Recombinant Photobacterium profundum LexA repressor (lexA)

Basic Research Questions

• What is the functional role of LexA repressor in bacterial SOS response?

  LexA functions as the master transcriptional repressor of the SOS response in bacteria. Under normal conditions, LexA dimers bind to specific DNA sequences (SOS boxes) in promoter regions of SOS genes, suppressing their expression. When DNA damage occurs, RecA protein becomes activated by binding to single-stranded DNA (ssDNA) and ATP, forming nucleoprotein filaments. These activated RecA filaments stimulate the autocleavage of LexA, leading to its dissociation from DNA and subsequent derepression of SOS genes . This coordinated response enables bacteria to repair DNA damage through various mechanisms including nucleotide excision repair, homologous recombination, and error-prone DNA synthesis .

• How can I clone and express recombinant Photobacterium profundum LexA in E. coli?

  To clone and express recombinant P. profundum LexA:

  1. Amplify the lexA coding region from P. profundum genomic DNA using primers designed to include appropriate restriction sites

  2. Clone the amplified fragment into an expression vector like pET-28a(+) to create a His6-tagged fusion protein

  3. Transform the construct into an expression strain such as E. coli Rosetta(DE3)

  4. Culture transformed bacteria in LB medium with appropriate antibiotics at 37°C

  5. Induce protein expression at OD600 of 0.8 with IPTG (typically 100 μM)

  6. Continue induction at lower temperature (16°C) for 19 hours to enhance solubility

  7. Harvest bacteria and disrupt cells by sonication in lysis buffer (300 mM NaCl, 50 mM NaH2PO4, 10 mM imidazole)

  8. Purify the His6-tagged LexA using Ni2+-NTA agarose beads

  9. Elute with lysis buffer containing 200 mM imidazole

  10. Dialyze against storage buffer (10 mM Tris-HCl, 1 mM EDTA, 80 mM NaCl, 4% glycerol, 20 mM β-mercaptoethanol, pH 7.5)

  11. Store at -80°C until use

• What methods can be used to verify LexA-DNA binding interactions?

  Electrophoretic Mobility Shift Assays (EMSAs) are the standard method to verify LexA-DNA binding:

  1. Generate DNA probes containing putative LexA-binding sites through PCR amplification or synthesized oligonucleotides

  2. Purify recombinant LexA protein as described in question 2

  3. Incubate purified LexA with labeled DNA fragments at varying protein concentrations

  4. Resolve the protein-DNA complexes on a native polyacrylamide gel

  5. Visualize shifts in DNA mobility that indicate protein binding

  For specificity validation, perform competition assays using unlabeled DNA fragments. Quantitative measurements of binding affinity can be obtained through surface plasmon resonance or fluorescence anisotropy techniques .

  For verification of binding sites in vivo, chromatin immunoprecipitation (ChIP) followed by sequencing or qPCR can identify genomic regions bound by LexA under different conditions .

• How does LexA regulation differ across bacterial species?

  LexA regulation exhibits considerable variation across bacterial species:

  Bacterial Group	LexA Binding Consensus	Notable Regulated Genes	Unique Features
  γ-Proteobacteria (E. coli)	CTGT(N)8ACAG	recA, uvrA, sulA, lexA	Core SOS response, >40 genes regulated
  α-Proteobacteria	GTTC(N)7GTTC	recA, lexA, parE, tag, comM, dnaE	Photoreactivation (splB) under SOS control
  β-Proteobacteria	CTGT(N)8ACAG-like	recA, lexA, hypA, hypB	Photoreactivation (splB) under SOS control, lack of recN regulation
  Vibrio species	CTGT(N)8ACAG-like	lexA, recA, imuA, topB, recG, mutH	Regulation of mismatch repair (mutH)
  Streptomyces	Custom motifs	lexA, tga (transglutaminase)	Dual role in SOS and morphological development

  These differences reflect adaptations to specific ecological niches and evolutionary pressures. When working with P. profundum LexA, researchers should note that marine bacteria often have regulatory adaptations for high-pressure environments that may affect LexA function .

Advanced Research Questions

• How does high hydrostatic pressure affect the SOS response mediated by LexA in P. profundum?

  High hydrostatic pressure (HHP) can induce the SOS response in marine bacteria like P. profundum, which has evolved to thrive in deep-sea environments. Methodological approaches to study this:

  1. Pressure treatment protocol: Subject bacterial cultures to sublethal pressures (75-100 MPa) for 15 minutes using specialized pressure chambers

  2. Gene expression analysis:

     • Use reporter systems like GFP fused to SOS-regulated promoters

     • Perform RT-qPCR of key SOS genes at various timepoints after pressure treatment

     • Conduct RNA-seq for genome-wide expression profiling

  3. Viability assessment: Compare survival rates between wild-type and SOS-deficient strains (ΔrecA, lexA1(Ind-)) following pressure treatment

  4. Functional consequences:

     • Measure DNA damage levels using comet assay or PFGE

     • Assess prophage induction rates if applicable

     • Quantify mutation frequencies following pressure exposure

  Research has demonstrated that pressure can trigger a genuine RecA-LexA dependent SOS response, with a 5-fold increase in expression of SOS genes observed at 100 MPa. This response is abolished in strains with non-cleavable LexA (lexA1) or lacking RecA, confirming the canonical SOS pathway activation by pressure .

