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Article

Structural Analysis of Bacillus subtilis Sigma Factors

1
Department of Chemistry, King’s College London, Britannia House, 7 Trinity Street, London SE1 1DB, UK
2
Department of Biological Sciences, Mount Holyoke College, 50 College Street, South Hadley, MA 01075, USA
*
Author to whom correspondence should be addressed.
These authors contributed equally to this work.
Microorganisms 2023, 11(4), 1077; https://doi.org/10.3390/microorganisms11041077
Submission received: 9 March 2023 / Revised: 16 April 2023 / Accepted: 17 April 2023 / Published: 20 April 2023
(This article belongs to the Special Issue Assembly, Structure, and Germination of Bacterial Spores)

Abstract

:
Bacteria use an array of sigma factors to regulate gene expression during different stages of their life cycles. Full-length, atomic-level structures of sigma factors have been challenging to obtain experimentally as a result of their many regions of intrinsic disorder. AlphaFold has now supplied plausible full-length models for most sigma factors. Here we discuss the current understanding of the structures and functions of sigma factors in the model organism, Bacillus subtilis, and present an X-ray crystal structure of a region of B. subtilis SigE, a sigma factor that plays a critical role in the developmental process of spore formation.

1. Introduction

Sigma (σ) factors are bacterial protein modules that plug into RNA polymerase (RNAP) to recruit the enzyme to specific programs of gene transcription via recognition of promoter DNA and the subsequent initiation of transcription [1,2]. The vast majority of σ factors are members of the σ70 protein superfamily, which is subdivided into four classes based upon their extent of conservation and the presence/absence of the conserved σ domains (σ1.1, σ2, σ3, and σ4 connected by flexible loop regions) that mediate interactions with RNAP and/or promoter DNA [3]. All bacteria employ an essential primary σ factor (Class I) that directs transcription of housekeeping genes [4]. Many bacteria also possess alternative σ factors (Classes II, III, and IV) that compete for binding to RNAP and redirect it to transcribe sets of genes required for adaptive responses [5]. Hence, the suite of genes expressed in a bacterial cell can be globally reprogrammed simply by manipulating the levels, activity, or availability of alternative σ factors [1].
The model organism, Bacillus subtilis, uses a set of well-characterised sigma factors to orchestrate different phases of its lifecycle [6]. As well as interacting with RNA polymerase, sigma factors can bind to many other proteins, including anti-sigma factors which prevent their binding to RNA polymerase in circumstances when their transcription programmes are not required [7]. There are also proteins that compete with sigma factors for binding to the same site on RNAP in another regulatory mechanism [8]. In isolation, sigma factors include several intrinsically disordered regions which allow the domains to wrap around protein partners including RNAP and anti-sigma factors [9]. This flexibility has precluded finding an experimental structure solution for most sigma factors; up until 2002, the only available Bacillus sigma factor structure was a stretch of fifty amino acids from SigF, derived from the extremophile Bacillus stearothermophilus. This was solved in complex with the anti-sigma factor SpoIIAB (PDB: 1L0O [10]) by X-ray crystallography to 2.9 Å resolution [10]. The first solved fragments of B. subtilis sigma factors only emerged in 2017 with domains from SigW (PDB: 5WUQ [11]) and SigA (PDB: 5MWW [12]), as outlined in Table 1. With the refinements supplied by AlphaFold2 [13], the predicted structures of all B. subtilis sigma factors are now publicly available in the AlphaFold Protein Structure Database [14].
