mRNA Interferase Bacillus cereus BC0266 Shows MazF-Like Characteristics Through Structural and Functional Study

Toxin–antitoxin (TA) systems are prevalent in bacteria and are known to regulate cellular growth in response to stress. As various functions related to TA systems have been revealed, the importance of TA systems are rapidly emerging. Here, we present the crystal structure of putative mRNA interferase BC0266 and report it as a type II toxin MazF. The MazF toxin is a ribonuclease activated upon and during stressful conditions, in which it cleaves mRNA in a sequence-specific, ribosome-independent manner. Its prolonged activity causes toxic consequences to the bacteria which, in turn, may lead to bacterial death. In this study, we conducted structural and functional investigations of Bacillus cereus MazF and present the first toxin structure in the TA system of B. cereus. Specifically, B. cereus MazF adopts a PemK-like fold and also has an RNA substrate-recognizing loop, which is clearly observed in the high-resolution structure. Key residues of B. cereus MazF involved in the catalytic activity are also proposed, and in vitro assay together with mutational studies affirm the ribonucleic activity and the active sites essential for its cellular toxicity.


Introduction
Toxin-antitoxin (TA) systems are prevalent in prokaryotes and function to regulate cellular growth in response to stress such as antibiotic exposure, nutrient starvation, heat shock, and DNA damage. Initially, TA systems were discovered as a part of the plasmid maintenance system, in which only the daughter cells harboring the vertically transferred TA operon can survive [1][2][3][4][5]. In addition to the primary maintenance function, TA systems are important for bacterial survival. The TA systems are involved in many cellular processes, including cell growth, cell persistence, cell dormancy, biofilm formation, antibiotic resistance, DNA replication, translation, cell division, cell wall synthesis, and cell apoptosis [6][7][8][9][10][11].
TA systems are a two-component system, composed of a toxin and an antitoxin usually sharing the same operon. Currently, TA systems can be categorized into six phenotypes (I-VI) by the nature of each component and the regulatory mechanisms of the antitoxins to its cognate toxins. In the case of RNA antitoxins, direct inhibition of mRNA encoding toxins and protein toxins constitute the type I and type III systems, respectively. Similarly, protein toxins are directly inhibited by their counterpart protein antitoxins in type II TA system. As for indirect regulatory mechanisms, protein antitoxins counteract the activity of protein toxins in type IV TA system and cleave mRNA encoding protein toxins in type V TA system. Lastly, in type VI TA system, protein toxins are degraded by specific proteases and lose their toxicity as a result of complex formation with protein antitoxins [12][13][14][15][16]. Typically, in type II TA system, the rather stable toxins have much more invariant characteristics than their labile and flexible cognate antitoxins. Upon degradation of antitoxins, free toxins can function in postsegregational cell killing, abortive infection, and even bacterial persistence. Thus, it has been regarded that bacterial persistence would indeed be closely related to type II TA systems due to the cellular process-specific nature of type II toxins [17][18][19][20][21]. One of the best characterized type II toxins is mRNA interferase MazF toxin, belonging to the MazEF family. Comprehensive bioinformatic analyses have shown that MazF is widely distributed in both Gram-positive and Gram-negative bacteria, allowing an in-depth understanding of the consequences of stress and MazF activation on the physiological effects of the bacteria [6,22,23]. In addition, understanding of the cutting specificity of MazF on its target RNA is well established. Specifically, the MazF toxin acts as a ribonuclease and cleaves mRNA in a sequence-specific and ribosome-independent manner, but in some cases also cleaves rRNA at specific sequences, exerting its toxicity [17,24,25].
Bacillus cereus is a Gram-positive, facultatively anaerobic, and rod-shaped bacterium that is well known for its association with foodborne illness [26]. Recently, the pathogenicity potential of the bacteria gained attention due to its ability in causing serious and fatal nongastrointestinal infections [27]. As TA systems are involved in several cellular responses such as stress response, bacterial persistence, and biofilm formation, there is a clear linkage between TA systems and the virulence of the bacteria [28]. For this reason, research was conducted on the putative MazF toxin, mRNA interferase BC0266.
Here, we report 2.0 Å X-ray crystal structure of mRNA interferase MazF as the first toxin structure in the TA system of B. cereus. Similar to that of other MazF toxins, B. cereus MazF adopts a PemK-like fold and also has an elongated loop between the β1 and β2 strands. Further sequence alignment with several other MazFs supported the key residues in ribonucleic catalysis. Finally, in vitro ribonuclease activity test together with site-directed mutational studies demonstrated that B. cereus MazF exhibits a clear ribonuclease activity and its key residues play a significant role in exerting its role as a ribonuclease.
In conclusion, we show that mRNA interferase BC0266 belongs to the MazEF family and is the first reported MazF toxin structure in the TA system of B. cereus. This is an important study to investigate the diversity of the TA system among many different bacterial strains.

