Recombinant Bacillus subtilis HTH-type transcriptional regulator immR (immR)

What is the structural organization of ImmR in Bacillus subtilis?

ImmR is a 127-residue intracellular protein (UniProt access code P96631) that functions as a transcriptional regulator. Its structural organization includes:

• An N-terminal DNA-binding domain (DBD) spanning residues M1-G63 with high confidence prediction (pLDDT = 96.7%)

• A compact pentahelical bundle (α1-α5) in the DBD, cohered by a central hydrophobic core

• Five helices connected by short linkers of 2-5 residues, with each helix approximately perpendicular to the preceding one

• Helices α2 and α3 corresponding to the "positioning helix" and "recognition helix" of the HTH motif engaged in double-stranded DNA binding

• A C-terminal domain (K64-E127) containing two large isolated α-helices (K64-K88 and E103-K126) predicted with lower confidence (pLDDT = 74.8%)

The crystal structure of the ImmR-DBD reveals a dimeric arrangement with an interface of 573 Ų, featuring nine hydrogen bonds and symmetric hydrophobic interactions between 11 residues of either molecule .

What is the role of ImmR in ICE Bs1 regulation?

ImmR plays a crucial role in regulating ICE Bs1, a 20 kb conjugative transposon in B. subtilis that contains over 20 genes and can be transferred to pathogenic bacteria:

• Acts as a transcriptional repressor that constitutively blocks transcription of ICE Bs1 genes by binding to six sites within the regulatory regions of both Pxis and PimmR promoters

• Ensures that a single stable copy of ICE Bs1 is maintained in the cell in a quiescent state under normal conditions

• Works in conjunction with the Int integrase and ImmA anti-repressor metallopeptidase to control ICE Bs1 mobility

• Prevents unnecessary excision and transfer of the transposon, which could be energetically costly to the cell

• Functions as part of a regulatory switch that activates only when DNA damage occurs or when potential recipient cells lacking the transposon are nearby

This regulation is critical because ICE Bs1 can be transferred to pathogenic bacteria like B. anthracis and L. monocytogenes, potentially contributing to the spread of genetic elements across species .

How do ImmR and ImmA function together as a regulatory system?

The ImmR-ImmA regulatory system operates as a precision switch controlling ICE Bs1 activity:

• ImmR binds to operator sites and represses transcription from both the Pxis and PimmR promoters

• ImmA is a metallopeptidase that acts as an anti-repressor, normally inactive under standard conditions

• When triggered by environmental signals (DNA damage or presence of potential recipient cells), ImmA becomes active

• Active ImmA specifically cleaves ImmR at the F95-M96 site in the linker between the two predicted C-terminal helices

• This proteolytic cleavage inactivates ImmR, causing the protein:DNA complex to dissociate

• With repression lifted, transcription of ICE Bs1 genes proceeds, leading to excision and transfer of the transposon

This mechanism provides a rapid and irreversible response to environmental signals, ensuring that horizontal gene transfer occurs only under appropriate conditions .

What structural similarities exist between ImmR and other transcriptional regulators?

ImmR shares significant structural homology with several transcriptional regulators in the "434 Cro family" of HTH-DBDs:

• Closest structural similarity is with SinR from B. subtilis, a repressor involved in sporulation inhibition

• Other structural relatives include C2 repressor of Salmonella bacteriophage P22 (PDB 1ADR), CylR2 of Enterococcus faecalis, and DdrO of Deinococcus geothermalis

• All these proteins share a similar arrangement of the first four helices in their DNA-binding domains

• Differences are primarily observed in the fifth helix, which varies in length and position

• SinR, CylR2, and DdrO display dimeric crystal structures equivalent to that of ImmR

The structural similarity to SinR is particularly notable, as it suggests ImmR may oligomerize to produce DNA-loop structures in a similar manner . The SinR complex structure has been used to construct a homology model for the DNA-complex of the ImmR-DBD dimer, indicating how the recognition helices would contact the DNA major groove .

How does horizontal gene transfer of ICE Bs1 impact bacterial communities?