All but one of the B. subtilis sigma factors belong to the σ70 factor family, with the only outlier, SigL, being a member of the σ54 factor family (see Table 1). In B. subtilis there are four sigma factors known to control sporulation—the process in which the bacteria become long-lived dormant spores to survive stress conditions (reviewed in [15,16]). This happens through a genetically choreographed sequence of events in which a cell divides asymmetrically and the smaller cell (forespore) is engulfed by the larger (mother cell), which ultimately lyses after supporting the spore through its metabolic shutdown and building it a sturdy outer shell. The sigma factor “puppeteers” involved in this process are SigF in the forespore and SigE in the mother cell at the early phases; then, these are replaced by SigG and SigK, respectively, as sporulation progresses [17]. Many of the remaining sigma factors are involved in the response to external and environmental conditions (e.g., acid stress), forming the group of extracytoplasmic function (ECF) sigma factors [18].
In earlier work we solved the structure of CsfB/Gin, an anti-sigma factor that acts on both SigG and SigE during sporulation [19,20]. Here we present an experimentally solved X-ray crystal structure of SigE residues 17–133 and review all of the available experimentally-solved and AlphaFold-predicted B. subtilis sigma factor structures.
Table 1. Sigma factor family members in B. subtilis (Data compiled from SubtiWiki [21], PDBe [22], AlphaFold Database [14] and other sources as indicated. For AlphaFold structures σ1 (turquoise), σ2 (slate blue), σ3 (olive), σ4 (raspberry)). Note the unlikely helix prediction for SigI (black).
Table 1. Sigma factor family members in B. subtilis (Data compiled from SubtiWiki [21], PDBe [22], AlphaFold Database [14] and other sources as indicated. For AlphaFold structures σ1 (turquoise), σ2 (slate blue), σ3 (olive), σ4 (raspberry)). Note the unlikely helix prediction for SigI (black).
σ FactorMolecular Weight (kDa)Domains & GroupExperimentally Solved B. subtilis StructuresFunction SummaryAlphaFold Structure Prediction
SigA42.80σ1, σ2, σ3, σ4
Group I
σ1.1 NMR 5mww [12]
Cryo-EM BmrR transcription activation complex 7ckq [23]
HousekeepingMicroorganisms 11 01077 i001
SigB29.99σ2, σ3, σ4
Group III
N/AStress response [24,25]Microorganisms 11 01077 i002
SigD29.32σ2, σ3, σ4
Group III
N/AChemotaxis & flagellar gene expression [26]. Expression of autolysin [27]Microorganisms 11 01077 i003
SigE27.55σ2, σ3, σ4
Group III
NMR σ2 chimera with GTAAAA 5or5 Early stages of sporulation (Mother cell only) [28]Microorganisms 11 01077 i004
SigF29.22σ2, σ3, σ4
Group III
N/AEarly stages of sporulation (Forespore only) [29]Microorganisms 11 01077 i005
SigG29.92σ2, σ3, σ4
Group III
N/ALate stages of sporulation (Forespore only) [30]Microorganisms 11 01077 i006
SigH25.30σ2, σ3, σ4
Group III
N/AExpression of genes associated with transition from growth phase to stationary phase. Initiation of sporulation [31]Microorganisms 11 01077 i007
SigI29.04σ2, σ3, σ4
Group III
N/AHeat shock response [32]Microorganisms 11 01077 i008
SigK33.00σ2, σ3, σ4
Group III
N/ALate stages of sporulation (Mother cell only) [33]Microorganisms 11 01077 i009
SigL49.54σ54 familyN/ACold shock response [34]. Assimilation of nitrogen sources/amino acid catabolism [35]Microorganisms 11 01077 i010
SigM19.26σ2, σ4
Group IV
N/AExtracytoplasmic function (ECF) [36].
Halophilic gene expression [37]
Microorganisms 11 01077 i011
SigV19.57σ2, σ4
Group IV
N/AECF.
Present during outgrowth from endospore, but knockout does not inhibit outgrowth [38]
Microorganisms 11 01077 i012
SigW21.57σ2, σ4
Group IV
Full length crystal structure with anti-sigma
factor RsiW 5wur [11], 5wuq [11];
crystal structure of σ4 bound to DNA 6jhe [39]
ECF.
Alkaline shock response [40]
Microorganisms 11 01077 i013
SigX23.03σ2, σ4
Group IV
N/AECF.
Regulation of peptidoglycan synthesis [41]?
Modification of cell envelope and resistance to antimicrobial peptides [42].
Microorganisms 11 01077 i014
SigY/yxlB21.21σ2, σ4
Group IV
N/AECF.
Production of and resistance to antibiotics (sublancin) through maintenance of Spβ prophage [43].
Microorganisms 11 01077 i015
SigZ20.57σ2, σ4
Group IV
N/AECFMicroorganisms 11 01077 i016
Xpf19.95σ2, σ4
Group IV
N/APositive control factor (PCF). Linked to PBSX prophage—induces bacterial death in response to DNA damage: “Bacterial suicide” [44]Microorganisms 11 01077 i017
YlaC20.78σ2, σ4
Group IV
N/AECF.
Resistance to oxidative stress [45]
Microorganisms 11 01077 i018
SigO-RsoA22.54; 9.00σ2, σ3; σ4
Group III
N/AResponse to acid stress [46]. Expression also induced by antibiotics that target the cell wallMicroorganisms 11 01077 i019

