New Results

The ChiS family DNA-binding domain contains a cryptic helix-turn-helix variant

Catherine A. Klancher, George Minasov, Ram Podicheti, Douglas B. Rusch, Triana N. Dalia, Karla J. F. Satchell, Matthew B. Neiditch, Ankur B. Dalia
doi: https://doi.org/10.1101/2020.11.18.389122

Abstract

Sequence specific DNA-binding domains (DBDs) are conserved in all domains of life. These proteins carry out a variety of cellular functions, and there are a number of distinct structural domains already described that allow for sequence-specific DNA binding, including the ubiquitous helix-turn-helix (HTH) domain. In the facultative pathogen Vibrio cholerae, the chitin sensor ChiS is a transcriptional regulator that is critical for the survival of this organism in its marine reservoir. We have recently shown that ChiS contains a cryptic DBD in its C-terminus. This domain is not homologous to any known DBD, but it is a conserved domain present in other bacterial proteins. Here, we present the crystal structure of the ChiS DBD at a resolution of 1.28 Å. We find that the ChiS DBD contains an HTH domain that is structurally similar to those found in other DNA binding proteins, like the LacI repressor. However, one striking difference observed in the ChiS DBD is that the canonical tight “turn” of the HTH is replaced with an extended loop containing a β-sheet, a variant which we term the “helix-sheet-helix”. Through systematic mutagenesis of all positively charged residues within the ChiS DBD, we show that residues within and proximal to the ChiS helix-sheet-helix are critical for DNA binding. Finally, through phylogenetic analyses we show that the ChiS DBD is found in diverse Proteobacterial proteins that exhibit distinct domain architectures. Together, these results suggest that the structure described here represents the prototypical member of the ChiS-family of DBDs.

Importance Regulating gene expression is essential in all domains of life. This process is commonly facilitated by the activity of DNA-binding transcription factors. There are diverse structural domains that allow proteins to bind to specific DNA sequences. The structural basis underlying how some proteins bind to DNA, however, remains unclear. Previously, we showed that in the major human pathogen Vibrio cholerae, the transcription factor ChiS directly regulates gene expression through a cryptic DNA binding domain. This domain lacked homology to any known DNA-binding protein. In the current study, we determined the structure of the ChiS DNA binding domain (DBD) and find that the ChiS-family DBD is a cryptic variant of the ubiquitous helix-turn-helix (HTH) domain. We further demonstrate that this domain is conserved in diverse proteins that may represent a novel group of transcriptional regulators.

Introduction

The intestinal pathogen Vibrio cholerae natively resides in the aquatic environment and can cause disease if ingested in the form of contaminated food or drinking water. In the aquatic environment, V. cholerae commonly associates with the chitinous surfaces of crustacean zooplankton (1). Chitin is an abundant source of carbon and nitrogen for marine bacteria, including V. cholerae (2, 3). In addition, chitin serves as a cue to induce horizontal gene transfer by natural transformation in this species (4). Thus, Vibrio-chitin interactions are critical for this facultative pathogen to thrive and evolve in its environmental reservoir.

Chitin is sensed in V. cholerae by the hybrid histidine kinase ChiS (5–7). In response to chitin, ChiS activates the expression of the chitin utilization program. This regulon includes the chb operon, which is required for the uptake and degradation of the chitin disaccharide chitobiose. In a recent study, we showed that unlike most histidine kinases, ChiS is capable of directly binding to DNA to regulate the expression of the chb operon (5). This finding was particularly surprising because ChiS is not predicted to encode a DNA-binding domain via primary sequence homology (BLAST (8)) or structural predictions (Phyre2 (9)). In the current study, we sought to understand the structural basis for ChiS DNA binding. To that end, we determined the structure of the ChiS DBD and found that it encodes a distinct variant of the canonical helix-turn-helix domain, which we term a “helix-sheet-helix”.

