02

Dec '23

Novel WYL domain-containing transcriptional activator acts in … – Nature.com

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Communications Biology volume 6, Article number: 1222 (2023)
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The WYL domain is a nucleotide-sensing module that controls the activity of transcription factors involved in the regulation of DNA damage response and phage defense mechanisms in bacteria. In this study, we investigated a WYL domain-containing transcription factor in Mycobacterium smegmatis that we termed stress-involved WYL domain-containing regulator (SiwR). We found that SiwR controls adjacent genes that belong to the DinB/YfiT-like putative metalloenzymes superfamily by upregulating their expression in response to various genotoxic stress conditions, including upon exposure to H2O2 or the natural antibiotic zeocin. We show that SiwR binds different forms of single-stranded DNA (ssDNA) with high affinity, primarily through its characteristic WYL domain. In combination with complementation studies of a M. smegmatis siwR deletion strain, our findings support a role of the WYL domains as signal-sensing activity switches of WYL domain-containing transcription factors (WYL TFs). Our study provides evidence that WYL TFs are involved in the adaptation of bacteria to changing environments and encountered stress conditions.
Mycobacteria are exposed to various environmental stressors such as temperature changes, nutrient limitation, genotoxic stress, and bacteriophage infection. To survive these stressors, bacteria employ a range of adaptive stress responses. Transcriptional regulation by a wide-spread class of bacterial transcription factors (TFs) called WYL domain-containing transcription factors (WYL TFs) was recently shown to be important in the context of DNA damage stress and for phage defense1.
Bacteria have evolved mechanisms to protect and repair their DNA to maintain the integrity of their genome. The SOS response, which is present in almost all bacteria, regulates gene expression in response to DNA damage. This response involves the repressor protein LexA and the recombinase A (RecA)2. When DNA damage occurs, RecA forms filaments with accumulating single-stranded DNA (ssDNA), leading to the auto-proteolytic cleavage of LexA3,4. This cleavage releases LexA repression on SOS genes by dissociation of LexA from the SOS box present in their promoter regions. In mycobacteria, a WYL TF called PafBC plays a crucial role as a key activator of the DNA damage response, upregulating more than 150 genes independently of LexA/RecA through a distinct mechanism5,6. PafBC is composed of the homologs PafB and PafC, which are encoded in the proteasome gene locus in an operon together with the prokaryotic ubiquitin-like protein (Pup) ligase PafA (proteasome accessory factor A)7,8. Proteasomal degradation is essential for a full recovery after DNA damage by degrading RecA and other DNA repair proteins5.
WYL TFs possess a characteristic domain organization, including an N-terminal winged helix-turn-helix (HTH) domain, followed by a WYL domain (named after a conserved Trp-Tyr-Leu motif), and in most cases, a third domain referred to as WCX (WYL extension) domain9. The WYL domain adopts an Sm-fold, where a long α-helix packs against a β-sheet sandwich, a fold frequently encountered in RNA-binding proteins9. Recent studies have revealed that PafBC is constitutively present and not induced upon DNA damage6. Instead, the WYL domain serves as a signal transducer by binding to ssDNA present during DNA damage, triggering a conformational change in PafBC that enables it to activate transcription of its target genes10. This activation occurs through a mechanism known as sigma adaptation, wherein PafBC inserts itself between the sigma factor of the mycobacterial housekeeping RNA polymerase holoenzyme and DNA, allowing transcriptional initiation from PafBC-dependent promoters10.
A study on DriD, a WYL domain-containing TF from Caulobacter crescentus, found that the WYL domain of DriD also binds ssDNA11. The crystal structure of the DriD homodimer, in the presence of a 9-nucleotide ssDNA, was determined using a truncated version lacking the HTH domain. Residues in PafBC previously shown to be crucial during DNA damage for rescue of cells lacking PafBC are conserved also in DriD and are involved in the interaction of the DriD WYL domain with ssDNA9. While DriD also regulates the SOS response regulator recA, its own regulon consists of only a few additional genes.
Although WYL domain-containing proteins are widespread in bacteria, only a limited number have been characterized to date. Recent studies have identified WYL domain-containing transcriptional repressors involved in the regulation of anti-phage systems12,13,14. CapW was identified upstream of operons involved in a cyclic oligonucleotide-based anti-phage signaling system (CBASS). The CBASS is upregulated upon phage infection and leads to cell death, limiting phage spread within the population12. In Acinetobacter and Escherichia fergusonii, a WYL domain-containing TF called BrxR acts as a repressor of the bacteriophage exclusion (BREX) system13,14. The BREX system protects cells from phage infection by inducing specific host DNA methylation, which inhibits phage replication through an unknown mechanism15. Interestingly, BrxR homologs have been found upstream of other bacterial immunity systems such as restriction-modification systems, CRISPR/Cas systems and other phage defense systems14.
In Mycobacterium smegmatis (Msm), another WYL domain-encoding gene exists, msmeg_1359, which we named stress-involved WYL domain-containing regulator (siwR). We show that SiwR forms a homodimeric transcription factor and, upon genotoxic stress, upregulates two genes encoding proteins of the DinB/YfiT-like putative metalloenzymes superfamily (DinB superfamily). We also performed in vivo characterization of an Msm ΔsiwR deletion mutant subjected to various sources of DNA damage and oxidative stress. Our findings provide insights into the roles of WYL TFs during stress.
Another WYL domain-containing protein in Msm, SiwR (MSMEG_1359), shares the same domain architecture with PafB and PafC9. It consists of an N-terminal wHTH domain (residues 13–65), connected via an extended linker region to a WYL domain (residues 146–215), followed by a WCX domain (residues 245–320) (Fig. 1a, b). The AlphaFold2-predicted structure16 of a SiwR dimer (Fig. 1b) displays an intertwined, two-fold symmetric topology, similar to previously studied TF structures with WYL domains9,10,11,12,13,14. The N-terminal HTH domains and the WXC domains participate in dimeric interactions, while the WYL domains are located at the periphery and do not directly interact with each other. Connecting the N-terminal HTH domains to the WYL domains is an extended linker region that crosses the two-fold axis.
a SiwR contains an N-terminal DNA binding module, the winged HTH domain (light brown), followed by a WYL (light green) and WCX (dark green) domain. b The structure predicted by AlphaFold2 for dimeric SiwR resembles the published structures of other WYL domain-containing TFs. The HTH domains (light brown) are connected to the WYL domains (light green) through a linker region (light gray). Like other WYL domains, the predicted SiwR WYL domain structure consists of an N-terminal α-helix followed by a five stranded, anti-parallel β-sheet. c The genomic locus of siwR (green) is shown for various Actinobacteria. The two genes msmeg_1357 and msmeg_1356 belong to the DinB superfamily and are located upstream of siwR. While msmeg_1357 belongs to the DinB_2 family (lavender), msmeg_1356 belongs to the DUF664 family (purple). In many Actinobacteria, siwR co-localizes with at least one gene of the DinB superfamily. In some Actinobacteria, a gene of the pfam18029 family (dark gray) is found upstream of siwR. This family contains proteins with a predicted glyoxalase-like domain. d The promoter region of msmeg_1357-56 comprises a 19 bp imperfect palindrome that is located upstream of the −10 promoter motif. e The genes msmeg_1357 and msmeg_1356 are encoded on the same mRNA and are therefore under control of the same promoter. Isolated RNA was reverse transcribed into cDNA using random primers followed by PCR analysis using primers amplifying the indicated regions (a, b). Msm Mycobacterium smegmatis, Tlu Terracoccus luteus, Cva Corynebacterium variabile, Arh Arthrobacter rhombi, Mrh Micromonospora rhizosphaerae, Ssp, Nfa Nocardia farcinica, Rpy Rhodococcus pyridinivorans, Shy Streptomyces hygroscopicus.
We performed a BLASTp search to identify the top 500 orthologs of SiwR in various organisms. SiwR orthologs were exclusively found in bacteria, particularly in the phylum Actinobacteria. Within the Mycobacterium genus, SiwR orthologs are predominantly present in rapid-growers (Supplementary Table 1). While slow-growing mycobacteria are associated with an intracellular lifestyle and pathogenicity, rapidly growing mycobacteria are mainly environmental bacteria17,18,19.