• What structural conformations does LexA adopt during different functional states, and how can they be studied?

  LexA exhibits distinct conformational states that dictate its function:

  1. DNA-binding conformation: Dimeric state where N-terminal domains engage specific DNA sequences

  2. Cleavage-competent conformation: Arrangement that exposes the Ala84-Gly85 peptide bond for autoproteolysis

  3. RecA-bound intermediate: Interaction with activated RecA nucleoprotein filament that facilitates self-cleavage

  Methodological approaches to study these conformations:

  Technique	Application	Advantages	Limitations
  X-ray crystallography	High-resolution structures of LexA alone or bound to DNA	Atomic-level details	Difficult to capture dynamic states
  Cryo-EM	Structure of LexA-RecA complexes	Can visualize larger assemblies	Lower resolution than crystallography
  FRET	Conformational changes in solution	Real-time dynamics	Requires fluorescent labeling
  HDX-MS	Protein dynamics and interface mapping	No size limitation, identifies flexible regions	Moderate resolution
  Site-directed mutagenesis	Function of specific residues	Can target specific interactions	Indirect structural information
  Molecular dynamics	Simulations of conformational transitions	Can model transitions between states	Computational approximations

  Recent structural insights reveal that LexA binding and cleavage sites on RecA are composite surfaces formed only in the active RecA filament, explaining how LexA cleavage is specifically regulated during the SOS response .

• How do specific mutations in LexA affect its ability to regulate recombination versus SOS response induction?

  Evolutionary Trace (ET) analysis has identified evolutionarily important surface amino acids involved in different RecA-LexA functional interactions. Specific mutations can differentially affect recombination and LexA cleavage functions:

  1. Experimental approach:

     • Generate targeted point mutations in predicted functional sites of recombinant LexA

     • Evaluate effects on DNA binding using EMSAs

     • Assess autocleavage rates in vitro with purified proteins

     • Test in vivo effects on SOS induction using reporter systems

     • Measure recombination frequencies using standard assays

  2. Key findings:

     • Mutations in specific surface clusters can disrupt either recombination or LexA cleavage

     • Some mutations specifically impair SOS induction while maintaining recombination function

     • Composite binding sites for LexA exist on the active RecA filament

  This separation of functions through specific amino acid determinants provides potential targets for developing antimicrobial compounds that selectively inhibit SOS-dependent mutagenesis (and thus antibiotic resistance development) without compromising essential recombination functions .

• How can I design a LexA-based heterologous gene regulation system in mammalian cells?

  The E. coli LexA repressor-operator system can be adapted for regulated gene expression in mammalian cells. Implementation methodology:

  1. Expression construct design:

     • Place LexA gene downstream of a strong constitutive promoter (e.g., RSVLTR)

     • Include eukaryotic polyadenylation and splice signals (e.g., from SV40)

     • Generate target constructs with reporter genes driven by mammalian promoters (e.g., HSV tk) containing synthetic LexA operator sequences

  2. System implementation:

     • Introduce the LexA expression vector into mammalian cells through transfection

     • Select stable transfectants expressing LexA protein

     • Test regulation by comparing expression of operator-containing versus control constructs

  3. Performance metrics:

     • Up to 10-fold repression of reporter gene expression has been achieved

     • Regulation efficiency depends on operator positioning and quantity

     • System functions in mouse cells and potentially other mammalian systems

  This approach provides an orthogonal gene regulation system for synthetic biology applications in eukaryotic cells, with the advantage of minimal cross-talk with endogenous regulators .

• What role does LexA play in regulating functions beyond canonical DNA repair in bacterial species?

  LexA regulation extends beyond canonical DNA repair to diverse cellular functions:

  1. Transglutaminase synthesis in Streptomyces:

     • LexA indirectly regulates transglutaminase (TGase) production in S. mobaraensis

     • LexA deletion significantly reduces TGase production

     • LexA activates expression of genes involved in protein synthesis (rplJ, sti)

     • Experimental approach: Compare TGase production in wild-type, ΔlexA, and complemented strains

  2. Morphological development:

     • LexA directly regulates morphological differentiation genes (whiB, ssgA, divIVA, ftsH)

     • Deletion of lexA impairs sporulation in Streptomyces

     • Method: RT-qPCR analysis at different development stages shows LexA-dependent expression

  3. Prophage induction:

     • High pressure induces λ prophage through RecA-LexA pathway

     • Up to 10^4-fold induction occurs after pressure treatment

     • Research approach: Measure phage titers following exposure to different stimuli

  4. Mismatch repair and photoreactivation:

     • LexA may regulate mutH (mismatch repair) in Vibrio species

     • splB (spore photoproduct lyase) is under LexA control in α- and β-Proteobacteria

     • Verification method: EMSA confirms LexA binding to regulatory regions

  These diverse functions highlight LexA's role as a master regulator that coordinates multiple cellular processes in response to stress conditions .