Results and Discussion

The C-terminus of ChiS (ChiS1024-1129) is sufficient to bind Pchb

Previous work from our group demonstrates that ChiS is a noncanonical hybrid histidine kinase that contains a DBD at its C-terminus (Fig. 1A) (5). In that study, we found that the C-terminal 106 amino acids of ChiS (ChiS1024-1129) was necessary and sufficient to bind to the chb promoter in vivo. We further showed that ChiS binds directly to two binding sites within the chb operon promoter (Pchb) to activate the expression of this locus. To confirm that ChiS1024-1129 was sufficient to bind DNA, we purified this domain and tested DNA-binding activity in vitro by electrophoretic mobility shift assays (EMSAs). We found that ChiS1024-1129 bound to a wildtype Pchb promoter probe, but not to a probe in which the two ChiS binding sites were mutated, suggesting that this domain is sufficient to bind to DNA in a sequence-specific manner (Fig. 1B and Fig. S1). Thus, based on our in vivo and in vitro analysis, we refer to ChiS1024-1129 as the ChiS DBD.

Figure S1.
Figure S1. Diagrams of EMSA probes used in this study.

A) Promoter map of chb with the region of Pchb used for the EMSAs shown in Figure 1B indicated. ChiS binding sites (CBSs) were left intact (black text, solid line) or mutated (white text, dotted line). B) Promoter map of chb with the region of Pchb used for the EMSA shown in Figure 4C and Figure S3. CBS 1 was mutated in all probes used.

Figure 1.
Figure 1. The C-terminus of ChiS (ChiS1024-1129) is sufficient to bind Pchb.

A) Diagram of the domain architecture for the hybrid histidine kinase ChiS. ChiS contains a histidine kinase (HK) domain, a receiver domain (Rec), a PAS domain, and a domain that does not have homology to known domains. Residues 1024-1129 were previously shown to be sufficient to bind Pchb in vivo (5). B) A fragment of the ChiS C-terminus (ChiS1024-1129) was purified and assessed for DNA binding activity by EMSA. Purified protein was incubated with the indicated Cy5-labeled 60 bp probes containing sequence from Pchb encompassing the two ChiS binding sites (CBSs). A promoter map with the region of the promoter used for EMSAs are diagrammed in Figure S1A. The concentration of ChiS used (from left to right) was 0 nM, 25 nM, 50 nM, 100 nM, 200 nM, and 400 nM. The probe sequence was WT (Pchb WT) or the CBSs were both mutated (Pchb Mutated). Data are representative of two independent experiments.

Identification of positively charged residues in the ChiS DBD that are critical for DNA binding and transcriptional activation of Pchb

As mentioned above, ChiS is not predicted to encode a DNA-binding domain based on in silico searches (i.e., BLAST (8) and Phyre2 (9)). To characterize interactions between the ChiS DBD and DNA, we first tried to identify residues important for DNA binding. The positively charged residues arginine (R) and lysine (K) commonly interact with the negatively charged DNA backbone (10). Thus, we mutated every R and K residue in the ChiS DBD to a glutamine (Q), to ablate their charge but maintain, to a reasonable extent, the steric properties of the side group.

To determine how these mutations affected ChiS activity, we introduced them into full-length FLAG-tagged ChiS (5), and assessed the ability of each mutant to bind to DNA in vivo (by chromatin immunoprecipitation, or ChIP) and to activate Pchb expression (using a Pchb-GFP reporter). We found that all mutations to the ChiS DBD reduced Pchb-GFP activation to varying degrees (Fig. 2). Most mutants were able to facilitate partial activation of Pchb and correspondingly partially enriched for Pchb by ChIP, indicating that they were binding to the promoter in vivo. Some mutants (R1068Q, R1074Q, K1078Q, R1090Q, and R1092Q) did not bind to Pchb DNA in vivo and resulted in complete loss of Pchb expression. Importantly, all mutants still produced ChiS protein as assessed by Western blot analysis (Fig. S2). Collectively, these data identify a subset of positively charged residues in the ChiS DBD that are critical for DNA binding and, subsequent transcriptional activation of the chb operon.