Sequence alignment revealed highly conserved regions in the HTH and WYL domains, whereas the C-terminal region exhibited more variability (Fig. S1a). Specifically, the HTH domain exhibited conserved charged residues across all 500 orthologs. Likewise the region between the HTH and the WYL domain contains highly conserved residues. In the WYL domain, the highly conserved residues consist mainly of charged and some hydrophobic residues. Previous studies demonstrated the significance of these residues, particularly the conserved arginines, in the PafBC-mediated activation of the DNA damage response9. Additionally, conserved residues in the WYL domain include the ssDNA-binding residues identified in DriD11. Notably, while no residue in the WCX domain showed strict conservation, several displayed high conservation among the majority of the top 500 orthologs.
Bioinformatic analysis of the gene neighborhood surrounding siwR in mycobacteria revealed the presence of a putative operon comprising two genes upstream of siwR in Msm, namely msmeg_1357 and msmeg_1356 (Fig. 1c, Supplementary Table 2). This gene arrangement is also observed in several other Actinobacteria. Notably, siwR is located on the plus strand, while msmeg_1357 and msmeg_1356 are situated on the minus strand. The gene products are classified within the DinB/YfiT-like putative metalloenzymes superfamily (hereinafter referred to as DinB superfamily). However, they belong to different families within this superfamily. MSMEG_1357 is classified in the DinB_2 family (pfam12867), which encompasses a group of putative enzymes. MSMEG_1356 belongs to the pfam04978 and DUF664 family, a family of proteins with unknown functions that contain multiple histidine residues potentially involved in metal binding. Some Actinobacteria possess an ortholog of MSMEG_1356, while others encode a protein belonging to the pfam18029 family. The latter family is characterized by a predicted glyoxalase-like domain. Interestingly, Streptomyces hygroscopicus possesses an ortholog of msmeg_1356 but lacks an msmeg_1357 ortholog.
An alignment of the roughly 60 bp sequence between siwR and msmeg_1357 in rapidly growing mycobacteria revealed the existence of a 19 bp imperfect palindrome within the promoter region of msmeg_1357, spanning from the −10 region to the −35 region (Fig. 1d, Fig. S1b). To investigate whether msmeg_1357 and msmeg_1356 form an operon, we isolated RNA to generate cDNA. Two specific primer pairs were then used, one spanning the coding region of msmeg_1357 and another spanning the coding regions of both genes. Amplification products were successfully obtained for both primer pairs, indicating that msmeg_1357 and msmeg_1356 are transcribed from a single mRNA (Fig. 1e).
Given the co-localization of siwR with the DinB superfamily genes and the presence of the 19 bp imperfect palindrome, we hypothesized that SiwR regulates the transcription of the adjacent dinB-like genes by binding to the palindromic region between siwR and msmeg_1357.
To investigate the assembly state and DNA binding properties of SiwR in vitro, we attempted to generate the wild-type, untagged version of SiwR through recombinant expression in E. coli. However, the native protein exhibited very low solubility, making it challenging to concentrate it to the required level for in vitro experiments. To overcome this issue, we explored various strategies, including the addition of tags, to enhance solubility. Eventually, we found that fusing a His6-TEV-Sumo-linker to the N-terminus of SiwR (referred to as Sumo-SiwR) improved the solubility of the protein. Analytical size exclusion indicated that Sumo-SiwR forms homodimers (Fig. S2).
Subsequently, we investigated the binding of SiwR to the intergenic region between siwR and msmeg_1357. In an electrophoretic mobility shift assay (EMSA), we titrated Sumo-SiwR to a FAM-labeled double-stranded DNA (dsDNA) fragment containing the promoter region of the msmeg_1357-56 operon. The observed concentration-dependent shift of the probe band on the gel revealed that SiwR bound to the DNA probe with a submicromolar affinity (Fig. 2a). To confirm the specificity of the observed binding, we conducted the same experiment in the presence of an excess of unspecific DNA (salmon sperm DNA). Even with the presence of the unspecific competitor dsDNA, a shift of the probe could still be observed (Fig. 2a).
a EMSAs were performed to analyze Sumo-SiwR binding to the msmeg_1357-56 operon promoter. Increasing concentrations of Sumo-SiwR were titrated to 10 nM FAM-labeled DNA containing the msmeg_1357-56 operon promoter sequence. A shift of the probe can be observed in presence and absence of an excess of unspecific competitor DNA (salmon sperm DNA). b The promoter sequence of the msmeg_1357-56 operon was divided into fragments a, b, c and d to narrow down the binding region of SiwR. Sumo-SiwR binds best to fragments c and d which both contain the 19 bp imperfect palindrome. c Upon scrambling the 19 bp imperfect palindrome (red nucleotides), binding of Sumo-SiwR is impaired. d The HTH domain is responsible for binding the msmeg_1357-56 promoter, as its deletion variant Sumo-SiwR ΔHTH does not cause a shift of the probe. In contrast, the Sumo-SiwR R204A-R207A WYL domain variant binds to the msmeg_1357-56 promoter. e Fluorescence anisotropy shows binding of Sumo-SiwR dimer to the FAM-labeled msmeg_1357-56 promoter region. The determined KD is 651 nM. Error bars represent data points of at least four independent fluorescent anisotropy measurements.
To further narrow down the specific interaction region, we performed EMSAs using dsDNA fragments that covered different segments of the promoter region (Fig. 2b). Four probes were tested: fragment a covered the 3’ half of the 62 bp upstream of the start codon, fragment b covered the 5’ half, and fragments c and d covered the middle region. Fragments a and b did not exhibit any shift under the experimental conditions, whereas probes c and d, which covered the middle part, showed almost complete shifting. These results indicate that the binding motif is located within the middle 30 bp region, which spans the region between the −35 and −10 element and includes the previously mentioned palindrome. Importantly, scrambling this sequence randomly, impairs the binding of Sumo-SiwR (Fig. 2c).
SiwR contains two potential nucleic acid binding domains, namely the HTH domain and the WYL domain. To demonstrate that the binding to the promoter region of msmeg_1357-56 is mediated by the HTH domains, we generated two additional Sumo-SiwR variants. One variant lacked the DNA binding HTH domain (Sumo-SiwR ΔHTH), while the other variant contained two point mutations in WYL domain residues that are responsible for nucleic acid sensing (Sumo-SiwR R204A-R207A). As expected, Sumo-SiwR ΔHTH did not bind the promoter region of msmeg_1357-56 (Fig. 2d). In contrast, Sumo-SiwR R204A-R207A exhibited comparable binding to the promoter region as Sumo-SiwR.
Finally, we assessed the interaction between SUMO-SiwR and the promoter fragment more quantitatively using the change in fluorescence anisotropy of the labeled DNA fragment upon binding of SUMO-SiwR. We determined a dissociation constant (KD) of 651 nM for the interaction between Sumo-SiwR and the promoter DNA (Fig. 2e). This falls within the range of measured affinities of other WYL TFs for their DNA targets, such as 65.9 nM for DriD, 300 nM for CapWSma, 1500 nM for CapWEco, and 1000 nM for CapWPae11,12. Additionally, our data demonstrate that, unlike the heterodimeric DNA damage response regulator PafBC, SiwR can bind to its operator even in the absence of any ligand6. Overall, our EMSA and fluorescence anisotropy experiments demonstrate that Sumo-SiwR binds the msmeg_1357-56 promoter region specifically.