2. Materials and Methods

2.1. Plasmids and Cloning

The gene for CsfBA48E, cloned into bacterial expression plasmid pNIC28 (which adds TEV-cleavable N-terminal His tag), and the SigE17–239 in bacterial expression plasmid pET28-TxrA (including His tag, thioredoxin fusion protein and TEV cleavage site) were used as described in [19]. For SigE17–239 designed for this study, a BamHI/XhoI-digested PCR fragment covering SigE codons 17–133 was ligated into BamHI/XhoI-digested pET28-TxrA plasmid (described above).

2.2. Protein Sequences

SigE17–239
MKLGLKSDEVYYIGGSEALPPPLSKDEEQVLLMKLPNGDQAARAILIERNLRLVVYIARKFENTGINIEDLISIGTIGLIKAVNTFNPEKKIKLATYASRCIENEILMYLRRNNKIRSEVSFDEPLNIDWDGNELLLSDVLGTDDDIITKDIEANVDKKLLKKALEQLNEREKQIMELRFGLVGEEEKTQKDVADMMGISQSYISRLEKRIIKRLRKEFNKMV
SigE17–133
MKLGLKSDEVYYIGGSEALPPPLSKDEEQVLLMKLPNGDQAARAILIERNLRLVVYIARKFENTGINIEDLISIGTIGLIKAVNTFNPEKKIKLATYASRCIENEILMYLRRNNKIR
CsfBA48E
MDETVKLNHTCVICDQEKNRGIHLYTKFICLDCERKVISTSTSDPDYEFYVKKLKSIHTPPLYS

2.3. Purification Buffers

Cell lysis buffer: 50 mM HEPES pH 7.5, 300 mM NaCl, 0.5 mM TCEP, 5% glycerol, 5 mM imidazole, 1 mg/mL lysozyme, 10 µg/mL DNaseI, 10 mM MgCl2, 2 cOmplete EDTA-free protease inhibitor cocktail tablets, and 2 mM PMSF.
HisTrap Buffer A: 50 mM HEPES pH 7.5, 300 mM NaCl, 0.5 mM TCEP, 5% glycerol, and 10 mM imidazole.
HisTrap Buffer B: 50 mM HEPES pH 7.5, 300 mM NaCl, 0.5 mM TCEP, 5% glycerol, and 250 mM imidazole.
SP Sepharose Buffer A: 50 mM Tris-HCl pH 8.0, 10 mM NaCl, and 0.5 mM TCEP.
SP Sepharose Buffer B: 50 mM Tris-HCl pH 8.0, 1 M NaCl, and 0.5 mM TCEP.

