Recombinant Bacillus cereus Putative ATP:guanido phosphotransferase BCE33L0076 (karG)

What is BCE33L0076 (karG) and what is its functional role in Bacillus cereus?

BCE33L0076 (karG) is a putative ATP:guanido phosphotransferase found in Bacillus cereus, belonging to the broader ATP:guanido phosphotransferase family of enzymes. These enzymes catalyze the reversible transfer of phosphate between ATP and various phosphagens. While the specific substrate of BCE33L0076 requires further characterization, related enzymes in this family catalyze reactions such as the transfer of phosphate from ATP to guanidoacetate, arginine, taurocyamine, and other phosphagens . In B. cereus, this enzyme likely plays a role in energy metabolism and phosphate transfer reactions that are critical for cellular function, particularly under certain metabolic conditions.

How is B. cereus used as a model organism for studying pathogenic Bacillus species?

B. cereus serves as an excellent model for studying pathogenic Bacillus species, particularly B. anthracis, due to their close evolutionary relationship. This approach allows researchers to overcome safety restrictions and regulations associated with studying true pathogens. Specific gene clusters in B. cereus (such as bc1531-bc1535) show high conservation with those in B. anthracis (ba1554-ba1558) and B. thuringiensis (bt1364-bt1368), indicating their critical roles across the Bacillus genus . This conservation enables researchers to study proteins from B. cereus as reliable proxies for understanding their counterparts in more dangerous pathogens.

What are the structural characteristics of ATP:guanido phosphotransferases?

ATP:guanido phosphotransferases contain a characteristic structural organization:

• C-terminal catalytic domain featuring a duplication where the common core consists of two beta-alpha-beta2-alpha repeats

• N-terminal domain with distinct structural features

• A substrate binding site located in the cleft between domains

• A highly conserved cysteine residue implicated in the catalytic activity

The enzyme family generally forms oligomeric structures, with tetrameric arrangements being common among some members. X-ray crystallography studies of related proteins such as rBC1531 have confirmed this tetrameric structure .

What is the recommended protocol for recombinant expression of BCE33L0076 (karG)?

Based on successful approaches with similar B. cereus proteins, the following methodology is recommended:

• Gene Amplification and Vector Construction:

  • Amplify the BCE33L0076 gene from B. cereus genomic DNA using PCR with primers designed to create appropriate restriction enzyme sites

  • Clone the PCR product into a modified pET expression vector (such as pET49bm) using restriction enzyme digestion and ligation

  • Transform the ligation product into E. coli DH5α for plasmid propagation

• Protein Expression:

  • Transform the verified expression plasmid into E. coli BL21(DE3) cells

  • Grow transformed cells in LB medium at 37°C until OD600 reaches 0.6-0.8

  • Induce protein expression with IPTG (typically 0.5 mM) and continue incubation at 18-20°C overnight

  • Harvest cells by centrifugation and proceed with cell lysis and protein purification

This approach has proven effective for related proteins and provides a starting point for BCE33L0076 expression.

What purification strategy is most effective for obtaining high-purity recombinant BCE33L0076?

A multi-step purification strategy is recommended:

• Initial Capture:

  • Immobilized metal affinity chromatography (IMAC) using Ni-NTA resin for His-tagged protein

  • Wash with buffer containing low imidazole concentrations (20-50 mM)

  • Elute with buffer containing high imidazole (250-300 mM)

• Tag Removal:

  • Perform a thrombin digestion trial to determine optimal enzyme:protein ratio

  • Scale up digestion based on trial results (typically 6 units of thrombin per mg of protein)

  • Verify tag removal by SDS-PAGE

• Final Polishing:

  • Size exclusion chromatography (SEC) to remove soluble aggregates and higher/lower molecular weight contaminants

  • Analyze fractions by SDS-PAGE to confirm purity (target >99% purity)

This approach typically yields approximately 8 mg of purified protein per liter of culture, which is sufficient for most research applications.