Next, we sought to determine the conditions that lead to the upregulation of MSMEG_1357 and MSMEG_1356 in Msm. Given that dinB-like genes were found to be upregulated upon DNA damage in Bacillus subtilis20, we investigated whether the transcript levels of msmeg_1357 and msmeg_1356 increase upon exposure to genotoxic and/or oxidative stress. We exposed Msm wild-type cells to various agents known to cause DNA damage, including mitomycin C (MMC), zeocin, diamide, H2O2 and UVC radiation. The quinone-containing antitumor drug MMC is a DNA alkylating and DNA cross-linking agent21, zeocin induces double-strand breaks22, and 254 nm UVC radiation generates cyclobutyl pyrimidine dimers23. To induce oxidative stress, we utilized H2O2, which engages in the Fenton reaction and creates hydroxyl radicals24, as well as diamide, a chemical oxidizing agent25,26. After treating the cells with each reagent for 30 min or exposing them to 15 mJ/cm2 UVC radiation, we extracted total RNA and performed RT-qPCR to quantify the mRNA levels for msmeg_1357 and msmeg_1356. Comparing these levels to those of untreated cells, we observed an increase of msmeg_1357 and msmeg_1356 transcript levels under all tested genotoxic conditions (Fig. 3a). The most substantial increase was observed following treatment with H2O2 or zeocin, exhibiting a change of more than 15–20-fold. However, it must be noted that upregulation is concentration-dependent, with higher upregulation observed for higher concentration of H2O2 (Fig. 3b). Interestingly, the upregulation of the PafBC-dependent genes recA and uvrA follows a similar pattern. The most substantial upregulation was observed upon treatment with 7 mM H2O2 and 5 µg/mL zeocin, while treatment with 80 ng/mL MMC or UV (15 mJ/cm2) showed a milder increase for recA and hardly any increase for uvrA transcript levels (Fig. S3). This suggests that the upregulation of msmeg_1357-56, recA, and uvrA upregulation occurs under similar cellular conditions.
a Msm SMR5 wild-type cells were grown to an OD600 of 0.5 at 37 °C and stressed with 10 mM diamide, 7 mM H2O2, 80 ng/mL MMC, 15 mJ/cm2 UV or 5 µg/mL zeocin for 30 min. RT-qPCR analysis revealed that msmeg_1357 (lavender) and msmeg_1356 (purple) transcript levels are upregulated among all tested genotoxic conditions but most strongly upon H2O2 and zeocin stress. Relative transcript levels were calculated by normalizing CT values of msmeg_1357 and msmeg_1356 against sigA. Accordingly, 2-ΔCT values were calculated and normalized to untreated cells. b Msm SMR5 wild-type cells were cultured as described before and treated with 0.2, 1 or 5 mM H2O2. RT-qPCR analysis shows that msmeg_1357 and msmeg_1356 transcript levels increase in a concentration dependent manner when cells are treated with H2O2. Relative transcript levels were calculated by normalizing CT values of msmeg_1357 and msmeg_1356 against sigA. Next, ΔCT of cells after H2O2 treatment were normalized against ΔCT values of cells before H2O2 treatment. Thus, 2-ΔΔCT values were calculated. Error bars represent the three biological replicates shown as individual data points.
In agreement with our results, a study that monitored global protein thiol-oxidation in Msm demonstrated the upregulation of msmeg_1357 and msmeg_1356 under NaOCl stress27. In addition, msmeg_1357 and msmeg_1356 transcript levels were shown to be elevated upon treatment with H2O25,28, with MMC5, and in a study analyzing the response to ciprofloxacin and UV treatment29.
To investigate the role of SiwR in the upregulation of msmeg_1357 and msmeg_1356 under genotoxic stress, we generated a siwR deletion strain (ΔsiwR) of Msm. As the stop codon of msmeg_1358 overlaps with the siwR stop codon (Fig. S4a), we deleted the coding region of siwR in a manner that the integrity of the msmeg_1358 stop codon remains intact, to not introduce polar effects. The ΔsiwR strain was constructed by homologous recombination using a suicide plasmid (Fig. S4a)30 and verified using PCR (Fig. S4b).
Next, we compared the upregulation of msmeg_1357 and msmeg_1356 mRNA levels in the Msm SMR5 wild-type versus the ΔsiwR strain when exposed to oxidative stress mediated by H2O2. Under standard conditions, the transcript levels of msmeg_1357-56 remained similar in both the Msm SMR5 wild-type and ΔsiwR cells (Fig. 4a). However, upon H2O2 exposure, the transcript levels of msmeg_1357-56 increased by 18–24-fold in wild-type cells but not in the ΔsiwR cells (Fig. 4a). This clearly indicates that SiwR acts as a transcriptional activator, upregulating msmeg_1357 and msmeg_1356 in response to oxidative stress caused by H2O2. Notably, the deletion of siwR did not affect the transcript levels of recA (Fig. 4a).
a Msm SMR5 wild-type and ΔsiwR cells were cultured as described before and stressed with 7 mM H2O2 for 30 min. The msmeg_1357 and msmeg_1356 transcript levels are not upregulated in the Msm ΔsiwR cells under standard condition or upon H2O2 treatment. Hence, SiwR is a transcriptional activator of the msmeg_1357-56 operon. The recA (light orange) transcript levels in Msm SMR5 wild-type and Msm ΔsiwR cells are similar. Relative transcript levels were calculated by normalizing CT values of recA, msmeg_1357 and msmeg_1356 against sigA. Thus, 2-ΔCT values were calculated and normalized to wild-type cells. b Msm ΔsiwR cells were complemented with siwR wild-type, empty vector, siwR R38A-R42A or siwR R204A-R207A under the native siwR promoter using an integrative plasmid. Upregulation of msmeg_1357 and msmeg_1356 upon 7 mM H2O2 treatment could be restored in Msm ΔsiwR cells complemented with siwR wild-type. Complementation of Msm ΔsiwR cells with empty vector, siwR R38A-R42A or siwR R204A-R207A did not restore msmeg_1357 and msmeg_1356 upregulation. This shows that the R38 and R42 residues in the HTH domain (light brown) as well as the R204 and R207 residues in the WYL domain (light green) are essential for upregulation of the msmeg_1357-56 operon. Relative transcript levels were calculated by normalizing CT values of msmeg_1357 and msmeg_1356 against sigA. Next, ΔCT of cells after H2O2 treatment were normalized against ΔCT values of cells before H2O2 treatment. Thus, 2-ΔΔCT values were calculated. Error bars represent the three biological replicates shown as individual data points.
To probe the involvement of nucleic acid binding by the HTH and WYL domains, we then proceeded to complement the ΔsiwR cells with various constructs, including an empty vector, a vector expressing wild-type SiwR, or expressing SiwR variants with point mutations in the HTH (R38A-R42A) or WYL (R204A-R207A) domains, respectively. We used an integrative plasmid carrying an attP site for chromosomal integration, along with a suicide plasmid carrying the L5 bacteriophage integrase gene to ensure stable integration31. Complementation of the ΔsiwR cells with wild-type SiwR, compared to complementation with empty vector, restored the induction of msmeg_1357 and msmeg_1356 upon oxidative stress (Fig. 4b). As expected, cells expressing the SiwR R38A-R42A mutant exhibited no upregulation of the msmeg_1357-56 operon, as this variant is no longer able to bind the promoter motif. Also, ΔsiwR cells transformed with SiwR R204A-R207A showed no upregulation of msmeg_1357 and msmeg_1356, thereby demonstrating the essential role of the WYL domain, and more specifically the conserved arginine residues in transcriptional activation. These results suggest that SiwR-binding to its operator alone is insufficient for transcriptional activation, and that signal sensing of DNA damage by the WYL domain is also required.