B. cereus MazF Adopts a PemK-Like Fold
Crystals of B. cereus MazF were diffracted to high resolution (2.0 Å) and the final structure was determined using the molecular replacement method with the MazF structure from Mycobacterium tuberculosis (PDB code 5XE2) [29]. The structure was refined to R work factor 18.9% and R free factor 22.5%. All 116 amino acids were present and lie within the Ramachandran favored region. Crystals had P3 1 21 space group with the unit cell dimensions of a = b = 60.648 Å, c = 76.247 Å, and α = β = 90.0 • , γ = 120 • . Detailed crystallographic data collection and refinement statistics are summarized in Table 1.
There are three α-helices and seven β-strands with the order of β-barrel arrangement in a monomer of B. cereus MazF ( Figure 1A). Among those β-strands, five β-strands (β1, β2, β3, β6, β7) and two β-strands (β4, β5) form a β-sheet antiparallel to each other. Two loops between the β1 and β2 strands, and the β3 and β4 strands are also clearly observed. The total solvent-accessible surface area of the monomeric structure is 6740 Å 2 , and the contact area between two monomers is 1440 Å 2 (21.4% per monomeric subunit). The crystal structure shows that the dimeric interface displays a concave structure covered by the neighboring loop ( Figure 1B). Calculations of surface and interface areas were conducted using the PISA server [30]. The oligomeric state of B. cereus MazF was predicted Toxins 2020, 12, 380 3 of 12 through size-exclusion chromatography with reference proteins in the Gel Filtration Calibration Kits (GE Healthcare, Chicago, IL, USA) ( Figure 1C). Because theoretical molecular weight of the B. cereus MazF monomer containing N-terminal His-tag is 15.1 kDa, it is reasonable that oligomeric state of B. cereus MazF is homodimer (30.2 kDa) as it was eluted at almost the same time as that of carbonic anhydrase (29 kDa).

Comparison of B. cereus MazF with MazF Homologs
To relate the structural characteristics of B. cereus MazF with mRNA interferase MazF, comparative analysis was performed with previously reported MazF structures. Illustrations of sequence alignment were displayed with Staphylococcus aureus MazF (PDB code 4MZM) [31], M. tuberculosis MazF3 (PDB code 5UCT), [32]; MazF4 (PDB code 5XE2) [29]; and MazF7 (PDB code 5WYG) [33] (Figure 2A). The results showed that two key residues of Arg25 and Thr48 involved in catalytic activity are well conserved among multiple MazFs. Interestingly, Lys19 is conserved in M. tuberculosis MazF4 instead of arginine [29], and Ser51 is conserved in M. tuberculosis MazF7 instead of threonine [33]. Yet, mutations of these two residues significantly impaired the activities of corresponding toxins, implying their role in ribonucleic catalysis [29,33]. Although considerable structural similarities are shared between the compared MazFs, there are notable differences in flexibilities and structural variations in their β1-β2 and β3-β4 loops ( Figure 2B). These loops are reported as a major binding site with its cognate antitoxin MazE and, therefore, it is supposed that the flexibilities of these loops have relations to interactions with its target RNA substrate depending on MazE binding [32,34,35].