Figure S2.
Figure S2. Mutations to the ChiS DNA binding domain does not prevent expression of ChiS.

Strains expressing the indicated ChiS-FLAG point mutations were assessed for expression by Western blot with anti-FLAG and anti-RpoA (loading control) antibodies. Asterisks above ChiS point mutants indicate the mutations found to be critical for the DNA binding activity of ChiS in Figure 2.

Figure 2.
Figure 2. Identification of positively charged residues in the ChiS DBD that are critical for DNA binding and transcriptional activation of Pchb.

All lysines and arginines in the ChiS DNA binding domain were individually mutated to a glutamine and ChiS was assessed for (1) transcriptional activation of a Pchb-GFP reporter (green bars; left Y-axis) and (2) ChiS binding to Pchb in vivo by chromatin immunoprecipitation (ChIP) (black bars; right Y-axis). ChiS can be activated with its native inducer, chitin, or by deletion of its periplasmic regulator, CBP; here, ChiS was activated artificially by deleting CBP. Data are the result of at least three independent biological replicates and are shown as the mean ± SD. Statistical markers indicated directly above bars indicate comparisons to the WT. Statistical comparisons were made by one-way ANOVA with Tukey’s Posttest. ***, p < 0.001; ** p < 0.01; NS, not significant.

Structure of the ChiS DNA binding domain reveals a variant of the helix-turn-helix

We next sought to determine the structure of the ChiS DBD to further explore how ChiS interacts with DNA. Since no structures for close sequence homologs were available in the Protein Data Bank (PDB) to serve as search models for molecular replacement, we used the Single-wavelength Anomalous Dispersion (SAD) technique to determine initial phases. Selenomethionine was used as the replacement for methionine. Anomalous data were collected from a single crystal (Tables S1 and S2). The crystal diffracted to 1.28 Å resolution and belonged to the orthogonal C2221 space group with unit cell parameters of a=51.91Å, b=78.61Å, c=72.37Å, α=β=γ=90.00°. There was one polypeptide chain in the asymmetric unit. The structure includes 105 out of 106 residues of the protein (1024 – 1128), two uncleavable residues of the purification tag, four sulfate ions (SO42−), one 2-(2-hydroxyethyloxy)ethanol molecule (PEG), two formic acids molecules (FMT) and 200 water molecules (HOH). Only the C-terminal E1129 was disordered in the structure and was not included in the final model.

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Table S1.

Data collection and processing

View this table:
Table S2.

Structure refinement

The structure of the ChiS DBD revealed that it contains a fold that is reminiscent of the canonical helix-turn-helix (HTH) used by diverse DNA-binding proteins (Fig. 3A-B). The basic HTH domain consists of a trihelical bundle where the second and third helices encompass the namesake “helix-turn-helix” (11). The two helices that compose the HTH are connected via a relatively short linker that forms a sharp turn, which is a characteristic feature of this domain. Helix 3 from the HTH is generally inserted into the major groove of DNA, thus forming the principle DNA-protein interface. Alignment of the trihelical bundle from ChiS with the DNA-bound structure of the LacI repressor (PDB: 1EFA (12); RMSD of modeled Cα carbons = 3.514) revealed a similar spatial arrangement for each helix (Fig. 3C). Notably, however, the ChiS HTH has an extended loop between helix 2 and helix 3 that forms a beta sheet (Fig. 3B-D). Structural insertion between these helices is not typical; thus, the sheet found here is a distinct variant of the HTH which we refer to as a “helix-sheet-helix”.

Figure 3.
Figure 3. Structure of the ChiS DNA binding domain reveals a variant of the helix-turn-helix.