2.4. Protein Expression & Purification

All SigE constructs were expressed in the T7 Express lysY/Iq E.coli strain from New England Biolabs (NEB C3013I). Cells were cultured at 37 °C 220 rpm in an LB growth medium until they reached an OD600 of 0.6. Alternatively, for downstream NMR studies SigE was expressed in an M9 Minimal medium supplemented with 0.7 g/L 15N-NH4Cl, and for carbon experiments also 2 g/L 13C-glucose. At OD600 0.6, the cells were induced via the addition of isopropyl β-d-1-thiogalactopyranoside (IPTG) to a final concentration of 0.5 mM. Following induction, the cells were incubated at 22 °C 220 rpm overnight to achieve the expression of SigE. Cells were harvested via centrifugation at 4000× g for 30 min and the pellets were snap frozen in liquid nitrogen prior to storage at −80 °C.
All SigE constructs were purified according to the following procedure. Cell pellets derived from 2 L of culture were resuspended in 30 mL lysis buffer. The pellets were thoroughly resuspended and then homogenized via ultrasonication on ice using an 80% amplitude and twelve cycles of 5 s “on” and 25 s “off”. Debris was removed from the lysate by ultracentrifugation at 105,000× g for 30 min and passed through a 0.2 µm filter. SigE constructs were purified via immobilized metal affinity chromatography (IMAC). The protein was applied to a HisTrap 5 mL FF column (Cytiva) that had been pre-washed and equilibrated with HisTrap Buffer A. HisTrap Buffer A was passed through the column until the 280 nm trace returned to baseline. At this point the protein was isolated via isocratic elution using the following steps: 4 column volumes (CV) 5% HisTrap Buffer B, 4 CV 10% HisTrap Buffer B, and 4 CV 100% HisTrap Buffer B. Fractions were analysed by SDS-PAGE; those containing SigE as identified through Coomassie staining were dialysed against HisTrap buffer A (containing no imidazole) in the presence of TEV protease overnight at 4 °C. The cleaved SigE was further purified via a reverse Ni-NTA step in which the material was applied to a pre-equilibrated HisTrap 5 mL FF column, although the flow-through was collected. The flow-through was concentrated using a VivaSpin centrifugal concentrator device to <5 mL and applied to a 120 mL Superdex 75 column that had been pre-equilibrated with SP Sepharose Buffer A. Fractions were analysed by SDS-PAGE and then those containing SigE were pooled. Due to the protein clinging to the Vivaspin concentrators at high concentrations, the final concentration step was performed using cation exchange chromatography (SP Sepharose). The protein was applied to a 1 mL HiTrap SP column pre-equilibrated with SP Sepharose Buffer A. The protein was then eluted with 100% SP Sepharose Buffer B into 1 mL fractions. The highest concentration fractions were dialysed against 1 L of the relevant buffer according to downstream usage.
CsfBA48E was produced as described [19]. In short, the protein was expressed in BL21(DE3)pLysS cells using an LB growth medium. Induction was accomplished by adding IPTG to 0.5 mM and incubating at either 37 °C for 4 h or 18 °C overnight. Protein purification was accomplished using IMAC and subsequent SEC.

2.5. X-ray Crystallography

All protein preparations were dialysed into 50 mM HEPES pH 7.5, 150 mM NaCl, and 0.5 mM TCEP prior to setting up crystallisation trials.
SigE17–133 formed large cuboid crystals in coarse screen condition SaltRx well H7 (0.5 M potassium thiocyanate, 0.1 M Tris pH 8.5) with a protein:liquor ratio of 1:1. These crystals were grown at 7 mg/mL at 16 °C and were discovered after 4 months. The crystals were cryoprotected using 5% glycerol in 3.33 M AmSO4. Data were collected at Diamond Light Source beamline I03 at a wavelength of 0.9795 Å with diffraction extending to 2.02 Å. Data were processed in space group C 2 2 21 with the unit cell dimensions: a = 8187, b = 164.94, c = 98.89, α = 90.00, β = 90.00, γ = 90.00. Indexing and integration were carried out using xia2 with DIALS [47], and POINTLESS and AIMLESS were used for the merging and scaling of the data [48]; all of this was conducted on ISpyB [49]. Data were cut to 2.38 Å based upon the CC1/2 [50]. The SIMBAD automated pipeline was used to ensure the data did not represent a crystal contaminant [51]. The MrBUMP [52] automated pipeline was used to solve the structure via molecular replacement using PDB entry: 3UGO [53] as a model. Refinement was carried out using Refmac5 [54] with non-crystallographic symmetry (NCS) applied and some automated model building was carried out in Coot [55]. Model building was also aided by PDBredo [56]. Final refinements were carried out in Phenix [57]. The final Rwork was 0.20 and the final Rfree was 0.25.

2.6. NMR

All protein was dialysed into 50 mM HEPES pH 7.5, 150 mM KCl, and 0.5 mM TCEP prior to NMR data collection.
For chemical shift perturbation studies, 1H-15N HSQC spectra were collected for 100 μM 15N-labelled SigE17–133 alone and in the presence of a 2-fold excess CsfBA48E. Spectra were collected at 298 K on a 700 MHz Bruker AVANCE NMR spectrometer equipped with a TXI cryoprobe. Incomplete triple resonance datasets were obtained for both 500 μM SigE17–133 alone and in complex with CsfBA48E using a 950 MHz spectrometer. All spectrometers were controlled using TopSpin 3. Data processing was performed using NMRPipe [58]/NMRDraw and analyzed using CcpNMR Analysis [59] v2.2.