Comparison of B. cereus MazF with MazF Homologs
To relate the structural characteristics of B. cereus MazF with mRNA interferase MazF, comparative analysis was performed with previously reported MazF structures. Illustrations of sequence alignment were displayed with Staphylococcus aureus MazF (PDB code 4MZM) [31], M. tuberculosis MazF3 (PDB code 5UCT), [32]; MazF4 (PDB code 5XE2) [29]; and MazF7 (PDB code 5WYG) [33] (Figure 2A). The results showed that two key residues of Arg25 and Thr48 involved in catalytic activity are well conserved among multiple MazFs. Interestingly, Lys19 is conserved in M. tuberculosis MazF4 instead of arginine [29], and Ser51 is conserved in M. tuberculosis MazF7 instead of threonine [33]. Yet, mutations of these two residues significantly impaired the activities of corresponding toxins, implying their role in ribonucleic catalysis [29,33]. Although considerable structural similarities are shared between the compared MazFs, there are notable differences in flexibilities and structural variations in their β1-β2 and β3-β4 loops ( Figure 2B). These loops are reported as a major binding site with its cognate antitoxin MazE and, therefore, it is supposed that the flexibilities of these loops have relations to interactions with its target RNA substrate depending on MazE binding [32,34,35]. Through early studies on E. coli MazEF, it was suggested that the β1-β2 loop in the MazF toxin undergoes conformational transition from disordered to ordered state upon binding to the MazE antitoxin [36]. During this transition, it is assumed that these loops are modulated by MazE, affecting the target RNA accessibility of MazF [36]. For instance, MazF4 and MazF7 toxins from M. tuberculosis with truncated β1-β2 loops had significant steric clashes within the β1-β2 loop region due to the high flexibility arising from the structurally unbound MazE antitoxin [29,33]. As for MazF3 toxin in M. tuberculosis, the length of the short β1-β2 loop may be insufficient to reach and recognize the downstream of the target RNA substrate [31,32]. Furthermore, it is predicted that absence of an elongated β1-β2 loop would cause even greater conformational transition of the β1-β2 loop, leaving the toxin structurally and functionally impractical. Therefore, it is suggested that target sequence specificity of truncated or short β1-β2 loops would be significantly reduced compared with MazFs Through early studies on E. coli MazEF, it was suggested that the β1-β2 loop in the MazF toxin undergoes conformational transition from disordered to ordered state upon binding to the MazE antitoxin [36]. During this transition, it is assumed that these loops are modulated by MazE, affecting the target RNA accessibility of MazF [36]. For instance, MazF4 and MazF7 toxins from M. tuberculosis with truncated β1-β2 loops had significant steric clashes within the β1-β2 loop region due to the high flexibility arising from the structurally unbound MazE antitoxin [29,33]. As for MazF3 toxin in M. tuberculosis, the length of the short β1-β2 loop may be insufficient to reach and recognize the downstream of the target RNA substrate [31,32]. Furthermore, it is predicted that absence of an elongated β1-β2 loop would cause even greater conformational transition of the β1-β2 loop, leaving the toxin structurally and functionally impractical. Therefore, it is suggested that target sequence specificity of truncated or short β1-β2 loops would be significantly reduced compared with MazFs having a long β1-β2 loop, such as that of B. cereus MazF. Therefore, it can be presumed that the long length of β1-β2 loop may be the most powerful factor that determines the target RNA specificity.
In contrast to the β1-β2 loop, β3-β4 loop shows an opposite conformational transition from ordered to disordered state upon binding to MazE antitoxin [29]. Furthermore, previous research has shown that aromatic residues located on the antitoxin MazEs are involved in essential MazEF  [29,34,35].
Thus, considering the E. coli and M. tuberculosis model proteins, the B. cereus MazE antitoxin, BC0265, likely contains C-terminal aromatic residues that might interact with the β3-β4 loop in B. cereus MazF. Because of high structural unpredictability and lack of structural information of B. cereus MazE and other MazE antitoxins in general, an accurate prediction of which aromatic residue will interact with its cognate toxin is difficult. However, assuming the model is correct, one of the aromatic residues in B. cereus MazE, Tyr63, Phe78, Tyr82 or His86, likely engages in a hydrophobic interaction during complex formation.