A common feature of WYL domains appears to be their ability to bind nucleic acids10,11,32,33. For instance, PafBC senses MMC-induced stress by binding ssDNA10. To explore the binding affinity of SiwR for different nucleic acid ligands, we carried out in vitro fluorescence anisotropy experiments. We first tested the interaction between SiwR and a polyT 20mer ssDNA fragment and observed that SiwR binds to the ssDNA probe with a dissociation constant (KD) of 182 nM (Fig. 5a). This affinity is consistent with the range previously determined for other WYL domain-containing proteins, such as the DriD-ssDNA interaction (KD = 130 nM)11. As anticipated, no binding was observed for the WYL domain variant Sumo-SiwR R204A-R207A.
a Binding of Sumo-SiwR to FAM-labeled polyT20 ssDNA was measured using fluorescence anisotropy. While a KD of 182 nM could be determined for the wild-type form of Sumo-SiwR, the WYL domain variant R204A-R207A showed no binding. b Overview of further nucleic acid molecules tested for Sumo-SiwR binding. Nucleotides in dark green show the conjugated FAM position. We tested a 12-nucleotide ssDNA piece (black) or equivalent RNA piece (pink), the same ssDNA with an 8-oxoguanine mutation (red), a 5’ tailed DNA piece (green), a 3’ tailed DNA piece (blue) and a DNA fork mimic (orange). c Sumo-SiwR binds to ssDNA containing oxidized guanine with a similar affinity (227 nM) as to the same nucleotide piece without oxidized guanine (178 nM). d SiwR binds other forms of nucleic acids such as 5’ tailed, 3’ tailed and fork DNA mimic, but not ssRNA. The observed KD values for the tailed or fork ssDNA molecules are higher than for simple ssDNA fragments. Each anisotropy datapoint was measured at least five times. Every KD was calculated under the assumption that both WYL domains of dimeric SiwR can bind ligand. Error bars represent data points of at least four independent fluorescent anisotropy measurements.
We further tested SiwR binding to a 12-nucleotide ssDNA fragment with a random sequence, to the same fragment containing an oxidized guanine mutation, to a DNA fork, and to a 5’ or 3’ tailed DNA construct (Fig. 5b). The inclusion of the oxidized fragment, featuring an 8-oxoguanine modification, was motivated by the fact that oxidized guanine is associated with oxidative stress34,35. Interestingly, the observed KD for the oxidized fragment (227 nM) was similar to that of the unoxidized ssDNA fragment (178 nM) (Fig. 5c). The dissociation constant for the 5’ tailed DNA (306 nM) was slightly higher than that for ssDNA (Fig. 5d), while the 3’ tailed DNA (702 nM) and the fork DNA mimic (898 nM) exhibited the highest KD of the tested oligonucleotides. This observation aligns with the behavior of the Pif1 helicase, which also exhibits strongest binding to free ssDNA33. We also examined the affinity of SiwR for RNA, as RspWYL1 was shown to bind ssRNA32. However, no ssRNA binding could be observed for SiwR (Fig. 5d).
Along with our complementation studies, this analysis shows that the SiwR WYL domain plays a role in ssDNA sensing during stress and is crucial for the transcriptional activation of the msmeg_1357-56 operon in vivo.
An alignment of the promoter region of msmeg_1357-56 among different actinobacterial species reveals a conserved GGA triplet at the −26 position of the msmeg_1357-56 promoter (Fig. 6a). Interestingly, the PafBC binding site in the PafBC-dependent promoters is also located at the −26 position and is involved in PafBC-mediated sigma adaptation10.
a Sequence alignment across actinobacterial species of the msmeg_1357-56 promoter region reveals conservation of a GGA triplet in the −26 region (gray area). b Δmsmeg1357-56 cells were complemented with an integrative plasmid containing the natural msmeg_1357-56 promoter (p0) and an integrative plasmid containing a scrambled −26 region (p-26). The cells were treated with 7 mM H2O2 and RT-qPCR analysis was performed. The msmeg_1357-56_int upregulation cannot be observed in cells carrying a mutated −26 motif in the msmeg_1357-56_int promoter. Relative transcript levels were calculated by normalizing CT values of msmeg_1357 and msmeg_1356 against sigA. Next, ΔCT values of msmeg_1357-56 of cells after H2O2 treatment were normalized against ΔCT values of cells before H2O2 treatment. Thus, 2-ΔΔCT values were calculated. Msm Mycobacterium smegmatis, Tlu Terracoccus luteus, Cva Corynebacterium variabile, Arh Arthrobacter rhombi, Mrh Micromonospora rhizosphaerae, Ssp Saccharopolyspora spinosa, Nfa Nocardia farcinica, Rpy Rhodococcus pyridinivorans, Shy Streptomyces hygroscopicus. Error bars represent the four biological replicates shown as individual data points.
To investigate the role of the −26 region in the upregulation of the msmeg-1357-56 operon, we first generated a Δmsmeg_1357-56 strain. We then complement this strain with an integrative plasmid carrying the msmeg_1357-56 operon along with its natural promoter and terminator sequences. However, we only achieved partial restoration of msmeg_1357-56_int upregulation under oxidative stress conditions (Fig. 6b). One possible explanation of this result is that the SiwR operator sequence was not eliminated during the knock-out procedure, leading to the presence of multiple binding sites for SiwR. Notably, when we complemented the knock-out strain with an integrative plasmid containing a scrambled −26 region, we observed no upregulation of the msmeg_1357-56_int operon. This indicates that, similar to PafBC, SiwR relies on the −26 region for transcriptional activation. It is plausible that the mechanism of transcriptional activation employed by SiwR is related to the PafBC-mediated sigma adaptation mechanism.
After demonstrating that SiwR is responsible for the upregulation of specific genes in response to genotoxic stress, our next objective was to examine whether the strain lacking the siwR gene would exhibit any phenotype under various genotoxic stresses. To assess the growth characteristics of the ΔsiwR strain, we conducted comparative growth experiments between this strain and the Msm SMR5 parent strain under standard culture conditions in 7H9 liquid medium. We observed no difference in the growth behavior of the knock-out strain compared to the parent strain (Fig. S5).
To compare the susceptibility of the knock-out strain and the parent strain to different genotoxic agents, we added 10 mM or 1 mM H2O2 to a growing culture (Fig. 7a, b) and continued monitoring the growth. The addition of 10 mM H2O2 immediately resulted in a decline, while the cells continued to grow when 1 mM H2O2 was added. Interestingly, we did not observe any difference in growth behavior between the Msm wild-type and ΔsiwR cells.
a, b Msm SMR5 wild-type and ΔsiwR cells were grown in 7H9 supplemented with 0.2% glycerol and 0.05% Tween-80 and grown for 6 h at 37 °C. The arrows indicate the time at which 1 mM or 10 mM H2O2 was added to the culture. For both Msm SMR5 wild-type and ΔsiwR cells, OD600 decreases upon addition of 10 mM H2O2 while the cells kept growing after addition of 1 mM H2O2. The error bars represent three independent biological replicates. Resazurin-based viability assays were performed with zeocin (c) or MMC (d). Msm SMR5 wild-type and ΔsiwR cells behaved similarly. The ΔpafBC ΔsiwR double knock-out cells behave like the ΔpafBC cells. The error bars represent three independent biological replicates.
Next, we analyzed the viability of the strains using a resazurin-based assay. The cells were incubated with various concentrations of MMC or zeocin for 24 h before adding resazurin, which is reduced to resorufin in metabolically active cells36,37. The ΔsiwR cells did not exhibit reduced viability compared to the Msm wild-type cells (Fig. 7c, d). In contrast, the ΔpafBC cells are hypersensitive to zeocin. We created a ΔpafBC ΔsiwR double knock-out to analyze whether PafBC might compensate for the loss of siwR. However, the double knock-out shows the same hypersensitivity as ΔpafBC cells. Our RT-qPCR data demonstrate that SiwR is involved in sensing certain sources of genotoxic stress. However, it appears that there is sufficient redundancy in the stress response to prevent an observable growth phenotype under the tested conditions.