3. Results

3.1. Construct Design

Initially we produced almost full-length B. subtilis SigE (residues 17–239, only missing the initial prosequence that maintains SigE in an inactive state before processing [60]), but we found that it degraded to a smaller domain that remained stable over time, as observed by SDS-PAGE and 2D NMR (Figure 1A). Smaller constructs were designed based on predicted domain boundaries, and SigE17–133 was the variant that successfully yielded diffracting crystals.
The first 27 residues of SigE are a pro-sequence that keeps the protein in the inactive state and ensures its localization to the mother cell [61]. These residues get cleaved during sporulation by SpoIIGA to activate the protein. The constructs used in this study lack the first 17 residues as this maintains activity in vivo without requiring processing by SpoIIGA [60].
The A48E mutation of CsfB was utilised as this variant is protected from proteolytic degradation while retaining anti-sigma factor function [19].

3.1.1. SigE17–133 NMR

The SigE17–133 construct displayed a relatively well-dispersed 1H-15N HSQC spectrum (Figure 1). Upon titration with unlabelled CsfBA48E (a previously published stability mutant [19]), many chemical shift perturbations were evident, confirming the interaction. Surprisingly, when bound to CsfB, SigE gave rise to better quality spectra despite the increased size of the complex from 13.3 kDa (SigE alone) to 20.8 kDa (13.3 kDa + 7.5 kDa, SigE plus CsfB) (Figure 1B). This is likely in part a consequence of SigE becoming increasingly ordered, leading to greater spectral distribution and reduced peak overlap, as well as possibly improved exchange characteristics. Unfortunately, triple resonance datasets were consistently of poor quality with many peaks missing, so it was not possible to obtain a backbone assignment for SigE17–133. Since the complex comprising 15N-labelled SigE17–133 and CsfBA48E displays sharper peaks than in the HSQC spectrum of isolated SigE17–133, we also collected a suite of triple resonance data for the complex on a 950 MHz spectrometer. However, this also proved inadequate for straightforward assignment.

3.1.2. SigE17–133 Structure Solution

Although the SigE17–133 construct was used for crystallisation, the structure we obtained (Figure 2A) was an ensemble of six almost identical (overlaying with RMSD from 0.164–0.310 Å over 52–67 atoms; Figure 2B) monomers each comprising residues 52–133, present in the asymmetric unit (deposited with PDB ID: 8B3Z). Crystallographic parameters are shown in Table 2. Crystals grew over a period of 4–6 months and the protein likely lost some N-terminal amino acids during this process. It is also possible that these residues were too flexible to give rise to discernible electron density. The structure is a classic four helix-turn-helix core found in all sigma factor σ2 domains and covers regions σ2.1 (55–78), σ2.2 (79–97), and σ2.3 (98–117) of SigE, which includes the binding sites for both CsfB and the −10 promoter DNA sequence for transcriptional activation (Figure 3A).
Dali searches identified the closest structural homology for B. subtilis SigE52–133 with E. coli RpoS (PDB: 5H6X [62], chain A, RMSD of 0.91 Å) also solved as an isolated domain, and E. coli RpoD (PDB: 4ZH3 [63], chain F, RMSD: 0.99 Å) and M. tuberculosis SigA (PDB: 6OY5 [64], chain F, RMSD: 1.16 Å) both solved as part of larger holoenzyme complexes. The solved structure aligns very well (RMSD: 0.51 Å) with the AlphaFold prediction for SigE (Figure 3B), with Alphafold providing a slight helix overprediction in the loop between two helices [65].

3.2. AlphaFold Prediction of B. Subtilis Sigma Factor Structures

All of the current AlphaFold-predicted structures for isolated B. subtilis sigma factors are shown in Table 1. AlphaFold predicts the core structured regions of the sigma factors with high levels of confidence. However, significant regions of the sigma factors are highly flexible in order to accommodate binding to partner proteins. Unsurprisingly, these regions are associated with less confident model building by AlphaFold and often feature unlikely helices [65] (see in particular the prediction for SigI), which is likely a feature of the artificial intelligence being mostly trained on crystal structures.