ICE Bs1 transfer has significant implications for bacterial population dynamics and evolution:

• ICE Bs1 can be transferred from B. subtilis to pathogenic bacteria including B. anthracis, B. licheniformis, and L. monocytogenes

• This conjugative transposon contributes to genome plasticity across bacterial species

• Mobile genetic elements like ICE Bs1 can facilitate the spreading of antibiotic resistance and virulence factors

• B. subtilis itself is generally considered safe (GRAS) but can occasionally cause food poisoning in immunocompromised patients

• The regulated transfer of ICE Bs1 balances potential benefits of gene acquisition with the energetic costs of conjugation

The precise regulation by ImmR-ImmA ensures that horizontal gene transfer occurs only under specific conditions, such as DNA damage or proximity to potential recipient cells .

What experimental approaches are most effective for expressing and purifying recombinant ImmR?

Based on successful crystal structure determination, the following approach has proven effective:

Production Protocol:

• Recombinant overexpression in Escherichia coli

• Optimization of expression conditions (temperature, induction time, media composition)

• Cell lysis and initial clarification of lysate

Purification Strategy:

• Two-step chromatography purification process

  • First step: likely affinity or ion exchange chromatography

  • Second step: size exclusion chromatography for final purity

• Buffer optimization to maintain protein stability

Crystallization Considerations:

• Full-length ImmR crystals diffracted poorly (7-8 Å)

• Focus on the DNA-binding domain (residues 1-63) for structural studies

• Crystals of the DBD diffracted to approximately 2 Å, though with high mosaicity and anisotropy

Quality Assessment:

• SDS-PAGE for purity evaluation

• Mass spectrometry for identity confirmation

• DNA-binding assays for functional verification

Researchers should note that while full-length ImmR was produced, the crystals contained only the DNA-binding domain, suggesting potential proteolysis during crystallization or inherent flexibility in the C-terminal domain .

How can researchers design experiments to study the proteolytic regulation of ImmR by ImmA?

Investigating the ImmR-ImmA proteolytic regulation requires systematic approaches:

In vitro Cleavage Assays:

• Express and purify both ImmR and ImmA recombinantly

• Establish conditions for reproducible cleavage (buffer composition, pH, temperature)

• Monitor cleavage using SDS-PAGE and Western blotting

• Quantify cleavage kinetics under various conditions

• Use mass spectrometry to confirm cleavage at the F95-M96 site

Mutational Analysis:

• Generate point mutations around the F95-M96 cleavage site

• Create alanine scanning mutations across potential recognition sequences

• Design cleavage-resistant variants for functional studies

• Develop truncation constructs to determine minimal recognition elements

Structural Approaches:

• Attempt co-crystallization of ImmA with substrate peptides

• Use hydrogen-deuterium exchange mass spectrometry to map interaction interfaces

• Apply solution NMR to study dynamics of the cleavage region

Cellular Assays:

• Develop fluorescent protein-based reporters for monitoring cleavage in vivo

• Design split-reporter systems to detect protein-protein interactions

• Use single-cell microscopy to observe spatial and temporal aspects of cleavage

Understanding this proteolytic switch is critical for elucidating how bacteria control horizontal gene transfer in response to environmental signals .

What methods can be used to characterize the DNA-binding properties of ImmR?

A comprehensive characterization of ImmR-DNA interactions requires multiple complementary techniques:

Biochemical Assays:

• Electrophoretic Mobility Shift Assays (EMSA) with:

  • Natural target sequences from ICE Bs1 regulatory regions

  • Systematic mutations to identify critical bases

  • Competition experiments to determine relative affinities

• DNase I footprinting to precisely map protected regions

• Fluorescence anisotropy for quantitative binding measurements

Structural Approaches:

• X-ray crystallography of ImmR-DNA complexes

  • Design DNA oligonucleotides based on known binding sites

  • Screen multiple crystallization conditions

  • Consider using both full-length ImmR and isolated DBD

• Homology modeling based on the SinR-DNA complex structure

• Molecular dynamics simulations of predicted complexes

Functional Analysis:

• Reporter gene assays using natural and mutated promoter sequences

• In vivo DNA binding studies using ChIP-seq

• Testing putative binding residues (T17-E20, N29-N31, S33-Y35, R37, Y39-D43)

Data Analysis and Integration:

• Develop binding motif models from experimental data

• Compare with binding sites of related regulators like SinR

• Map identified binding sites to genomic context

The strong structural similarity to SinR provides a valuable framework for these studies, as the DNA-binding mechanism is likely conserved between these related transcriptional regulators .