Arg25 and Thr48 Act as Key Residues in Catalytic Activity
To assure that two key residues Arg25 and Thr48 are critical in catalytic activity, comparative analysis with B. subtilis MazF complexed with RNA (PDB code 4MDX) [34] ( Figure 3A) and E. coli MazF complexed with DNA substrate analog (PDB code 5CR2) [35] (Figure 3B) was performed. Subsequently, in silico model of B. cereus MazF complexed with RNA and DNA was generated by superimposition of B. cereus MazF structure onto the 4MDX (left) and 5CR2 (right), respectively ( Figure 3C).

B. cereus MazF Shows Ribonuclease Activity by Two Key Residues
MazF is a ribonuclease that is known to cleave RNA substrate [41]. Toxins such as VapC in the Type II TA system require metal ions as a cofactor to exhibit ribonuclease activity, but in the case of MazF, cofactor metal is not necessarily needed [42,43]. To demonstrate that B. cereus MazF shows ribonuclease activity like other MazF toxins, in vitro ribonuclease activity test was performed ( Figure  4A). In this assay, a synthetic RNA strand of unknown sequence is used as substrate. A fluorophore is covalently attached to one end of the RNA strand while the quencher is located at the other end. If this RNA is cleaved by a ribonuclease, the quencher is detached and as a result fluorescence is detected depending on the amount of cleaved RNA. The result from in vitro ribonuclease assay showed an elevation of RFU (resulting fluorescence unit) as a function of time. B. cereus MazF exhibits In the in silico model of B. cereus MazF in complex with RNA and DNA substrate, Arg25 and Thr48 residues were well organized with RNA and DNA substrate ( Figure 3C). Moreover, mutational study on B. subtilis MazF Arg25 and Thr48 resulted in complete inactivation of toxicity of MazF [34]. From these results, it can be assumed that Arg25 and Thr48 from B. cereus would also be involved in RNA catalysis. In addition, according to earlier work performed on MazF toxins, Arg25 is predicted to Toxins 2020, 12, 380 7 of 12 act as a general acid/base during RNA catalysis and Thr48 may serve as a charge stabilizer in transition state during RNA substrate binding [35,37,38].
Interestingly, charge distributions observed in the front surfaces of three proteins ( Figure 3A-C) are similar but slightly different from each other, mainly due to the differences in the charged amino acid content. In E. coli MazF, negative-charged front surface is mostly formed by Asp16, Asp18, and Glu24 in the β1-β2 loop (residues 16 to 27). However, corresponding residues are substituted with different amino acids in both B. subtilis and B. cereus MazF. Because these front surfaces are known as the binding region of cognate antitoxin MazE, each MazE is thought to have a surface potential specific to its cognate MazF toxin to effectively bind and neutralize toxin surfaces. This seems to be the one of strong reasons why a specific toxin is neutralized only by its cognate antitoxin, although considerable structural similarities are observed within the same family of TA system [29,39,40].

B. cereus MazF Shows Ribonuclease Activity by Two Key Residues
MazF is a ribonuclease that is known to cleave RNA substrate [41]. Toxins such as VapC in the Type II TA system require metal ions as a cofactor to exhibit ribonuclease activity, but in the case of MazF, cofactor metal is not necessarily needed [42,43]. To demonstrate that B. cereus MazF shows ribonuclease activity like other MazF toxins, in vitro ribonuclease activity test was performed ( Figure 4A). In this assay, a synthetic RNA strand of unknown sequence is used as substrate. A fluorophore is covalently attached to one end of the RNA strand while the quencher is located at the other end. If this RNA is cleaved by a ribonuclease, the quencher is detached and as a result fluorescence is detected depending on the amount of cleaved RNA. The result from in vitro ribonuclease assay showed an elevation of RFU (resulting fluorescence unit) as a function of time. B. cereus MazF exhibits a ribonuclease activity showing time and dose dependency, and hence shares the same functional activity with MazF toxins.