What biophysical techniques are most informative for characterizing BCE33L0076?

Based on approaches used for related enzymes, the following techniques provide valuable insights:

X-ray crystallography is particularly valuable for generating atomic-level insights into protein structure and has been successfully applied to related proteins such as rBC1531 .

What enzymatic assays can be used to measure BCE33L0076 (karG) activity?

Based on the enzymatic function of ATP:guanido phosphotransferases, several assays can be employed:

• Coupled Enzyme Assays:

  • ADP production can be coupled to NADH oxidation via pyruvate kinase and lactate dehydrogenase

  • Measure decrease in NADH absorbance at 340 nm

  • Reaction buffer: 50 mM HEPES pH 7.5, 100 mM KCl, 10 mM MgCl₂, 1 mM ATP, varying concentrations of guanido substrate

• Direct Pyrophosphate Detection:

  • Measure released pyrophosphate (PPi) using colorimetric or fluorescent PPi detection kits

  • Reaction conditions: 50 mM Tris-HCl pH 7.5, 5 mM MgCl₂, 1 mM ATP, guanido substrate

• Radiometric Assays:

  • Use [γ-³²P]ATP to track phosphate transfer to guanido substrates

  • Separate products by thin-layer chromatography or precipitation methods

When testing BCE33L0076, it's important to screen multiple potential substrates, including arginine, guanidoacetate, creatine, and other phosphagens to determine its preferred substrate.

How does substrate specificity of BCE33L0076 compare to other ATP:guanido phosphotransferases?

ATP:guanido phosphotransferases show distinct substrate preferences:

BCE33L0076 (karG) from B. cereus likely shows specificity patterns similar to bacterial arginine kinases, though complete kinetic characterization is needed to confirm its preferred substrate and catalytic efficiency. Comparative activity assays using different substrates would provide valuable insights into its function within the B. cereus metabolic network .

What factors influence the catalytic activity of BCE33L0076?

Several factors can influence the enzymatic activity of BCE33L0076:

• pH Dependence:

  • Optimal pH typically ranges from 7.0-8.5 for most ATP:guanido phosphotransferases

  • Activity drops significantly outside this range due to ionization states of catalytic residues

• Divalent Cation Requirements:

  • Mg²⁺ is essential for catalysis (typically 5-10 mM)

  • Mn²⁺ can sometimes substitute but with altered kinetics

  • Other divalent cations (Ca²⁺, Zn²⁺) typically inhibit activity

• Temperature Effects:

  • Activity increases with temperature up to an optimal point (typically 30-50°C)

  • Higher temperatures lead to protein denaturation and activity loss

• Redox Sensitivity:

  • The conserved catalytic cysteine residue makes these enzymes sensitive to oxidation

  • Reducing agents like DTT or β-mercaptoethanol can protect activity

• Allosteric Regulation:

  • Product inhibition by ADP and phosphorylated guanido compounds

  • Potential for regulation by cellular metabolites specific to B. cereus

Systematic investigation of these factors provides insights into the physiological role of BCE33L0076 within B. cereus metabolism.

How can structural studies of BCE33L0076 inform drug development against pathogenic Bacillus species?

Structural studies of BCE33L0076 can provide valuable insights for drug development:

• Conservation Analysis:

  • Mapping conserved regions between BCE33L0076 and homologs in pathogenic Bacillus species

  • Identifying structural features unique to pathogenic variants as potential drug targets

• Active Site Architecture:

  • High-resolution structural determination of the active site configuration

  • Identification of critical residues for substrate binding and catalysis

  • Structure-guided design of inhibitors that can selectively target pathogenic variants

• Virtual Screening Approaches:

  • Using resolved structures for in silico screening of compound libraries

  • Molecular dynamics simulations to identify transitional states suitable for drug targeting

• Fragment-Based Drug Discovery:

  • Crystallographic fragment screening to identify chemical scaffolds with binding affinity

  • Structure-guided optimization of hit compounds

The close evolutionary relationship between B. cereus and B. anthracis makes BCE33L0076 studies particularly valuable for understanding corresponding proteins in these pathogens, potentially leading to novel therapeutic approaches for anthrax and related diseases .