How can structure-guided engineering be applied to create chimeric regulators based on ImmR?

Applying structure-guided engineering to create functional chimeric regulators requires precise domain definition and strategic design:

Domain Analysis and Selection:

• Define domain boundaries based on:

  • Crystal structure of ImmR-DBD (residues 1-63)

  • AlphaFold predictions for full-length ImmR

  • Comparison with homologous regulators like SinR

• Identify potential fusion partners with compatible structures

• Select domains that provide desired input sensing capabilities

Design Principles:

• Maintain intact secondary structure elements at fusion junctions

• Design appropriate linkers between domains:

  • Flexible glycine-serine linkers for minimal interference

  • Natural linkers from related proteins for functional compatibility

• Preserve critical functional residues:

  • DNA-binding residues in the HTH motif

  • Dimerization interface residues

  • Regulatory sites including the F95-M96 cleavage site

Optimization Strategies:

• Create and test multiple variants with:

  • Different domain boundaries

  • Various linker compositions

  • Strategic mutations at domain interfaces

• Apply directed evolution to select for optimal function

• Use structure-guided mutagenesis to fine-tune performance

Case Study Guidance:
The successful engineering of chimeric MerR-family regulators in B. subtilis provides a useful model :

• A constant DNA-binding domain from a Gram-positive donor was combined with variable metal-binding domains

• This approach ensured compatibility with the host's transcriptional machinery while harnessing diverse input sensing capabilities

• Structure-guided mutagenesis improved the functionality of hybrid proteins

This approach can be adapted for ImmR-based chimeras, potentially creating novel regulators responsive to different environmental signals while maintaining compatibility with B. subtilis gene expression machinery.

What biophysical techniques are most informative for studying dynamic aspects of ImmR function?

Understanding the dynamic aspects of ImmR function requires specialized biophysical techniques:

Solution-Based Methods:

• Nuclear Magnetic Resonance (NMR) spectroscopy:

  • Maps residue-specific dynamics

  • Detects conformational changes upon DNA binding

  • Identifies flexible regions not resolved in crystal structures

  • May be limited to individual domains due to size constraints

• Small-Angle X-ray Scattering (SAXS):

  • Provides low-resolution shape information in solution

  • Detects large-scale conformational changes

  • Complements crystallographic data

  • Useful for studying full-length ImmR where crystal structure is unavailable

Single-Molecule Techniques:

• Single-molecule FRET:

  • Monitors distances between labeled positions

  • Reveals conformational heterogeneity in the population

  • Can detect dynamic transitions between states

  • Useful for studying ImmR-DNA interactions in real-time

• Optical tweezers or magnetic tweezers:

  • Measure mechanical properties of ImmR-DNA complexes

  • Detect force-induced conformational changes

  • Can probe the energy landscape of binding and unbinding

Thermodynamic and Kinetic Methods:

• Isothermal Titration Calorimetry (ITC):

  • Measures thermodynamic parameters of binding

  • Determines stoichiometry and binding constants

  • Quantifies enthalpy and entropy contributions

• Surface Plasmon Resonance (SPR):

  • Measures association and dissociation kinetics

  • Determines equilibrium binding constants

  • Enables analysis of complex formation under various conditions

Advanced Mass Spectrometry:

• Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS):

  • Maps protein regions involved in DNA binding

  • Identifies regions with altered solvent accessibility

  • Detects conformational changes upon binding or proteolysis

• Native Mass Spectrometry:

  • Determines oligomeric states

  • Confirms complex formation with DNA

  • Detects protein modifications

These complementary approaches can provide a comprehensive understanding of how ImmR transitions between different functional states during its regulatory cycle .