Conclusions
We conducted an in-depth characterization and investigation on putative mRNA interferase MazF in B. cereus. High-resolution (2.0 Å) structure was obtained by X-ray crystallography, and overall structural folding of B. cereus MazF indicated β-barrel arrangement containing two independent β-sheets identical to that of MazF toxins. Through comparison analysis with previously reported structures of MazFs, amino acid residues Arg25 and Thr48 in B. cereus MazF were well conserved with other MazFs and were also confirmed as key residues in the catalytic activity of B. Furthermore, two single and one double site-directed mutagenesis on the two key catalytic residues of Arg25 and Thr48 were performed ( Figure 4B). The results of the mutational studies showed that single mutations of either Arg25 or Thr48 resulted in severe reductions in the ribonuclease activity Toxins 2020, 12, 380 8 of 12 of toxin while double mutations of both Arg25 and Thr48 resulted in close to none ribonuclease activity. Therefore, in agreement with the results of B. subtilis MazF, Arg25 and Thr48 are key residues in B. cereus MazF, which is a characteristic observed consistently in MazF toxins.

Conclusions
We conducted an in-depth characterization and investigation on putative mRNA interferase MazF in B. cereus. High-resolution (2.0 Å) structure was obtained by X-ray crystallography, and overall structural folding of B. cereus MazF indicated β-barrel arrangement containing two independent β-sheets identical to that of MazF toxins. Through comparison analysis with previously reported structures of MazFs, amino acid residues Arg25 and Thr48 in B. cereus MazF were well conserved with other MazFs and were also confirmed as key residues in the catalytic activity of B. cereus MazF. Also, β1-β2 and β3-β4 loops were clearly observed in B. cereus MazF structure and their role in the recognition of target RNA substrate depending on the binding of its cognate antitoxin was discussed. From previously conducted studies, it can be presumed that the β1-β2 loop acts as a gateway to binding of its target RNA substrate and cognate antitoxin and the β3-β4 loop also acts as a major interaction site in antitoxin-binding via van der Walls interactions. Lastly, ribonuclease activity test via in vitro assay together with site-directed mutational studies on Arg25 and Thr48 showed a decrease in its ribonuclease activity, which was consistent in other MazF toxins. Altogether, our study provides a unified structural and functional basis that mRNA interferase BC0266 is indeed a MazF toxin. This is the first determined MazF and toxin structure in type II TA system of B. cereus. Our work may be a rational basis for understanding the TA systems regarding the stress-responsive toxin MazF and the general regulatory mechanisms of TA systems.

Cloning and Transformation
The BC0266 gene was amplified by polymerase chain reaction (PCR). The following primers were used in PCR: forward, 5 -GGAATTCCATATGATGATTGTAAAACGCGGC-3 ; reverse, 5 -CCGCTCGAGTTAAAAATCTATTAGTCCTAAAC-3 . Nde1 and Xho1 were used as restriction enzymes, and the cutting sites are underlined. The PCR products and pET28a vector were double-cleaved by the same restriction enzymes and ligated to each other. For purification, residual N-terminal His tag (MGSSHHHHHHSSGLVPRGSH) was attached to the gene of B. cereus MazF and was transformed into E.coli DH5α competent cells (Novagen, Madison, WI, USA). As the next steps, these cloned plasmids were re-transformed into E. coli Rosetta2 (DE3) pLysS competent cells (Novagen, Madison, WI, USA).

Protein Expression and Purification
Transformed cells were grown in Luria broth (LB) at 37 • C until the OD 600 reached to 0.5. Overexpression of target protein was induced by 0.5 mM isopropyl 1-thio-B-D-galactopyranoside (IPTG). Expressed cells were further incubated for 4 h at 37 • C. These cells were harvested by centrifugation at 11,355× g. Harvested cells were suspended in a buffer A (20 mM Tris-HCl, pH 7.9, and 500 mM NaCl) containing 5% glycerol and lysed by ultrasonication. Lysed cells were centrifugated for 1 h at 28,306× g. After centrifugation, the supernatant containing soluble moiety of protein was loaded on a Ni 2+ affinity chromatography column (Bio-Rad, Hercules, CA, USA), which was pre-equilibrated with buffer A. Loaded protein was washed with buffer A containing 50 mM imidazole to remove impurities. Then, the remaining protein, bound to the Ni 2+ column, was eluted using an imidazole gradient (100−800 mM) and fractionized. The purity of eluted protein in each fraction was checked using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS−PAGE). Finally, selected high-purity protein was loaded on a size-exclusion chromatography column HiLoad 16/600 Superdex 200 prep-grade column (GE Healthcare, Chicago, IL, USA), pre-equilibrated with final buffer (20 mM Tris, pH 8.0, and 500 mM NaCl). Final eluted protein was concentrated to 20 mg/mL using an Amicon Ultra centrifugal filter unit (Millipore, Burlington, MA, USA). The purity of the final protein solution was verified by SDS-PAGE. Mutants of B. cereus MazF were expressed and purified by the same procedure as native ones.