The term dinB-like genes refers to DNA damage-inducible genes that were initially described in Bacillus subtilis. These genes are part of the SOS regulon and are believed to play a role in the DNA damage repair response20. It is important to note that the DinB superfamily should not be confused with DinB polymerases and other enzymes involved in DNA repair38. The DinB superfamily is predominantly found in bacteria and is most commonly observed in Proteobacteria, Actinobacteria, Firmicutes and Bacteroidetes (Fig. 8a). Among these, Actinobacteria have the highest number of dinB-like genes per species, with an average of 17 dinB-like genes encoded per actinobacterial species. In contrast, proteobacterial species typically encode an average of three dinB-like genes. Despite their wide-spread presence, the role of these genes remains poorly understood. Msm encodes 21 proteins belonging to the DinB superfamily, while the slow-growing human pathogen Mtb encodes 11 such proteins (Supplementary Tables 3, 4). The DinB superfamily encompasses ten families, of which four different families (Fig. 8b) are present in Msm and three in Mtb. Additionally, some proteins do not belong to the DinB superfamily but possess characteristic domains associated with the DinB superfamily, such as the mycothiol-dependent maleylpyruvate isomerase (MDMPI) domain39,40.
a Taxonomic distribution of all proteins belonging to the DinB superfamily. Even though Actinobacteria only make up 23% of species with DinB superfamily genes, 43% of DinB superfamily sequences can be found within Actinobacteria. b The DinB superfamily encompasses 10 families of which the DinB_2 and DUF664 families together make up one third. The largest family consists of the CHP03083-86 families that is almost exclusively found in Actinobacteria. Similarly, the DUF664 family consists of proteins that are mostly found in Actinobacteria. The DinB_2 family contains proteins that are present especially in Proteobacteria, Firmicutes and Bacteroidota. c Heatmap of sequence identities (blue gradient) and structural similarities (gray gradient) of the 21 DinB superfamily genes in Msm. The sequence identity and structural similarity correlate. d RT-qPCR reveals that among the 21 DinB superfamily genes in Msm only five are noticeably upregulated upon DNA damage. However, most show upregulation upon diamide treatment.
MSMEG_1357 and MSMEG_1356 belong to different families within the DinB superfamily; MSMEG_1357 belongs to the DinB_2 family, while MSMEG_1356 belongs to the DUF664 family (Supplementary Table 3). The DUF664 family of proteins is predominantly found in Actinobacteria, while DinB_2 proteins are mainly present in Proteobacteria, Firmicutes and Bacteroidetes (Fig. 8b). An alignment of the 21 proteins belonging to the DinB superfamily in Msm reveals limited sequence similarity among them, which is typical for members of this superfamily (Fig. 8c, blue portion of the heatmap). Some of those 21 proteins display homology to each other (for example MSMEG_1357 and MSMEG_3910; MSMEG_1356 and MSMEG_3923; MSMEG_0336 and MSMEG_6923; MSMEG_0682 and MSMEG_0441; MSMEG_5349 and MSMEG_6779). The other half of the heatmap (gray) shows the structural similarity calculated in a DALI (distance matrix alignment)41 structure comparison based on AlphaFold2 models (Fig. 8c). Overall, sequence similarity and structural similarity correlate. Next, we analyzed changes in mRNA transcript levels of all 21 DinB superfamily genes upon exposure to various stress conditions (Fig. 8d). While most DinB superfamily genes exhibit some level of upregulation in response to diamide treatment, aside from msmeg_1357-56, the majority of the DinB superfamily genes are not upregulated upon H2O2, MMC, UV, or zeocin treatment. Exceptions are msmeg_3910 and msmeg_6249, that encodes EgtB (ergothioneine synthesis gene B). In contrast to msmeg_1356-57, the strongest upregulation of the egtB gene is observed in response to diamide treatment. Ergothioneine is a low-molecular-weight thiol synthesized by mycobacteria and plays a role in protecting the bacteria against oxidative stress.
The sequence alignment revealed relatively low sequence identity (15%) between MSMEG_1357 and MSMEG_1356 (Fig. 8c, Fig. S6a). However, the sequence alignment shows homology between the only other DinB_2 protein in Msm (MSMEG_3910) and MSMEG_1357 (32% sequence identity), and between MSMEG_1356 and one of the other DUF664 proteins (MSMEG_3923) (31% sequence identity) (Fig. S6a). Interestingly, msmeg_3910 transcript levels follow the same pattern as msmeg_1357-56 upon exposure to the tested stresses. However, the transcript levels of msmeg_3923 remain unchanged (Figs. 8d, S6b). As observed for msmeg_1357-56, the upregulation of msmeg_3910 is dependent on the concentration of the stress-inducing agents (Fig. S6c). It is worth noting, that msmeg_3910 is part of the PafBC regulon5. When complementing ΔsiwR cells with an empty vector compared to wild-type siwR, no difference in msmeg_3910 levels was observed (Fig. S6d), indicating that SiwR is not responsible for the upregulation of msmeg_3910 under stress. To test whether functional redundancy between msmeg_3910 and msmeg_1357-56 could explain why we did not observe a growth phenotype for the ΔsiwR knockout, we generated a ΔsiwR Δmsmeg_3910 double knock-out. However, the double knockout did also not display a phenotype in the MIC assay with zeocin in comparison to wild-type or ΔsiwR cells (Fig. S7). Nevertheless, given the large number of DinB superfamily genes, further functional redundancies might exist.
The Alphafold2-predicted structures of MSMEG_1357 and MSMEG_1356 exhibit the characteristic four-helix bundle fold commonly observed in the DinB superfamily38,40,42 (Fig. S6e). A DALI search revealed structural homology (Z-score > 10.0) with other members of the DinB superfamily, such as ClbS, DfsB, DR0053, BtsA, and EgtB (Supplementary Table 5, 6) (Fig. S8)43,44,45,46,47. However, functional insight from these structures is limited to the shared feature of a characteristic histidine triad suggestive of metal binding. Most studied DinB superfamily proteins form homodimers45,46 with just a few exceptions43,44,48. The recombinantly expressed MSMEG_1357 and MSMEG_1356 also eluted at the positions of their respective homodimers in size exclusion analysis (Fig. S6f), and circular dichroism analysis supported the prevalence of alpha-helical structure in both proteins (Fig. S6g).
These preliminary findings align with the existing literature on the DinB superfamily. However, further investigations are necessary to unravel the functions of these proteins in rapidly growing mycobacteria.
WYL TFs are a class of bacterial TFs that play important roles in combatting DNA damage and phage infection in bacteria1. They modulate gene expression in response to stresses by nucleotide-sensing through their WYL domains. However, while the identified phage defense regulators are transcriptional repressors, those identified to date as associated with genotoxic stress act as activators5,11.
Our study establishes SiwR as an activating WYL TF that promotes the operon under its control in response to genotoxic agents. Unlike other known activating WYL TFs, SiwR does not regulate the canonical SOS response. Instead, it upregulates a pair of genes, msmeg_1357 and msmeg_1356, that together form an operon and are located directly adjacent to SiwR. We observed the most potent SiwR-mediated upregulation of msmeg_1357-56 upon exposure to zeocin or H2O2 and less in response to MMC, although all three robustly triggered the induction of the msmeg_1357-56 operon. We therefore suggest to name this the “DNA damage-inducible by SiwR” operon (dinS).
We surmise that the different genotoxic insults ultimately lead to production of a common DNA stress signal in the form of an ssDNA ligand that is sensed by the WYL domains of both SiwR and PafBC. In agreement with this notion, we observed a similar gene induction profile for PafBC-dependent genes, exemplified by recA and uvrA in our study, under the different stress reagents. H2O2 engages in Fenton chemistry leading to production of HO radicals that can oxidize base as well as ribose moieties of the DNA24,49,50,51,52. This gives rise to lesions such as 8-hydroxyguanine that can pair with adenine, potentially causing mutations34,35. We found that SiwR binds ssDNA containing 8-hydroxyguanine with similar affinity as unmodified ssDNA, demonstrating that the response is triggered by presence of ssDNA rather than specific lesions. This hypothesis is further supported by our observation that the SiwR WYL domains bind to nucleic acids containing ssDNA regions, regardless of their form—individual ssDNA pieces, single-stranded overhangs, or forks. In contrast, ssRNA or dsDNA is not recognized by the WYL domain of SiwR. However, it is known that diverse WYL domains can bind different ligands. The PIF1 helicase, which contains a WYL domain, favors ssDNA, 5’-tailed and fork substrates33, whereas WYL1 binds ssRNA32. It must be noted that WYL1 differs from the other characterized WYL domain-containing TFs by featuring a distinct ribbon-helix-helix domain instead of a wHTH domain. The DNA damage regulators PafBC and DriD both bind ssDNA10,11, but the ligands for BrxR and CapW remain unidentified. As previously stated, the WYL domain possesses an Sm-fold, a characteristic fold present also in the bacterial RNA chaperone Hfq53,54. Interestingly, the location of the RNA/ssDNA binding site is conserved between Hfq and ssDNA-binding WYL TFs, with the involved arginine residues located in the β4/β5 loop in PafBC and DriD9,54,55. Despite the structural similarity among WYL domains, they can interact with different ligands and thus respond to different signals.
