaction in the pathway. I hypothesize that the 5'-untranslated region of PRAIC may indirectly affect the production of IMP biosynthetic pathway proteins or their intermediate substrates during conditions of abundant adenine or guanine. Establishing the potential effect of the UTR on production of the proteins involved in the pathway and their enzymatic products will give an indication of a possible feed-back effect. This aim is intended to evaluate the extent to which the 5'-UTR of PRAIC influences the overall IMP pathway under different purine nucleotide conditions.
Experimental Design: The chromosomal 5'-UTR will be inactivated to investigate its effect on production of IMP pathway intermediate proteins and their substrates. The TargeTron Gene Knockout System kit from Sigma-Aldrich (St. Louis, Mo) will be used to insert a 1.5-2 kb DNA fragment specifically in the middle of the 5'-UTR of PRAIC to inactivate it. This technique uses retargeted group II introns to insert efficiently into a target gene (Chen et al., 2005; Perutka et al., 2004; Frazier et al., 2003; Karberg et al., 2001). Group II introns insert themselves via the activity of an RNA-protein complex expressed from a single plasmid, pJir750ai (Chen et al., 2005; Zhong et al., 2003). Using four unique primers that will be designed based on the UTR sequence, a PCR reaction will be performed to re-target the intron by primer-mediated mutation at several positions spanning a 350 bp region. This 350 bp PCR product will be double-digested along with the shuttle plasmid pJir750ai using HindIII and BsrGI. The insert and plasmid will be ligated and propagate in E. coli DH5α cells. After confirmation, C. botulinum wild type strain LNT01 will be transformed with the shuttle plasmid carrying the re-targeted intron specific for the UTR. The knocked out gene will be confirmed by colony PCR and sequencing using gene specific primers that flank the insertion sites or a gene and intron specific primers to amplify across gene-intron junctions.
Single colonies of the mutants and wild type (as control) will be grown overnight anaerobically in a TPGY broth at 37oC. The overnight cultures will be diluted 1:100 in a fresh media and incubated for different time periods with and without adenine or guanine nucleotides at different concentrations. To evaluate the relative transcript amounts of the proteins, mRNA (for both mutants and wild type) will be isolated and reverse transcribed to obtain cDNA. Primers with fluorescent-labeled taqman designed to amplify the genes for phosphoribosylaminoimidazole carboxylase, adenylosuccinate lyase, IMP cyclohydrolase, and triose phosphate isomerase (as control and normalization) with cDNA as template. The amount of the corresponding intermediate products produced, 4-carboxyaminoimidazole ribonucleotide (CAIR), aminoimidazole carboxamide ribonucleotide (ACR), and IMP respectively, due to the enzymatic activity of the proteins will be determined. These will be done according to established procedure. For CAIR, the absorbance of the culture supernatant will be measured at 260nm with an extinction coefficient of 10500 M-1 cm-1 (Meyer et al., 1992; Casey and Lowenstein, 1987; Casey et al., 1986). To determine the amount of ACR, cleavage of 5'-phosphoribosyl-4-(N-succinocarboxamide)-5-aminoimidazole by adenylosuccinate lyase to produce ACR will be assayed by a decrease in absorbance at 282 nm minus 320 nm using a difference in the extinction coefficient of 10 mM-1 cm-1 (Schultz and Lowenstein, 1976). The amount of IMP will be determined by monitoring the enzymatic hydrolysis of phosphoribosyl-formamido-carboxamide (PFC) by IMP cyclohydrolase spectrophotometrically at 250 nm using a difference in the extinction coefficients of PFC and IMP of 5710 M-1 cm-1 (Mueller and Benkovic, 1981; Rayl et al., 1996; Ikegami et al., 1985).
To confirm the effect of the 5'-UTR inactivation on the IMP pathway, the mutants will also be complemented with a plasmid carrying an active 5'-UTR of PRAIC. The complemented mutants will be treated under the same conditions as the 5'-UTR mutants and amounts of the proteins with their intermediate products analyzed for a potential rescue.
Expected Results: The amounts of mRNA transcripts of the two IMP pathway proteins (adenylosuccinate lyase and IMP cyclohydrolase) are expected to be unaffected in the UTR mutants regardless of purine nucleotide conditions. However, the transcript level of PRAIC is expected to be reduced or even transcription lost entirely in the UTR mutants in the presence or absence of purine nucleotides. PRAIC mRNA and its intermediate product (4-carboxyaminoimidazole ribonucleotide) amounts in the wild type are expected to be high in the absence of purine nucleotides and low when sufficient purine is present. Little or no 4-carboxyaminoimidazole ribonucleotide is expected in the UTR mutant and this would likely affect the subsequent reactions in the pathway leading to cessation of production of ACR and IMP.
Potential problems/Alternative Approaches: C. botulinum has proven difficult for genetic manipulation due to lack of appropriate technology. As an alternative, conjugative transfer of the constructs will be done by first transforming conjugative E. coli strain CA434 with the constructs. The E. coli transformants will then be mated with C. botulinum for the constructs to be transferred by conjugation. The quantitative RT-PCR proposed may not work properly due to degradation of the RNA or incomplete cDNA synthesis that may produce truncated copies of the genes. Northern blot hybridization (for the RNA) and Western blot analysis (for proteins) will be used as an alternative approach. The IMP pathway is the major route for de novo purine biosynthesis but there is also a purine salvage pathway that recycles purines. It is likely that there may not be sufficient purines from the purine salvage pathway required by the mutants to survive. Hence, the mutants may not survive due to inactivation of the inosine monophosphate that supplies essential purine nucleotides. Adequate purines will be exogenously added to the medium should this situation occur.
Until recently, proteins have been known to be solely responsible for most of the regulatory and structural requirements of life. RNAs have now also been shown to possess the capability to perform such functions. Riboswitches are complex, metabolite-binding RNA elements found at the non-coding regions of mRNA. Allosteric modulations in the riboswitch induced as a result of metabolite binding are utilized to regulate a variety of essential metabolic pathways genes in archaea, prokaryotes, and eukaryotes. Such regulatory functions of riboswitches are attractive targets for antimicrobial agents to inhibit essential cellular processes. This project proposes to examine the role of a predicted purine riboswitch located at the 5' end the of phosphoribosylaminoimidazole carboxylase gene. The aptamer region, expression platform, and the cognate purine nucleotide specific for this riboswitch will be identified. Structural modulations that occur after metabolite binding and the effect of this riboswitch on the production of IMP will also be investigated. The significance of this study is that it will provide data, which will help in understanding the role of regulatory RNAs in the regulation of purine nucleotides in C. botulinum. This knowledge may be critical for the development of new cellular antimicrobial therapeutic agents to control C. botulinum. This study is unique because it will be the first to characterize a riboswitch in a serious human pathogen.