4. Discussion

B. subtilis is the best studied Gram positive bacterium and is widely used as a model organism to investigate bacterial cell and developmental biology [16]. Gaining a greater understanding of the processes that underpin genetic regulation in this model system has broader ramifications for antibiotic development and understanding hospital superbugs. In order to do this, however, we require biophysical and structural insight into the behaviour of the multitude of different sigma factors that modulate gene expression [1].
With the advent of AlphaFold, we are now able to access reliable models for the individual domains of most B. subtilis sigma factors [14]. The positioning of the connecting loops, especially when wrapped around binding partners in large assemblies, is the next structural frontier, and is well on the way to being cracked both experimentally through large high resolution cryo-EM structures [66] and computationally with AlphaFold multimer [67], which is becoming more and more sophisticated at a rate of knots. These developments are unprecedented given the high flexibility of sigma factors and the difficulty associated with the expression and purification of many of them.
Here, we have presented a crystal structure of the SigE sigma factor from B. subtilis and have compared its structure to those others solved experimentally or predicted by AlphaFold. It shares a similar structure with the other members of the σ70 family in B. subtilis. Whilst there are few experimentally solved structures of the sigma factors and their domains in B. subtilis, there is wider coverage of the various domains from sigma factors across bacterial species. These structures, combined with the models from AlphaFold, provide a good overall picture of how sigma factors operate to regulate gene expression in bacteria.
Of those B. subtilis sigma factor structures that have been experimentally solved (summarised in Table 1), two were determined via NMR (5MWW [12] and 5OR5 (unpublished)), three were achieved using X-ray crystallography (5WUR [11], 5WUQ [11], and 6JHE [39]), and there was a single available structure of a complex solved by cryo-EM (7CKQ [23]). These systems showcase the relative strengths and weaknesses of each biophysical technique and expose different insights into the respective sigma factors. The NMR structure of the σ1.1 domain from SigA (5MWW [12]) revealed that the domain was unexpectedly compact and, surprisingly, showed similarity to the δ domain of the RNAP [12]. The overlay of this NMR structure also matched well with the AlphaFold model of SigA; the terminal regions of the NMR construct were unsurprisingly heavily disordered, but the core helices of the domain aligned well (Figure 4A). The match between the SigA AlphaFold model and the cryo-EM structure was also good; however, there appeared to be some movement in the cryo-EM structure, which was likely the result of SigA being incorporated into the BmrR-RNAP-DNA complex [23] (Figure 4B). This structure illuminates the sigma factor in its broader context in a way that would likely not be feasible with any other technique. The unpublished NMR structure of the σ2 domain of SigE (5OR5 [12]) was likewise a good match, with the corresponding region in the full length AlphaFold model; however, bigger differences are seen around the turns (Figure 4C). This is fairly unsurprising as these regions are typically modelled with lower confidence by AlphaFold [65], whilst NMR ensembles are well-suited to explore the conformational space of highly dynamic regions. Similarly, the AlphaFold model of SigW overlaps extremely well with the crystal structure of the SigW bound to its anti-sigma factor partner protein, RsiW (5WUR) [11] (Figure 4D). This can likely be explained by AlphaFold being trained predominantly on a library of crystal structures, and so it may be biased towards rigid, well-ordered structures. The crystal structure of the σ4 domain of SigW bound to the −35 region of DNA (6JHE [39]) also mapped well onto the AlphaFold model (Figure 4E).
These few examples of experimentally determined sigma factor structures from B. subtilis serve to highlight how well AlphaFold generally handles these highly dynamic systems. This also suggests that the AlphaFold models of those sigma factors lacking experimentally determined structures have excellent utility so long as they are interpreted with caution due to AlphaFold’s propensity to occasionally overbuild helices, most notably observed in the case of SigI (see Table 1). The study of sigma factors will likely also reap the rewards from the ascendancy of cryo-EM, which is better able to peer into more complex and dynamic systems than crystallography. This is exemplified by the cryo-EM structure 7CKQ [23] of the BmrR-RNAP-DNA complex; as time goes on we expect to see many further structures of sigma factors in this DNA-bound context. These combined advances in experimental and computational structural biology will hopefully rapidly translate into corresponding advances in our understanding of bacterial molecular biology.

Author Contributions

Conceptualization, R.L.I., K.M.C., N.J.E. and A.H.C.; methodology, K.M.C. and N.J.E.; validation, N.J.E., J.H.T., J.M.H. and B.A.H.; formal analysis, K.M.C., N.J.E., J.H.T. and R.L.I.; investigation, K.M.C. and N.J.E.; resources, R.L.I.; data curation, N.J.E.; writing—original draft preparation, R.L.I.; writing—review and editing, all authors; visualization, J.H.T. and R.L.I.; supervision, R.L.I.; project administration, R.L.I.; funding acquisition, R.L.I. and A.H.C. All authors have read and agreed to the published version of the manuscript.