How can computational approaches enhance understanding of ImmR structure-function relationships?

Computational methods offer powerful tools for exploring ImmR structure-function relationships:

Structural Prediction and Analysis:

• AlphaFold and related AI tools:

  • Have successfully predicted ImmR structure with high confidence for the DBD (pLDDT = 96.7%)

  • Can model full-length protein including more flexible C-terminal regions

  • Enable predictions of protein-protein interfaces

• Molecular Dynamics (MD) simulations:

  • Reveal dynamic behavior not captured in static crystal structures

  • Predict effects of mutations on stability and function

  • Model conformational changes upon DNA binding or proteolytic cleavage

  • Simulate dimerization and higher-order oligomerization

DNA-Binding Prediction:

• Docking approaches:

  • Predict ImmR-DNA complex structures

  • Screen multiple potential binding sites

  • Evaluate energetics of binding

• Sequence-based methods:

  • Identify potential binding motifs from genomic data

  • Compare with binding sites of related regulators

  • Predict effects of promoter mutations on regulation

Network Analysis:

• Systems biology approaches:

  • Model the complete ImmR-ImmA regulatory circuit

  • Predict system behavior under various conditions

  • Identify potential interactions with other cellular processes

Evolutionary Analysis:

• Sequence conservation mapping:

  • Identify functionally important residues across related species

  • Trace the evolution of ImmR and related regulators

  • Predict co-evolution with interaction partners

• Comparative genomics:

  • Analyze distribution and conservation of ICE Bs1 and related elements

  • Identify potential horizontal gene transfer events

  • Map evolutionary relationships between regulatory systems

The successful use of AlphaFold predictions to facilitate structure determination demonstrates the value of these computational approaches . Integration of computational methods with experimental data provides a more comprehensive understanding of ImmR function in regulating horizontal gene transfer.

What are the current challenges in developing synthetic regulatory circuits based on ImmR?

Developing synthetic regulatory circuits based on ImmR faces several key challenges:

Structural and Functional Challenges:

• Defining optimal domain boundaries for chimeric regulators

• Ensuring proper folding and stability of engineered proteins

• Maintaining appropriate oligomerization for function

• Preserving DNA binding specificity while altering regulatory inputs

• Balancing expression levels for optimal circuit performance

Regulatory Design Considerations:

• Engineering the proteolytic switch mechanism:

  • Maintaining the F95-M96 cleavage site functionality

  • Ensuring specific recognition by proteases

  • Controlling the kinetics of the switch

• Promoter engineering:

  • Designing optimal spacing between -10 and -35 elements

  • Incorporating multiple binding sites for cooperative regulation

  • Preventing unwanted crosstalk with host regulatory systems

System Integration Challenges:

• Compatibility with host machinery:

  • Ensuring proper interaction with B. subtilis RNA polymerase

  • Avoiding interference with essential cellular processes

  • Managing metabolic burden of synthetic circuit components

• Logic gate design:

  • Creating multi-input regulatory systems

  • Implementing AND/OR/NOT logic operations

  • Ensuring tight regulation with minimal leakiness

Solution Approaches:

• Modular design principles as demonstrated with MerR regulators:

  • Using constant DNA-binding domains with variable sensing domains

  • Standardized output modules like luciferase reporters

• Structure-guided engineering:

  • Using protein structure predictions to guide domain fusion

  • Rational mutagenesis to improve compatibility between domains

• Logic gate implementation:

  • Two-component regulatory systems for multi-input sensing

  • AND gate design similar to the SigO-RsoA σ-factor system in B. subtilis

The chimeric MerR-family regulator work provides a valuable template for addressing these challenges, demonstrating that domain swapping can create functional synthetic regulators with novel specificities while maintaining compatibility with the host transcriptional machinery .

Structural Parameters of ImmR DNA-Binding Domain

Putative DNA-Binding Residues Based on Homology with SinR

Structural Homologs of ImmR Identified by Dali Search