• How can comparative genomics approaches be utilized to identify the complete LexA regulon in P. profundum?

  Comparative genomics provides powerful tools for identifying the complete LexA regulon:

  1. Methodological workflow:

     • Extract LexA-binding motifs from experimentally validated binding sites

     • Develop position-specific scoring matrices or hidden Markov models

     • Scan genomic sequences for putative binding sites

     • Filter candidates based on conservation, position relative to genes, and score thresholds

     • Validate selected candidates experimentally

  2. Implementation for P. profundum:

     Analysis Step	Specific Methods	Expected Outcomes
     Motif definition	Align known binding sites from related species	P. profundum-specific LexA-binding motif
     Genome scanning	Use tools like MEME, FIMO, or custom scripts	Initial set of potential binding sites
     Conservation analysis	Compare to other Photobacterium and Vibrio species	Evolutionarily conserved binding sites
     Functional categorization	GO term enrichment, pathway analysis	Processes regulated by LexA
     Experimental validation	EMSA, ChIP-seq, reporter assays	Confirmed direct targets

  3. Expected regulon composition:

     • Core SOS genes (recA, lexA, uvrA)

     • DNA repair and recombination genes

     • Error-prone polymerases

     • Cell division inhibitors

     • Species-specific genes related to deep-sea adaptation

  In Vibrio species, this approach has identified non-canonical LexA targets including topB, recG, and genes of unknown function. For P. profundum, particular attention should be paid to genes involved in high-pressure adaptation that may be uniquely regulated by LexA in this deep-sea bacterium .

Methodological Questions

• What are the best techniques for analyzing LexA cleavage kinetics under different conditions?

  Several techniques can effectively analyze LexA cleavage kinetics:

  1. In vitro cleavage assays:

     • Purify recombinant LexA and RecA proteins

     • Prepare activated RecA filaments (RecA + ssDNA + ATP or ATP-γ-S)

     • Incubate LexA with activated RecA under different conditions

     • Sample reaction at timed intervals

     • Analyze by SDS-PAGE to quantify intact and cleaved LexA fragments

     • Calculate cleavage rates using densitometry

  2. Real-time monitoring:

     • Use FRET-based reporters with fluorophores on LexA domains

     • Monitor fluorescence changes during cleavage

     • Enable continuous kinetic measurements

  3. In vivo cleavage monitoring:

     • Create reporter systems (e.g., LexA-GFP fusions)

     • Expose cells to DNA damaging agents or high pressure

     • Track cleavage through western blotting or live-cell imaging

     • Correlate with expression of SOS genes

  4. Variables to investigate for P. profundum:

     Parameter	Range for Testing	Expected Effect
     Hydrostatic pressure	0.1-100 MPa	Increased cleavage at higher pressures
     Temperature	4-37°C	Temperature dependence reflecting habitat
     pH	6.0-8.0	Possible adaptation to marine environment
     Salt concentration	0.1-0.6 M NaCl	Marine adaptation effects

  When studying P. profundum LexA, it's essential to consider that optimal cleavage conditions may differ from those of E. coli due to adaptations to the deep-sea environment .

• How can I develop a high-throughput screening system to identify inhibitors of LexA activity?

  A comprehensive high-throughput screening approach for LexA inhibitors:

  1. Primary screening assays:

     • FRET-based assay: Develop a FRET reporter with fluorophores positioned to detect LexA cleavage or conformational changes

     • AlphaScreen: Measure LexA-DNA binding through proximity-based signal generation

     • Fluorescence polarization: Detect changes in LexA binding to fluorescently labeled DNA probes

  2. Secondary validation:

     • EMSA: Confirm effects on LexA-DNA binding

     • In vitro cleavage assay: Validate inhibition of RecA-mediated LexA cleavage

     • Reporter strains: Test compounds in bacteria with SOS-responsive reporters

  3. Compound libraries to screen:

     • Natural product collections

     • Fragment-based libraries

     • Structure-based virtual screening hits

     • Marine-derived compounds (potentially relevant for P. profundum LexA)

  4. Counter-screens and selectivity:

     • Test for general RecA inhibition

     • Assess effects on other self-cleaving proteases

     • Evaluate bacterial growth inhibition

     • Test mammalian cell toxicity

  Targeting LexA offers a strategy to combat bacterial antibiotic resistance by preventing SOS-induced mutagenesis. Inhibitors could serve as adjuvants to conventional antibiotics, reducing the development of resistance without directly affecting bacterial viability .