A) Domain architecture of the ChiS DNA binding domain. The primary sequence of the ChiS DBD (S1024-S1128) is shown. Helices are depicted as cylinders, while sheets are depicted as arrows. B) Crystal structure of the ChiS DNA binding domain. The structural elements are colored coded as depicted in the primary sequence in B. C) Alignment of the ChiS trihelical bundle (rainbow) with the LacI triehlical bundle bound to the LacI operator site (PDB: 1EFA; pink). Alignment of alpha carbons gave an RMSD of 3.514. D) Cartoon representations of the trihelical bundle from LacI and ChiS. Helices are labeled with nomenclature presented in Aravind, et. al. (11). E) Structure of the ChiS trihelical bundle colored to represent B-factor. Helices found in the helix-sheet-helix motif (H2 and H3) are indicated.

Alignment of the ChiS DBD to LacI also revealed that the sheet within the ChiS helix-sheet-helix domain runs along the major groove (Fig. 3C, 4A), though it sterically conflicts with the DNA bases. This may suggest that the ChiS DBD takes on a slightly different conformation when bound to DNA. Consistent with this idea, the beta sheet has the highest B-factor (a measure of structural motion) in the ChiS DBD structure, indicating that it is relatively flexible (Fig. 3E). Thus, we speculate that this beta sheet is stabilized in the major groove when the ChiS DBD is bound to DNA. The unique helix-sheet-helix feature of the ChiS C-terminal domain may also explain why it was not previously identified as a DBD by structure prediction algorithms like Phyre2.

Figure 4.
Figure 4. ChiS may bind to intrinsically bent DNA.

A) Model of the ChiS trihelical bundle bound to double-stranded DNA from the alignment shown in Figure 3C. Side chains for the residues critical for DNA binding (R1068, R1074, K1078, R1090, and R1092) are shown and indicated. B) Diagram of the 7 distinct 230 bp probes used in C. ChiS binding site 1 (CBS 1) was mutated (white text) and ChiS binding site 2 (CBS 2) was left intact (black text). CBS 2 was shifted by 30 bp between each probe. C) The DNA probes diagrammed in B were labeled with Cy5 and separated by native PAGE in the absence of ChiS protein.

ChiS may bind to intrinsically bent DNA

Above, we identified five residues (R1068, R1074, K1078, R1090, and R1092) that were critical for the ChiS DBD to bind to DNA. Mapping these residues onto the ChiS DBD structure revealed that all five residues were found within the trihelical bundle that forms the helix-sheet-helix (Fig. 4A), which is consistent with this domain playing a critical role in DNA binding. Specifically, these residues were located in the beta sheet of the helix-sheet-helix (R1068), helix 3 (R1074, K1078), and helix 1 (R1090, R1092).

Most residues critical for DNA binding activity (R1068, R1074, K1078, R1090) were in close proximity to DNA on our modeled alignment; however, one residue (R1092), was distant from the DNA (Fig. 4A). Many transcription factors bend DNA upon binding to their target site (13, 14). Thus, one possible explanation for the critical role of R1092 is that the Pchb promoter is bent when bound by ChiS, which would allow for R1092 to come into close contact with DNA. To test this idea, we carried out a classic in vitro gel mobility shift assay to test DNA bending (15). This assay operates on the basis that the location of a bend within a DNA molecule alters its mobility during native PAGE analysis (16, 17). DNA probes that contain a bend in the middle of the probe exhibit the lowest mobility, while probes with the bend closer to one end show the highest mobility. Thus, we designed seven DNA probes of equal length that gradually shifted the position of the ChiS binding sites within the chb promoter (Fig. 4B and Fig. S1). First, we ran these probes in the absence of ChiS protein and found that they ran at different mobilities where the probes with the ChiS binding sites in the middle exhibited the lowest mobility (Fig. 4C). This suggested that the chb promoter likely has an intrinsic bend that is centered around the ChiS binding sites. The mobility pattern observed for these DNA probes did not change when incubated with the purified ChiS DBD (Fig. S3), suggesting that binding of the DNA probe by ChiS does not further bend the promoter. We propose that the chb promoter has an intrinsic bend, which may allow residues in the ChiS DBD, like R1092, to directly interact with DNA. The intrinsic bend found in the chb promoter may increase the affinity of ChiS for this region of DNA; indeed, DNA bending has been shown to increase the affinity of certain transcription factors for their DNA binding site (18).