MSMEG_1357 and MSEMG_1356, categorized as DinB superfamily members, belong to the DinB_2 and DUF664, families, respectively. The function of this superfamily, which includes several thiol S-transferases, remains largely enigmatic. Distinct enzymes like mycothiol S-transferases (MST) and bacillithiol S-transferases (BST) fall under the DUF664 and DinB_2 families, respectively. Glutathione S-transferases (GST) belong to either of these families. MST MSMEG_0887 shares 16% sequence identity with MSMEG_1356, suggesting a potential role for the latter as a mycothiol-dependent enzyme.
Most DinB superfamily proteins form homodimers, though exceptions exist, such as ClbS, which functions as a monomer and confers colibactin resistance to colibactin-producing bacteria by ring-opening that detoxifies the compound. Interestingly, ClbS and the structurally related toxin precursor protein DfsB, lack the typical metal binding motif of the DinB superfamily43,48. While DfsB mostly exists as a monomer, some species encode both monomeric and dimeric DfsB variants. Upon subtilisin cleavage, DfsB becomes a potent toxin, possibly functioning similarly to cationic antimicrobial peptides due to its high basic and aromatic residue content44. However, neither DfsB nor ClbS, which both belong to the DUF1706 family, are present in Msm or Mtb.
The gene msmeg_1356 was reported amongst the group of genes conserved in rapidly growing mycobacteria but absent in slow-growing species, implying its necessity in conditions unique to free-living mycobacteria like Msm, a soil bacterium coexisting with other bacteria, fungi, nematodes, protists and invertebrates56,57. Soil composition varies greatly, with Proteobacteria, Acidobacteria, Bacteroidota, Chloroflexi and Actinobacteria as predominant phyla, their abundance fluctuating based on factors like nutrient availability, soil depth, salinity and pH58,59. Msm likely faces multiple stressors simultaneously, including antimicrobial compounds from organisms like Streptomyces, which comprises 90% of soil Actinobacteria60. This might explain why we saw no phenotype for the siwR deletion strain under our tested stress conditions. Actinobacteria’s adaptability to harsh conditions8,61 suggests that regulation of the dinS operon through SiwR is crucial under conditions not easily mimicked in the lab.
WYL TFs are known in bacterial immunity contexts and are frequently associated with CRISPR-Cas loci, R-M systems, CBASS and BREX systems1,12,13,14,62,63. While SiwR’s involvement in bacterial immunity cannot be ruled out, its role as a transcriptional activator contrasts with other WYL TFs functioning as repressors in this context. Nonetheless, the accumulation of nucleic acids during phage infection could possibly trigger SiwR-mediated transcriptional activation, given some bacterial CBASS are activated in response to DNA damaging agents64. In that scenario, the CBASS are repressed by CapH which is cleaved by CapP upon its activation through CapP-ssDNA binding. This two-protein transcriptional regulator module was found upstream of hundreds of CBASS but also associated with DISARM and Pycsar anti phage operons. This highlights that ssDNA binding modules can be engaged under various scenarios.
WYL TFs have emerged as regulators in phage defense or protection against alien DNA elements1. To date, no WYL TF has been described to regulate phage defense in Actinobacteria. However, the current knowledge on bacterial immune systems is strongly biased towards Proteobacteria65, leaving Actinobacteria’s anti-phage mechanisms understudied. DefenseFinder identifies four anti-phage systems in Msm66, none closely associated with a WYL domain-containing protein. However, considering the unidentified enzymatic activity of the proteins expressed by the dinS operon, we cannot exclude a potential role in the context of phage defense.
Our study presents SiwR, a stress-induced regulator with a WYL domain, that functions as a transcriptional activator under genotoxic conditions, primarily regulating the dinS operon. We demonstrate that the WYL domain of SiwR binds various ssDNA forms with high affinity in vitro, and that the ssDNA binding residues are crucial for transcription activation in vivo. These findings enhance our understanding of the broad family of WYL domain-containing TFs. Future research should explore whether SiwR regulates other genes beyond the dinS operon, whether it operates through sigma adaptation like the PafBC transcriptional activator, and should shed light on the cellular roles of the proteins expressed by the dinS operon, to better comprehend the biological process controlled by SiwR. Another interesting question to address is why the dinS operon is not part of the large PafBC regulon, given that PafBC and SiwR respond to the same stress conditions. A dedicated regulator for the dinS operon suggests that this operon has to be regulated independently of the PafBC regulon under certain circumstances.
SiwR orthologs in mycobacteria were identified using the Integrated Microbial Genomes (IMG) database at the DOE Joint Genome Institute (https://img.jgi.doe.gov) based on the SiwR (Gene ID 639739896) protein sequence. Each hit was manually inspected to ensure members of the PafBC branch were not included mistakenly due to the high similarity. We conducted a cassette search to identify organisms that contain a gene upstream of siwR belonging to the DinB_2 superfamily. Hits were manually inspected.
Total RNA was isolated from Msm SMR5 wild-type cells and treated with DNase (Turbo DNase, Invitrogen) prior to serving as a template for cDNA synthesis. 100 ng random primers, 500 ng RNA, 10 mM dNTP mix were mixed and heated to 65 °C for 5 min. After a quick incubation on ice, DTT was added to a final concentration of 10 mM as well as 1x First-Strand buffer (Thermo Fisher Scientific). Additionally, 20 U of RiboLock RNase inhibitor (Thermo Fisher Scientific) was added prior to addition of 200 U of reverse transcriptase (SuperScript RT, Thermo Fisher Scientific). Non-reverse transcriptase controls were run in parallel. cDNA synthesis was carried out under the following temperature protocol: 25 °C for 10 min, 42 °C for 50 min, 70 °C for 15 min. The resulting cDNA was used as a template in a PCR reaction with OneTaq polymerase (NEB) using two different primer pairs for amplification of either msmeg_1357 or msmeg_1357 to msmeg_1356. The PCR products were analysed on a 1% agarose gel in 1x TAE and visualized with SYBR safe nucleic acid gel stain. All oligo nucleotides used in this study can be found in Supplementary Table 7.
Sumo-SiwR fusion protein was expressed from a codon-optimized pET21-His6-TEV-Sumo-SiwR plasmid that was synthesized by Twist Bioscience. Based on this construct, the constructs Sumo-SiwR ΔHTH and Sumo-SiwR R204A-R207A were generated using the Q5 Site-Directed Mutagenesis kit (NEB) followed by a one-step DpnI digest, kinase and T4 ligation reaction using the KLD enzyme mix (NEB) (Supplementary Table 7). Plasmids were sequenced using the Microsynth T7 primer (Microsynth). The IPTG-inducible plasmids were transformed into E. coli Rosetta (DE3) cells (Invitrogen). Bacteria were grown for 4 h at 37 °C, induced with 0.25 mM IPTG and further incubated overnight at 18 °C. Cells were harvested and lysed by sonication (2x 1 min, 10 s on/20 s off) in lysis buffer (50 mM HEPES-KOH pH 7.0, 300 mM NaCl, 5 mM MgCl2, 0.5 mM TCEP, 1 mM PMSF, 1 mM EDTA) containing cOmplete protease-inhibitor cocktail (Roche). Lysates were cleared by centrifugation for 45 min at 20,000 rpm at 4 °C. Then, NaCl was added to the cleared lysates to a final concentration of 1 M to disrupt protein-DNA interactions. Lysates were subjected to a buffer-equilibrated Ni2+ affinity chromatography column using a 5 mL IMAC Sepharose 6 FF column (Cytiva). Impurities were removed by washing four column volumes of lysate buffer containing 10 mM imidazole and 1 M NaCl followed by a second wash step with two column volumes lysate buffer containing 50 mM imidazole and 1 M NaCl. Next, His-tagged Sumo-SiwR fusion protein was eluted with four column volumes of lysate buffer containing 250 mM imidazole and 1 M NaCl. The elution fractions were then dialyzed against buffer H (50 mM HEPES-KOH pH 7.0, 250 mM NaCl, 0.5 mM TCEP, 1 mM EDTA, 5 mM MgCl2) and loaded onto a HiPrep Heparin FF 16/10 column (Cytiva). The column was washed with 10 column volumes of buffer H before a linear gradient of buffer H with 1 M NaCl was applied. SDS-PAGE analysis revealed that all Sumo-SiwR constructs elute at about 0.5 M NaCl. After buffer exchange using PD-10 desalting columns (Cytiva) proteins were concentrated to 1–2 mg/mL using Amicon Ultra 10 K centrifugal filtration devices (Merck Millipore; 3500 × g, 10 min intervals, 4 °C). Final buffer was 50 mM HEPES-KOH pH 7.0, 250 mM NaCl, 0.5 mM TCEP. Protein concentration was determined spectrophotometrically by measuring absorbance at 280 nm and using the calculated extinction coefficients for the reduced proteins. Finally, proteins were flash-frozen in liquid nitrogen and stored at −20 °C until further use.