What are the evolutionary implications of BCE33L0076 conservation across Bacillus species?

The conservation of BCE33L0076 across Bacillus species provides insights into bacterial evolution:

• Phylogenetic Analysis:

  • The gene cluster containing BCE33L0076 is highly conserved across B. cereus, B. anthracis, and B. thuringiensis

  • This conservation suggests essential metabolic functions predating the divergence of these species

• Horizontal Gene Transfer Assessment:

  • Analysis of codon usage bias and GC content can reveal potential horizontal gene transfer events

  • Understanding whether this gene was acquired or represents core Bacillus metabolism

• Functional Adaptation:

  • Comparing subtle sequence variations across species may reveal adaptations to different ecological niches

  • Correlation of sequence changes with phenotypic differences between species

• Structural Conservation:

  • Despite sequence variations, the core structural elements of ATP:guanido phosphotransferases remain highly conserved

  • This structural conservation highlights the fundamental importance of their catalytic mechanism

Understanding these evolutionary patterns helps contextualize BCE33L0076 within the broader adaptive strategies of Bacillus species and may reveal why this enzyme has been maintained throughout Bacillus evolution.

What strategies can address solubility issues when expressing recombinant BCE33L0076?

Solubility challenges are common when expressing recombinant proteins. For BCE33L0076, consider these approaches:

• Expression Optimization:

  • Lower induction temperature (16-20°C)

  • Reduce IPTG concentration (0.1-0.3 mM)

  • Use auto-induction media instead of IPTG induction

  • Test different E. coli expression strains (BL21(DE3), Rosetta, ArcticExpress)

• Fusion Tag Selection:

  • Test solubility-enhancing fusion partners (MBP, SUMO, TrxA)

  • Compare N-terminal vs. C-terminal tag placement

  • Optimize linker length between tag and target protein

• Buffer Optimization:

  • Screen various pH conditions (pH 6.0-9.0)

  • Test different salt concentrations (100-500 mM NaCl)

  • Add stabilizing additives (5-10% glycerol, 1 mM EDTA, 1-5 mM DTT)

  • Include osmolytes (0.5-1 M sorbitol, 0.5-1 M arginine)

• Co-expression Strategies:

  • Co-express with molecular chaperones (GroEL/GroES, DnaK/DnaJ)

  • Co-express with natural binding partners if known

For BCE33L0076, approaches used successfully with rBC1531 provide a good starting point, including expression at 18°C overnight after IPTG induction and purification in Tris buffer with 150 mM NaCl .

How can researchers distinguish between BCE33L0076 activity and endogenous E. coli phosphotransferase activity?

Distinguishing BCE33L0076 activity from endogenous E. coli enzymes requires careful experimental design:

• Negative Controls:

  • Use purified tag only (without BCE33L0076) expressed from the same vector

  • Include lysate from E. coli containing empty expression vector

  • Compare activity between heat-inactivated and active BCE33L0076

• Substrate Specificity:

  • Test activity with substrates not utilized by E. coli enzymes

  • Compare activity profiles across multiple substrates to identify unique patterns

• Inhibitor Profiling:

  • Identify specific inhibitors of BCE33L0076 that don't affect E. coli enzymes

  • Use site-directed mutagenesis to create catalytically inactive BCE33L0076 as control

• Kinetic Analysis:

  • Determine detailed kinetic parameters that can distinguish BCE33L0076

  • Compare with published values for E. coli enzymes

• Mass Spectrometry:

  • Use mass spectrometry to track isotope-labeled substrates and products

  • Confirm reaction products are specifically generated by BCE33L0076

These approaches ensure that observed activity can be confidently attributed to BCE33L0076 rather than endogenous E. coli enzymes.