Figure S3.
Figure S3. ChiS protein does not further bend the Pchb promoter.

The probes shown in Figure S1B were incubated in the absence (No ChiSDBD) or presence (+ChiSDBD) of 400 nM ChiSDBD.

The ChiS family DNA binding domain is associated with variable domain arrangements in diverse proteins

Above, we show that the ChiS DBD represents a cryptic variant of an HTH domain. As noted previously, the ChiS DBD is found in proteins other than homologs of ChiS (5). To more fully catalog proteins that contain this domain, we generated a profile Hidden Markov Model (HMM) to the ChiS DBD and screened for its presence among eubacterial genomes. A profile HMM is a position-specific scoring system that can effectively encode the variation in a training set of representative peptide sequences, and then find similar sequences from a much larger and more distantly related dataset compared to tools that do not require training, such as BLAST (19, 20).

This analysis revealed that the ChiS DBD is present in diverse Proteobacterial genomes (Spreadsheet S1). The vast majority of hits from our search were direct homologs of ChiS (3242/3829 = 84.7%), however, many proteins exhibited distinct domain architectures (587/3829 = 15.3%) (Fig. 5A). Strikingly, the ChiS DBD was found exclusively at the C-terminus in all of these proteins and was commonly associated with sensory domains (Fig. 5A). Furthermore, the helix-sheet-helix is highly conserved across these diverse proteins (Fig. 5B, Spreadsheet S1), and even the most dissimilar ChiS DBD homolog (MAC43155.1, bit score of 43.5; 22.6% identical, 43.4% similar to the ChiS DBD) still threaded (9) remarkably well onto the trihelical bundle of the ChiS DBD structure (RMSD of modeled Cα carbons = 0.002) (Fig. S4). Thus, it is tempting to speculate that ChiS is the founding member for a new group of DNA-binding transcription factors whose activity are regulated by diverse sensory inputs.

Figure S4.
Figure S4. The most dissimilar ChiS DBD homolog threads onto the trihelical bundle of the ChiS DBD structure.

The sequence of the ChiS-family DBD from MAC43155.1 was threaded onto the crystal structure of the ChiS DBD using Phyre2 (9). Alignment of alpha carbons gave an RMSD of 0.002.

Figure 5.
Figure 5. The ChiS family DBD is found in diverse proteins with distinct domain architectures among Proteobacterial genomes.

A) Diagrams of the most abundant protein architectures containing the ChiS family DBD. Protein domains shown are HAMP, Histidine Kinase (HK), Receiver (Rec), Per-Arnt-Sim (PAS), Tetratricopeptide Repeat (TPR), 7 Transmembrane Receptors with Diverse Intracellular Signaling Modules (7TMR-DISM), and the ChiS family DNA binding domain (ChiS DBD). For a complete list of hits containing the indicated architectures see Spreadsheet S1. B) Alignment of the primary sequences of the ChiS family DBD in the indicated proteins are shown. Residues in black are identical, while those in gray are similar. The sequence for helix 1 (H1), helix 2 (H2) and helix 3 (H3) are boxed in teal.

Materials & Methods

Bacterial strains and culture conditions

All V. cholerae strains used in this study are derived from the El Tor strain E7946 (21). V. cholerae strains were grown in LB medium and on LB agar supplemented when necessary with carbenicillin (20 μg/mL), kanamycin (50 μg/mL), spectinomycin (200 μg/mL), and/or trimethoprim (10 μg/mL). See Table S3 for a detailed list of mutant strains used in this study.

View this table:
Table S3. Strains used in this study.