Funding

RLI’s work on Bacillus subtilis has been funded by BBSRC grants: BB/N006267/1; BB/R006091/1; BB/S006877/1. AHC was supported by National Institutes of Health grants DP2 GM105439 and R15 GM101559. The authors would like to thank Diamond Light Source for beamtime (proposal mx13597; King’s College London BAG). NMR experiments were performed at the Centre for Biomolecular Spectroscopy, King’s College London, established with a Capital Award from the Wellcome Trust. This work was supported by the Francis Crick Institute through provision of access to the MRC Biomedical NMR Centre. The Francis Crick Institute received core funding from Cancer Research UK (FC001029), the UK Medical Research Council (FC001029), and the Wellcome Trust (FC001029). The 950 MHz NMR facility at the University of Oxford was funded by the Wellcome Trust Joint Infrastructure Fund and the E. P. Abraham Fund.

Data Availability Statement

We have deposited the X-ray crystal structure of SigE17-133 in the Protein Data Bank in Europe [22] with PDB ID: 8B3Z.

Acknowledgments

The authors thank the staff of beamline I03 at Diamond Light Source for assistance with X-ray diffraction data collection and Andrew Atkinson for help with NMR experiments at the Centre for Biomolecular Spectroscopy, King’s College London. We thank J.M. Pérez-Cañadillas (Rocasolano Physical Chemistry Institute, Madrid, Spain) for providing a modified version of the pET28 vector and plasmid encoding TEV protease.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. NMR spectra of B. subtilis SigE. (A) Overlaid 1H-15N HSQC spectra of 15N-labelled SigE17–239 (blue) and SigE17–133 (orange) constructs. The broad peaks indicate that the former had likely aggregated and/or degraded. The substantial overlap with the peaks of the latter construct, and the lack of many additional peaks, imply that the SigE17–239 sample now resembles SigE17–133. (B) 1H-15N HSQC spectra of 15N-labelled SigE17-133 alone (turquoise) and in the presence of a two-fold excess CsfBA48E (red). Chemical shift perturbation clearly indicates interaction between the two proteins.
Figure 1. NMR spectra of B. subtilis SigE. (A) Overlaid 1H-15N HSQC spectra of 15N-labelled SigE17–239 (blue) and SigE17–133 (orange) constructs. The broad peaks indicate that the former had likely aggregated and/or degraded. The substantial overlap with the peaks of the latter construct, and the lack of many additional peaks, imply that the SigE17–239 sample now resembles SigE17–133. (B) 1H-15N HSQC spectra of 15N-labelled SigE17-133 alone (turquoise) and in the presence of a two-fold excess CsfBA48E (red). Chemical shift perturbation clearly indicates interaction between the two proteins.
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Figure 2. Crystal structure of SigE. (A) View of the crystallographic asymmetric unit showing six copies of SigE residues 52–133, each representing a classic helix-turn-helix domain. (B) Alignment of the six units of SigE 52–133 from the asymmetric unit. This overlay shows some minor differences between the different biological units (in the same colours as shown in (A), with slight structural deviations in the flexible loop regions).
Figure 2. Crystal structure of SigE. (A) View of the crystallographic asymmetric unit showing six copies of SigE residues 52–133, each representing a classic helix-turn-helix domain. (B) Alignment of the six units of SigE 52–133 from the asymmetric unit. This overlay shows some minor differences between the different biological units (in the same colours as shown in (A), with slight structural deviations in the flexible loop regions).
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Figure 3. Structural alignments of SigE. (A) Structural overlay of SigE52–133 crystal structure (cream) with Thermus aquaticus RNAP sigma factor A (light blue; PDB: 3UGO [53]) bound to a −10 promoter element ssDNA oligo (TACAAT). The structures align with RMSD: 0.99 over 72 residues, indicating how SigE52–133 likely interacts with the −10 promoter in B. subtilis. (B) SigE52–133 crystal structure (cream) overlaid with the AlphaFold model of full-length SigE (light blue) from B. subtilis (UniProt ID: P06222). Regions of helix overprediction (residues 77–80, 104–106) by AlphaFold are indicated in red.