What are common pitfalls in crystallization of BCE33L0076 and how can they be overcome?

Protein crystallization is often challenging. For BCE33L0076, consider these solutions to common issues:

Based on experience with rBC1531, successful crystallization might include vapor diffusion methods at 15-20°C with protein concentrations around 10-15 mg/ml. Co-crystallization with ATP analogs or substrate molecules can also improve crystal formation by stabilizing the protein in a defined conformation .

How can CRISPR-Cas9 technology be applied to study BCE33L0076 function in B. cereus?

CRISPR-Cas9 technology offers powerful approaches for studying BCE33L0076 function:

• Gene Knockout Studies:

  • Generate precise BCE33L0076 deletion mutants in B. cereus

  • Assess growth phenotypes under various conditions to determine essential functions

  • Compare wild-type and knockout strains using metabolomics to identify accumulated substrates

• CRISPRi Applications:

  • Use CRISPR interference to tune down BCE33L0076 expression without complete knockout

  • Create conditional knockdowns to study function in specific growth phases

  • Generate an expression gradient to identify threshold levels needed for various functions

• Gene Editing:

  • Introduce point mutations to study specific catalytic residues

  • Create chimeric enzymes to investigate domain-specific functions

  • Engineer tagged versions for in vivo localization studies

• Multiplex Studies:

  • Simultaneously modify BCE33L0076 and related genes to study pathway interactions

  • Create double mutants to identify synthetic lethal interactions

  • Study epistatic relationships with other enzymes in phosphagen metabolism

These CRISPR-based approaches provide unprecedented precision for dissecting BCE33L0076 function within its native context in B. cereus.

What potential biotechnological applications exist for recombinant BCE33L0076?

Recombinant BCE33L0076 has several potential biotechnological applications:

• Enzymatic ATP Regeneration:

  • Use in coupled enzymatic reactions requiring ATP regeneration

  • Application in biosensors for detecting guanido compounds

  • Component in cell-free protein synthesis systems

• Biocatalysis:

  • Production of phosphorylated compounds for pharmaceutical applications

  • Stereoselective phosphorylation reactions

  • Green chemistry approaches to phosphate transfer reactions

• Structural Biology Tools:

  • Model system for studying ATP-binding proteins

  • Template for engineering novel phosphotransferases with altered specificity

  • Platform for inhibitor screening and drug discovery

• Diagnostic Applications:

  • Development of assays for detecting pathogenic Bacillus species

  • Creation of antibody-based detection systems

  • Component in biosensors for environmental monitoring

These applications leverage the catalytic properties and structural features of BCE33L0076 for diverse biotechnological purposes beyond basic research.

How might systems biology approaches enhance our understanding of BCE33L0076 function?

Integrative systems biology approaches can provide comprehensive insights into BCE33L0076 function:

• Multi-omics Integration:

  • Combine transcriptomics, proteomics, and metabolomics data from BCE33L0076 mutants

  • Create genome-scale metabolic models incorporating BCE33L0076 activity

  • Identify regulatory networks controlling BCE33L0076 expression

• Flux Analysis:

  • Use ¹³C metabolic flux analysis to track carbon flow through pathways connected to BCE33L0076

  • Quantify changes in metabolic fluxes under different conditions

  • Model how BCE33L0076 activity affects global metabolic homeostasis

• Network Analysis:

  • Map protein-protein interaction networks involving BCE33L0076

  • Identify metabolic neighbors and functional partners

  • Characterize the role of BCE33L0076 in metabolic network robustness

• Comparative Systems Biology:

  • Compare systems-level function across different Bacillus species

  • Correlate BCE33L0076 activity with ecological adaptations

  • Understand how pathway architecture varies between pathogenic and non-pathogenic species

These approaches place BCE33L0076 within its broader biological context, moving beyond isolated biochemical characterization to understand its role in the complex adaptive strategies of Bacillus species.