Generating mutant strains

V. cholerae mutant constructs were generated using splicing-by-overlap extension exactly as previously described (22). See Table S4 for all of the primers used to generate mutant constructs in this study. Mutant V. cholerae strains were generated by chitin-dependent natural transformation and cotransformation exactly as previously described (23). Mutant strains were confirmed by PCR and/or sequencing.

View this table:
Table S4. Primers used in this study.

Cloning, protein production and purification

The chiS1024-1129 (VC0622) construct was cloned into an AmpR pET15b-based vector using the FastCloning method (24). This vector appended a TEV cleavable 6x His tag onto the N-terminus of ChiS1024-1129. Vector and inserts were amplified using the primers listed in Table S4. The plasmid was transformed into E. coli BL21(DE3) (Magic) cells (25) and the protein was expressed in M9 media (High Yield M9 Se-Met media, Medicilon Inc.). The starting overnight culture was grown in LB medium supplemented with 130 μg/mL ampicillin and 50 μg/mL kanamycin at 37°C and 220 rpm. The next day, M9 medium supplemented with 200 μg/mL ampicillin and 50 μg/mL kanamycin was inoculated with the overnight culture (1:100 dilution) and incubated at 37°C and 220 rpm. Protein expression was induced at OD600=1.8-2.0 by the addition of 0.5 mM isopropyl β-d-1-thiogalactopyranoside and the culture was further incubated at 25°C, 200 rpm for 14 hours (26). The cells were harvested by centrifugation at 6,000 xg for 10 minutes, resuspended to 0.2 g/mL in lysis buffer (50 mM Tris pH 8.3, 0.5 M NaCl, 10% glycerol, 0.1% IGEPAL CA-630) and frozen at −30°C until purification.

Frozen pellets were thawed and sonicated at 50% amplitude, in a 5s on, 10s off cycle for 20 min at 4°C. The lysate was clarified by centrifugation at 18,000 xg for 40 minutes at 4°C and the supernatant was collected. The protein was purified in one step by IMAC followed by size exclusion chromatography using ÅKTAxpress system (GE Healthcare) as previously described with some modifications (27). The cell extract was loaded into a His-Trap FF (Ni-NTA) column with loading buffer (10 mM Tris-HCl pH 8.3, 500 mM NaCl, 1 mM Tris (2-carboxyethyl) phosphine (TCEP), 5% glycerol) and the column was washed with 10 column volumes of loading buffer and 10 column volumes of washing buffer (10 mM Tris-HCl pH 8.3, 1 M NaCl, 25 mM imidazole, 5% glycerol). Protein was eluted with elution buffer (10 mM Tris pH 8.3, 500 mM NaCl, 1 M imidazole), loaded onto a Superdex 200 26/600 column, separated in loading buffer, collected, and analyzed by PAGE. The 6x His tag was cleaved with recombinant TEV protease in a ratio of 1:20 (protein:protease) overnight at room temperature. The cleaved protein was separated from uncleaved protein, recombinant TEV protease, and 6x His tag peptide by Ni-NTA-affinity chromatography using loading buffer followed by loading buffer with 25 mM imidazole. The cleaved protein was collected in the flow-through in both the loading buffer and the loading buffer with 25 mM imidazole. Both fractions were analyzed by PAGE for 6x His tag cleavage, concentrated to 6-8 mg/mL, and set up for crystallization.

Crystallization, data collection, structure solution and refinement

The protein from both fractions (collected in flow through and in 25 mM imidazole) was set up at 6-8 mg/mL in loading buffer containing 0 or 500 mM NaCl as 2 μL crystallization drops (1 μL protein: 1 μL reservoir solution) in 96-well plates (Corning) using commercial Classics II, PACT and JCSG+ (QIAGEN) crystallization screens. Diffraction quality crystal of the protein collected with 25 mM imidazole grown from the condition with 0.2 M lithium sulfate, 0.1 M Bis-Tris, pH 5.5, 25%(w/v) PEG 3350 (Classics II, #74) was flash frozen in liquid nitrogen for data collection.