MSMEG_1356 and MSMEG_1357 were expressed from a pET21-His6-TEV-MSMEG_1356 or pET21-His6-TEV-MSMEG_1357 plasmid. The IPTG-inducible plasmids were transformed into E. coli Rosetta (DE3) cells (Invitrogen). Bacteria were grown for 4 h at 37 °C, induced with 0.5 mM IPTG and further incubated overnight at 18 °C. Cells were harvested and lysed in lysis buffer (50 mM HEPES-KOH pH 7.0, 300 mM NaCl, 5 mM MgCl2, 0.5 mM TCEP, 1 mM PMSF, 1 mM EDTA) containing cOmplete protease-inhibitor cocktail (Roche) using a Microfluidizer M110-L device (Microfluidics; 5 passes; 7000–12,000 psi chamber pressure). Lysate was cleared by centrifugation (20,000 rpm, 30 min, 4 °C, SS34 rotor). Lysates were subjected to Ni2+ affinity chromatography using a buffer-equilibrated 5 mL IMAC Sepharose 6 FF column (Cytiva). Impurities were removed by washing with four column volumes of lysate buffer containing 10 mM imidazole followed by a second wash step with two column volumes of lysate buffer containing 50 mM imidazole. Next, His6-TEV-MSMEG_1357 was eluted with 250 mM imidazole while His6-TEVM-MSMEG_1356 was eluted with 100 mM imidazole. The elution fractions were dialyzed against buffer D (50 mM HEPES-KOH pH 7.0, 150 mM NaCl, 0.5 mM TCEP) and loaded onto a Superdex 75 10/300 GL 24 mL gel filtration column (Cytiva). Both proteins eluted as homodimers. Protein concentration was determined spectrophotometrically by measuring absorbance at 280 nm and using the calculated extinction coefficients for the reduced proteins. Finally, proteins were flash-frozen in liquid nitrogen and stored at −20 °C until further use.
All purified proteins were subjected to an LC-MS analysis at the Functional Genomics Center Zurich (FGCZ) to ensure the integrity of the sample.
SiwR (40 µM protomer), MSMEG_1357 (100 µM protomer), or MSMEG_1356 (100 µM protomer) were incubated in buffer GF (50 mM HEPES-KOH pH 7.0, 150 mM NaCl, 0.5 mM TCEP, 1 mM EDTA) at 37 °C for 20 min. Then, the sample was applied onto a Superdex 200 Increase 10/300 GL column (Cytiva) and run at 0.5 mL/min at room temperature in buffer GF. Absorbance at 280 nm, 260 nm, and 230 nm was recorded and peak fractions were analyzed by SDS-PAGE.
To generate a marker-less deletion of siwRsiwR), the suicide plasmid pGOAL19 was generated containing the 1500 bp up- and downstream chromosomal region of siwR, as well as the first and last three amino acids of SiwR (Supplementary Table 1). The PCR products were designed with an overhang for ligation by cutting the plasmid with XmnI and were then ligated using Gibson assembly (NEBuilder HiFi DNA Assembly Master Mix, NEB). NEB5α cells were transformed with the Gibson assembly reaction mix, plated on LB plates containing hygromycin (100 µg/mL), and successful transformants were sequenced (Microsynth). Msm SMR5 cells were transformed with the suicide plasmid for allelic exchange mutagenesis30. Briefly, 200 µL of electro-competent Msm SMR5 cells were transformed with 2.0 µg suicide plasmid by electroporation (2.5 kV). Electroporated cells were immediately recovered in 5 mL shaking cultures of 7H9 medium complemented with 0.2% glycerol and 0.05% Tween-80 for 4 h at 37 °C, after which cells were plated on 7H10 plates supplemented with 0.5% glycerol and hygromycin (50 µg/mL). Following 3 days of incubation at 37 °C, single-crossover (SCO) colonies were identified by 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-gal) underlay (200 µL of 0.4% X-gal in DMSO were spread underneath the agar). SCOs turn blue after an overnight incubation at 37 °C due to the expression of β-galactosidase encoded on the suicide plasmid. SCOs were picked and grown in 5 mL 7H9 supplemented with 0.2% glycerol and 0.05% Tween-80 as well as hygromycin (50 µg/mL) to an OD600 of 0.7. Then, the cells were plated on 7H10 plates supplemented with 0.5% (v/v) glycerol and 2% (w/v) sucrose in 1:10 and 1:100 dilutions. Expression of the Bacillus subtilis levansucrase SacB, encoded on the suicide plasmid, is lethal in the presence of sucrose. After incubation at 37 °C for 3 days the X-gal underlay was repeated as described as a second round of selection. Double crossover (DCO) cells should stay white, as the suicide plasmid should be lost during homologous recombination. DCO cells were screened through colony PCR as well as sequencing of a PCR product generated with primers annealing 2500 bp up- and downstream of siwR. In addition, deletion of siwR was verified through RT-PCR. The ΔsiwR Δmsmeg_3910 and ΔpafBC ΔsiwR double knock-outs were made as described above however, instead of Msm SMR5 cells, ΔsiwR or ΔpafBC cells were used as parent strain.
For ΔsiwR complementation studies, various integrative plasmids were cloned. The pattp plasmids were amplified without the ccdB gene through PCR. The insert siwR was amplified from genomic Msm DNA containing the 60 bp upstream region and 100 bp downstream region of the siwR gene. The insert and plasmid backbone were then ligated using Gibson assembly (NEBuilder HiFi DNA Assembly Master Mix, NEB). Half of the Gibson assembly reaction mix was transformed into NEB5α cells and plated onto LB plates containing apramycin (50 µg/mL). Transformants were verified through sequencing (Microsynth). Based on this integrative plasmid (pattp-siwR) multiple variants were amplified: a siwR variant with point mutations in the HTH domain (pattp-siwR R38A, R42A) and a siwR variant with point mutations in the WYL domain (pattp-siwR R204A, R207A). The constructs were produced using the Q5 Site-Directed Mutagenesis kit (NEB) followed by one-step DpnI digest, kinase and T4 ligation reaction using KLD enzyme mix (NEB) and transformed into NEB5α cells and plated onto LB plates containing apramycin (50 µg/mL). Again, transformants were verified through sequencing (Microsynth). Finally, electro-competent ΔsiwR mutant cells were co-transformed (2.5 kV) with 200 ng of the pattp integrative plasmids and 200 ng of a plasmid expressing the L5 integrase. Transformed cells were recovered immediately in 5 mL 7H9 medium supplemented with 0.2% glycerol and 0.05% Tween-80 and plated onto 7H10 plates supplemented with 0.5% glycerol and apramycin (25 µg/mL).