Crystallization, Data Collection, and Processing
The final protein solution was screened for crystallization using crystallization kit Wizard (Rigaku Reagents, Bainbridge Island, WA, USA) and Index (Hampton Research, Aliso Viejo, CA, USA). Protein solution (1 µL) was mixed with 1 µL of each buffer solution in the crystallization kit. Crystals of B. cereus MazF were grown in the transparent 96-well plate using sitting-drop vapor diffusion method at 20 • C. Only one crystallized well contained the hit solution of 100 mM Sodium Citrate, pH 5.6, and 1.0 M ammonium phosphate monobasic. Cryo-protection was achieved by addition of 20% glycerol to the hit solution. After cryo-protection, crystals were immediately cooled in liquid nitrogen prior to data collection. The data were collected using an Rayonix MX-300 HE CCD detector at BL44XU of the Spring-8, Japan. All raw data of crystal were scaled and processed by XDS [44]. A set of data from a hit crystal was used to solve the structure at 2.00 Å resolution and to refine 7BXY (code name of B. cereus MazF). PHENIX [45] was first used to do molecular replacement and automatically build the model, and COOT [46] was utilized to yield the starting model for refinement. The R work /R free values [47] of the final model were obtained using REFMAC and PHENIX [45,48]. The validation of overall geometry was achieved using MolProbity [49]. PyMOL (PyMOL Molecular Graphics System, Version 1.2r3pre, Schrödinger, LLC, NY, USA) was used to generate figures in this study.

Mutiple Sequence Alignment
To conduct sequence alignment of B. cereus MazF and four MazFs whose structures have been identified, the sequence information of five proteins was browsed using Uniprot [50]. Alignments of amino acid residues were carried out using Clustal W [51] and visualized using ESPript 3.0 [52]. The consensus value was set to 0.85 and %Equivalent was used in similarity mode. In visualization, structural information of B. cereus MazF was used as top secondary structures and the description and sequence numbering on the topside correspond to B. cereus MazF.

In Vitro Ribonuclease Assay
To confirm the ribonuclease activity of B. cereus MazF, an RNase Alert Kit (IDT, Coralville, IA, USA) was purchased and used following the manufacturer's protocol. Equal aliquot RNA substrate (5 µL) was interacted with 2 µM, 4 µM, 8 µM, and 16 µM concentrations of B. cereus MazF in a final purification buffer. For ribonuclease activity test, 10 µM aliquots of wild-type B. cereus MazF and its mutants were prepared. Then, the resulting fluorescence units (RFU) were detected using a SPECTRAmax GEMINI XS spectro-fluorometer (Marshall Scientific, Hampton, NH, USA) at 37 • C by emission fluorescence at 520 nm upon excitation at 490 nm. Assay setting was performed on 384-well opaque plate, and all of experiments were performed in triplicate.

Site-Directed Mutagenesis
The mutations in B. cereus MazF (R25A, T48A, R25A+T48A) were performed using B. cereus MazF in pET28a (+) as template. Single-site mutations were performed in one step, and double-site mutations were performed in a step-wise manner. Reaction components were mixed and subjected to PCR machine guided by the manufacturer's protocol (EZchange Site-Directed Mutagenesis Kit, Enzynomics, Daejeon, Korea). Each plasmid was transformed into E. coli XL10-Gold competent cells (Agilent Technologies, Santa Clara, CA, USA), and the resulting inserted genes were verified through DNA sequencing.