Figure 3. Structural alignments of SigE. (A) Structural overlay of SigE52–133 crystal structure (cream) with Thermus aquaticus RNAP sigma factor A (light blue; PDB: 3UGO [53]) bound to a −10 promoter element ssDNA oligo (TACAAT). The structures align with RMSD: 0.99 over 72 residues, indicating how SigE52–133 likely interacts with the −10 promoter in B. subtilis. (B) SigE52–133 crystal structure (cream) overlaid with the AlphaFold model of full-length SigE (light blue) from B. subtilis (UniProt ID: P06222). Regions of helix overprediction (residues 77–80, 104–106) by AlphaFold are indicated in red.
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Figure 4. Structural alignments of experimentally solved (partial) B. subtilis sigma factors (cream) with the equivalent AlphaFold models (light blue): (A) Overlay of the NMR structure of σ1.1 domain of SigA (5MWW) [12] with the AlphaFold model of SigA. (B) SigA structure excised from the cryo-EM (7CKQ [23]) structure of the BmrR transcription activation complex [23] overlaid with the full-length AlphaFold model of SigA. (C) Overlay of the NMR structure of SigE σ2 domain (5OR5; unpublished) with the equivalent AlphaFold model of SigE. (D) 2.6 Å crystal structure of SigW (5WUR [11]) excised from the co-crystal complex with the anti-sigma factor RsiW [11] overlaid with the AlphaFold model of SigW; structured regions are a near perfect match. (E) Overlay of the 3.1 Å crystal structure (6JHE [39]) domain bound to the −35 element DNA [39] (hidden) with the AlphaFold model of full-length SigW.
Figure 4. Structural alignments of experimentally solved (partial) B. subtilis sigma factors (cream) with the equivalent AlphaFold models (light blue): (A) Overlay of the NMR structure of σ1.1 domain of SigA (5MWW) [12] with the AlphaFold model of SigA. (B) SigA structure excised from the cryo-EM (7CKQ [23]) structure of the BmrR transcription activation complex [23] overlaid with the full-length AlphaFold model of SigA. (C) Overlay of the NMR structure of SigE σ2 domain (5OR5; unpublished) with the equivalent AlphaFold model of SigE. (D) 2.6 Å crystal structure of SigW (5WUR [11]) excised from the co-crystal complex with the anti-sigma factor RsiW [11] overlaid with the AlphaFold model of SigW; structured regions are a near perfect match. (E) Overlay of the 3.1 Å crystal structure (6JHE [39]) domain bound to the −35 element DNA [39] (hidden) with the AlphaFold model of full-length SigW.
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Table 2. Crystallographic parameters.
Table 2. Crystallographic parameters.
ProteinSigE
BeamlineDiamond Light Source I03
Data processing xia2 dials
Resolution Range63.39–2.379 (2.464–2.379) Å
Space GroupC 2 2 21
Unit Cell81.953 165.143 98.930
90.00 90.00 90.00
Total Reflections 356785 (16578)
Unique Reflections 27360 (1349)
Multiplicity 13.0 (12.3)
Completeness 100 (99)%
Mean I/Sigma(I) 13.4 (1.8)
Wilson B-factor48.27
R-meas0.131 (2.143)
CC1/20.999 (0.851)
Reflections used in refinement27115
Reflections used for Rfree1332
Final Rwork0.198
Final Rfree0.256
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Collins, K.M.; Evans, N.J.; Torpey, J.H.; Harris, J.M.; Haynes, B.A.; Camp, A.H.; Isaacson, R.L. Structural Analysis of Bacillus subtilis Sigma Factors. Microorganisms 2023, 11, 1077. https://doi.org/10.3390/microorganisms11041077

AMA Style

Collins KM, Evans NJ, Torpey JH, Harris JM, Haynes BA, Camp AH, Isaacson RL. Structural Analysis of Bacillus subtilis Sigma Factors. Microorganisms. 2023; 11(4):1077. https://doi.org/10.3390/microorganisms11041077

Chicago/Turabian Style

Collins, Katherine M., Nicola J. Evans, James H. Torpey, Jonathon M. Harris, Bethany A. Haynes, Amy H. Camp, and Rivka L. Isaacson. 2023. "Structural Analysis of Bacillus subtilis Sigma Factors" Microorganisms 11, no. 4: 1077. https://doi.org/10.3390/microorganisms11041077

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