6-Carboxyfluorescein (FAM) labeled DNA (see Supplementary Table 1) was annealed with a complementary DNA fragment (Microsynth). Annealing was achieved by heating the two complementary DNA fragments in presence of annealing buffer (50 mM Tris-HCl pH 7.5, 50 mM NaCl, 1 mM EDTA) to 95 °C and lowering the temperature by 1 °C every minute until 20 °C. Then, 5 nM FAM-labeled dsDNA was incubated with 50 nM to 1600 nM protein in presence of binding buffer (5 mM HEPES-KOH pH 7.0, 30 mM NaCl, 2.5% Ficoll 400 K, 1% glycerol) for 15 min at 37 °C. The samples were then loaded onto a 1% agarose 0.5x TB gel and run for 10 min in 0.5x TB buffer at 200 V at room temperature. Finally, FAM-labeled DNA was visualized using the Azure Sapphire imager (Azure Biosystems).
Generally, all Msm cells were grown in 7H9 supplemented with 0.2% glycerol and 0.05% Tween-80 unless stated otherwise. For RT-qPCR analysis, cells were grown to OD600 = 0.5 and treated with either MMC (80 ng/mL), zeocin (200 µg/mL), H2O2 (0.2–5 mM), diamide (10 mM) or exposed to UVC (10 mJ/cm2). After 30 more min at 37 °C, cells were harvested (4000 x g, 15 min, 4 °C) and pellets stored at −20 °C. All experiments were performed in triplicates or quadruplicates.
In addition, Msm cells were stressed in liquid culture with 1 mM or 10 mM H2O2. For this, cells were diluted to an OD600 of 0.05–0.01 and grown for 6 h at 37 °C whilst shaking (150 rpm). Then H2O2 was added and growth further monitored. Each experiment was performed at least three times.
Growth curves were obtained using a spectrophotometer. Pre-cultures were diluted to an OD600 of 0.005 in 7H9 supplemented with 0.2% glycerol and 0.05% Tween-80 in 25 mL in 100 mL Erlenmeyer flasks. The flasks were incubated at 37 °C whilst shaking at 150 rpm. The OD600 was measured at the indicated time points.
7H9 medium supplemented with 0.2% glycerol and 0.05% Tween-80 containing zeocin or MMC at the indicated concentration was inoculated with Msm cells to an OD600 of 0.005. After a 24-h incubation at 37 °C, resazurin was added to a final concentration of 20 µg/mL. The plates were then incubated at room temperature for 3 days. Reduction of resazurin to resorufin by oxidoreductases in metabolically active cells causes a color change. Resorufin absorbance at 605 nm was measured and raw values were normalized within each dataset.
First, RNA was extracted using the SPLIT RNA extraction kit (Lexogen) according to the manufacturer’s instruction. Briefly, cell pellets were resuspended in 400 µL IB buffer and lysed by bead-beating for 30 s at 5 ms−1 (Omni International). Lysates were then centrifuged (20,000 × g, 15 min, 4 °C) and the soluble fraction transferred to a 2 mL phase-lock tube. Subsequently, 400 µL phenol solution pH 4.3 were added and the phase-lock tubes inverted 5 times. 150 µL AB buffer were added and mixed by pipetting before adding 200 µL 1-bromo-3-chloropropane and vortexing the tubes. After a 2 min incubation at RT, the samples were centrifuged (12,000 × g, 2 min, RT) and the upper phase decanted into a 2 mL tube. 1.75x volumes of 2-propanol were added to the upper phase and mixed by vortexing. Then, the samples were loaded onto the provided purification columns and centrifuged (12,000 × g, 2 min, RT) and washed with buffer WB. Finally, the samples were eluted with EB buffer. To get rid of DNA, the RNA was treated with Turbo DNAse (FisherScientific) for 30 min at 37 °C. DNase was inactivated using DNase inactivation reagent and separated from the RNA by centrifugation (10k xg, 90 s RT). RNA was then transcribed to cDNA using the SuperScriptII reverse transcriptase (Invitrogen). One qPCR reaction contained 10 µL KAPA SYBR (Roche), 2.5 µL of 5 µM HPLC-purified oligos (Microsynth) and 10 ng cDNA with a final volume of 20 µL in white 96 well PCR plates (BioRad). Each reaction was performed in technical triplicates. The PCR reaction consists of 35 cycles of 10 s at 95 °C, 10 s at 60 °C, 12 s at 72 °C (BioRad CFX96). To ensure sample integrity, melting curves were measured after the 35 cycles by increasing the temperature to 95 °C. Negative controls such as non-template (NTC) and non-reverse-transcriptase (NRT) controls were run in parallel. The housekeeping genes sigA and rpoB were used as reference genes. CT values were automatically calculated by the BioRad software and the ΔCT or ΔΔCT method were used to calculate gene expression ratios where appropriate.
Various forms of ssDNA as well as ssRNA were used for anisotroby binding experiments (Supplementary Table 1). Also, annealed, FAM labeled dsDNA promoter motif and Sumo-SiwR were used for anisotropy binding experiments. Two complementary ssDNA strands were annealed in 10 mM HEPES-KOH pH 7.5, 50 mM NaCl, 1 mM EDTA by heating to 95 °C for 10 min and then decreasing the temperature by 1 °C every minute until 25 °C is reached. 10 nM of the FAM-dsDNA was mixed with increasing concentrations of Sumo-SiwR in black 96-well half-plates in a reaction volume of 30 µl (5 mM HEPES-KOH pH 7, 30 mM NaCl, 5 mM DTT). The reaction was incubated for 15 min at 37 °C. Then, the intensity of the emission parallel and perpendicular to the excitation polarization was measured using a Biotek Synergy 2 plate reader (excitation: 490 nm, emission 520 nm). The anisotropy was calculated using the following formula (r=frac{{I}_{parallel }-{I}_{perp }}{{I}_{parallel }+2* {I}_{perp }}). The different concentrations of Sumo-SiwR were plotted against the anisotropy and the curve was fitted using the following formula for the Sumo-SiwR-promoter interaction:
For the ssDNA-Sumo-SiwR interaction studies, the curve was fitted using the following formula:
For evaluation of secondary structure content 1 mg/mL of protein was measured on a Jasco J-710 spectropolarimeter in a Hellma QS Macro Cell cuvette with a 1 mm path length. Three scans from wavelength 205 nm–260 nm were taken and the mean residue ellipticity was calculated using the following formula θMRW = (frac{theta * 100* M}{c* d* n}). (θ = measured ellipticity, M = molecular weight in g/mol, c = protein concentration in g/L, d = thickness of cuvette in cm, n = number of amino acids).
Analysis and graphs were performed in the GraphPad Prism 9.3.1 software. Data are represented as mean with standard deviation derived from biological replicates as described in the respective figure legends.
Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.
All data are contained in the paper or in the associated supplementary information files. Source data underlying graphs and uncropped images of gels are provided in Supplementary Data 1.
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We thank the Weber-Ban group members for critically reading the manuscript and fruitful discussions. The authors are supported by the Swiss National Science Foundation (SNSF Grant 310030_185250 and 310030_215606). We are also grateful for the support of the Functional Genomics Center Zurich with acquisition and evaluation of mass-spectrometry data. We thank Markus Seeger for providing the pFLAG_attP plasmid backbone for the integrative plasmid work.
Institute of Molecular Biology and Biophysics, ETH Zurich, 8093, Zurich, Switzerland
Lena Maria Leone Keller, Kim Flattich & Eilika Weber-Ban
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L.M.L.K. and E.W.B. conceived and planned the study. L.M.L.K. and K.F. performed experiments and analyzed the data. L.M.L.K. and E.W.B. wrote and revised the manuscript.
Correspondence to Eilika Weber-Ban.
The authors declare no competing interests.
Communications Biology thanks Stephane Rety and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Primary Handling Editors: Jonathan Perreault, Christina Karlsson-Rosenthal and Tobias Goris.
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Keller, L.M.L., Flattich, K. & Weber-Ban, E. Novel WYL domain-containing transcriptional activator acts in response to genotoxic stress in rapidly growing mycobacteria. Commun Biol 6, 1222 (2023). https://doi.org/10.1038/s42003-023-05592-6
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