The crystals were screened, and data were collected at the Life Sciences-Collaborative Access Team (LS-CAT) beamline F at the Advanced Photon Source (APS) of the Argonne National Laboratory. A total of 300 diffraction images were indexed, integrated and scaled using HKL-3000 (28). The structure was determined with the HKL3000 structure solution package using anomalous signal from selenomethionine (Se-Met). The initial model went through several rounds of refinement in REFMAC v. 5.8.0258 (29) and manual corrections in Coot (30). The water molecules were generated using ARP/wARP (31) and ligands were added to the model manually during visual inspection in Coot. Translation–Libration–Screw (TLS) groups were created by the TLSMD server and TLS corrections were applied during the final stages of refinement. MolProbity was used for monitoring the quality of the model during refinement and for the final validation of the structure. The structure was deposited to the Protein Data Bank (https://www.rcsb.org/) with the assigned PDB code 7KPO.

Electrophoretic mobility shift assay (EMSA)

Binding reactions contained 10 mM Tris HCl pH 7.5, 1 mM EDTA, 10 mM KCl, 1 mM DTT, 50 μg/mL BSA, 0.1 mg/mL salmon sperm DNA, 5% glycerol, 1 nM of a Cy5 labeled DNA probe, and purified ChiS DBD at the indicated concentrations (diluted in 10 mM Tris pH 7.5, 10 mM KCl, 1 mM DTT, and 5% glycerol). Reactions were incubated at room temperature for 20 minutes in the dark, then electrophoretically separated on polyacrylamide gels in 0.5x Tris Borate EDTA (TBE) buffer at 4°C. Gels were imaged for Cy5 fluorescence on a Typhoon-9210 instrument. Cy5-labeled Pchb probes were made by Phusion PCR, where Cy5-dCTP was included in the reaction at a level that would result in incorporation of 1–2 Cy5 labeled nucleotides in the final probe as previously described (22).

Measuring GFP reporter fluorescence

GFP fluorescence was determined essentially as previously described (34). Briefly, single colonies were picked and grown in LB broth at 30°C for 18 hours. Cells were then washed and resuspended to an OD600 of 1.0 in instant ocean medium (7 g/L; Aquarium Systems). Then, fluorescence was determined using a BioTek H1M plate reader with excitation set to 500 nm and emission set to 540 nm.

Chromatin immunoprecipitation (ChIP)-qPCR assays

ChIP assays were carried out exactly as previously described (5). Briefly, overnight cultures were diluted to an OD600 of 0.08 and then grown for 6 hours at 30°C. Cultures were crosslinked using 1% paraformaldehyde, then quenched with a 1.2 molar excess of Tris. Cells were washed with PBS and stored at −80°C overnight. The next day, cells were resuspended in lysis buffer (1x FastBreak cell lysis reagent (Promega), 50 μg/mL lysozyme, 1% Triton X-100, 1 mM PMSF, and 1x protease inhibitor cocktail; 100x inhibitor cocktail contained the following: 0.07 mg/mL phosphoramidon (Santa Cruz), 0.006 mg/mL bestatin (MPbiomedicals/Fisher Scientific), 1.67 mg/mL AEBSF (DOT Scientific), 0.07 mg/mL pepstatin A (Gold Bio), 0.07 mg/mL E64 (Gold Bio)) and then lysed by sonication, resulting in a DNA shear size of ~500 bp. Lysates were incubated with Anti-FLAG M2 Magnetic Beads (Sigma), washed to remove unbound proteins, and then bound protein-DNA complexes were eluted off with SDS. Samples were digested with Proteinase K, then crosslinks were reversed. DNA samples were cleaned up and used as template for quantitative PCR (qPCR) using iTaq Universal SYBR Green Supermix (Bio-Rad) and primers specific for the genes indicated (see Table S4 for primers) on a Step-One qPCR system. Standard curves of genomic DNA were included in each experiment and were used to determine the abundance of each amplicon in the input (derived from the lysate prior to ChIP) and output (derived from the samples after ChIP). Primers to amplify rpoB served as a baseline control in this assay because ChiS does not bind this locus. Data are reported as ‘Fold Enrichment’, which is defined as the ratio of Pchb / rpoB found in the output divided by the same ratio found in the input.

Western blot analysis

Strains were grown as described for ChIP assays, pelleted, resuspended, and boiled in 1x SDS PAGE sample buffer (110 mM Tris pH 6.8, 12.5% glycerol, 0.6% SDS, 0.01% Bromophenol Blue, and 2.5% β-mercaptoethanol). Proteins were separated by SDS polyacrylamide gel electrophoresis, then transferred to a PVDF membrane, and probed with rabbit polyconal α-FLAG (Sigma) or mouse monoclonal α-RpoA (Biolegend) primary antibodies. Blots were then incubated with α-rabbit or α-mouse HRP conjugated secondary antibodies, developed using Pierce ECL 529 Western Blotting Substrate (ThermoFisher), and imaged on a ProteinSimple Fluorchem E instrument.

Bioinformatic identification of eubacterial proteins with putative ChiS DBD domains

The DBD sequence segments from the protein sequences of seven ChiS DNA binding domain homologs (THB81618.1, OGG93021.1, OUR95018.1, WP_084205767.1, ODU31202.1, WP_070993003.1, WP_078715702.1) (5) were aligned using MUSCLE version 3.8.31 (35). The resulting multiple sequence alignment was turned into a profile HMM which was searched against the eubacterial subset (taxonomy id: 2) of NCBI non-redundant protein sequence database using HMMER version 3.2.1 (http://hmmer.org/), requiring the alignment length to be at least 90. Among the hits, proteins sequences tagged “partial” in their FASTA headers were excluded. Domain architectures for the remaining hits were obtained from the NLM conserved domain database (36). Any protein hits with regions aligned to the DNA binding domain HMM overlapping with known annotated functional domains were excluded. The resulting ChiS DBD homolog protein sequences were clustered using cd-hit ver. v4.8.1-2019-0228 (37) (parameters: -M 0 -g 1 -s 0.8 -c 0.4 -n 2 -d 500). Clusters identified by cd-hit were further grouped together by manually analyzing the domain architecture of hits as shown in Fig. 5A. Only clusters containing 10 or more representatives were grouped, while the remaining proteins were left unassigned. For a list of all proteins containing a putative ChiS DBD, see Spreadsheet S1.

Acknowledgements

We thank Dipankar Sen, Julia van Kessel, and Ryan Chaparian for helpful discussions. We thank Olga Kiryukhina, Grant Wiersum, Ivgeniia Dubrovska, and Ludmilla Shuvalova for assistance on protein crystallization. This work was supported by grant R35GM128674 from the National Institutes of Health (to ABD) and, in part, with Federal funds from the Department of Health and Human Services. National Institutes of Health, National Institute of Allergy and Infectious Diseases under Contract No. HHSN272201700060C (to KJFS). This research used resources of the Advanced Photon Source, a U.S. Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under Contract No. DE-AC02-06CH11357. Use of the LS-CAT Sector 21 was supported by the Michigan Economic Development Corporation and the Michigan Technology Tri-Corridor (Grant 085P1000817). This research was supported in part by Lilly Endowment, Inc., through its support for the Indiana University Pervasive Technology Institute.

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Posted November 18, 2020.
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Supplementary Material

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The ChiS family DNA-binding domain contains a cryptic helix-turn-helix variant
Catherine A. Klancher, George Minasov, Ram Podicheti, Douglas B. Rusch, Triana N. Dalia, Karla J. F. Satchell, Matthew B. Neiditch, Ankur B. Dalia
bioRxiv 2020.11.18.389122; doi: https://doi.org/10.1101/2020.11.18.389122
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