Recombinant Bdellovibrio bacteriovorus GTPase obg (obg)

What is Bdellovibrio bacteriovorus and why is its Obg GTPase of scientific interest?

Bdellovibrio bacteriovorus is a small Deltaproteobacterium (0.3-0.4 μm wide, 0.8-1.2 μm long) distinguished by its unique ability to prey on other Gram-negative bacteria . The Obg protein from B. bacteriovorus is an essential GTPase belonging to the TRAFAC class OBG-HflX-like GTPase superfamily . It has garnered scientific interest because it plays critical roles in cell cycle control, stress response, ribosome biogenesis, and morphogenesis control, particularly in bacteria that undergo differentiation . Given B. bacteriovorus' predatory lifestyle and unique biphasic life cycle (consisting of a free-living attack phase and an intraperiplasmic growth phase), its Obg GTPase may have specialized functions related to predation and intraperiplasmic growth regulation .

What are the structural domains and key functional regions of B. bacteriovorus Obg?

The Obg protein from B. bacteriovorus, like other Obg proteins, consists of three main domains:

• N-terminal glycine-rich domain (Obg fold): A conserved domain characteristic of Obg proteins

• Central G domain: Contains five conserved motifs (G1-G5) responsible for nucleotide binding and hydrolysis

• C-terminal domain: Less conserved, likely involved in protein-specific functions

The G domain shows Ras-like folds with five α-helices and six-stranded β-sheets. Each G motif has specific functions:

• G1: Responsible for alpha and beta interactions for guanine nucleotide binding

• G2: Binding and coordinating Mg²⁺

• G3 and G4: Performs canonical hydrolytic activity

• G5: Acts as a recognition site for guanine nucleotides

The complete sequence of B. bacteriovorus Obg consists of 343 amino acids with a molecular mass of approximately 37.3 kDa .

What biochemical properties characterize B. bacteriovorus Obg?

B. bacteriovorus Obg is characterized by:

• Moderate affinity binding of GTP, GDP, and possibly (p)ppGpp

• High nucleotide exchange rates

• Relatively low GTP hydrolysis rate compared to other GTPases

• Cycling between GTP-bound "on" state and GDP-bound "off" state to control various cellular processes

These properties make it distinct from many other bacterial GTPases and suggest a role in signaling or regulatory functions rather than primarily catalytic ones.

What methods can be used to investigate the role of Obg in B. bacteriovorus predation?

To investigate Obg's role in B. bacteriovorus predation, researchers can employ several approaches:

• Site-directed mutagenesis: Create specific mutations in the Obg gene (similar to those described for MglA in search result ) to identify key residues essential for function. Focus on conserved residues in G domains that correspond to known activating mutations in Ras-like GTPases (e.g., G21V, L22V equivalents) .

• Predation efficiency assays: Compare wild-type and Obg-mutant strains for their ability to prey on Gram-negative bacteria using:

  • Double-layer agar plaque assays (measuring plaque formation over time)

  • Co-culture experiments monitoring prey population decline via OD600 measurements

  • Microscopic analysis of predation dynamics

• Gene expression analysis: Examine Obg expression during different stages of the predatory cycle using:

  • qRT-PCR

  • RNA-Seq comparing attack phase vs. growth phase expression

  • Proteomics to monitor Obg protein levels

• Protein localization studies: Use fluorescent protein fusions or immunofluorescence microscopy to track Obg localization during predation, similar to approaches used for studying MglA localization .

• GTPase activity assays: Measure GTP hydrolysis rates of purified recombinant Obg under various conditions to understand its biochemical regulation.

How can I express and purify functional recombinant B. bacteriovorus Obg protein?

Expression and purification of functional B. bacteriovorus Obg can be achieved through the following protocol:

• Cloning:

  • Amplify the obg gene (WP_011166147.1) from B. bacteriovorus genomic DNA

  • Clone into an appropriate expression vector with a histidine or other affinity tag

  • Verify sequence integrity

• Expression system selection:

  • E. coli BL21(DE3) or similar strain is commonly used

  • Consider codon optimization for improved expression

  • Use autoinduction media or IPTG-inducible systems

• Expression conditions optimization:

  • Test different temperatures (16-30°C)

  • Vary induction times (3-16 hours)

  • Test different inducer concentrations

• Purification steps:

  • Lyse cells in appropriate buffer (typically 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10% glycerol)

  • Perform affinity chromatography (Ni-NTA for His-tagged protein)

  • Consider ion exchange chromatography as a second purification step

  • Perform size exclusion chromatography for highest purity

• Protein activity verification:

  • Assess GTP binding using fluorescent GTP analogs

  • Measure GTPase activity using colorimetric phosphate release assays

  • Verify proper folding using circular dichroism spectroscopy

Commercially available recombinant B. bacteriovorus Obg can be obtained from vendors for controls or when in-house expression is challenging .

How do mutations in conserved GTPase domains affect Obg function?

Mutations in conserved GTPase domains can significantly alter Obg function, as demonstrated by studies on related GTPases. The effects vary depending on the specific domain and residue affected:

• G1 domain (P-loop) mutations:

  • Mutations equivalent to G21V in Ras (G12V) typically decrease GTP hydrolysis rates 7-fold or more

  • These mutations create a constitutively active form that remains GTP-bound longer

  • In B. bacteriovorus, such mutations may affect predation efficiency by disrupting normal cycling between active/inactive states

• Switch region mutations:

  • Mutations in switch I and II regions (equivalent to those in the G2/G3 region) can render the protein insensitive to GAP proteins

  • This affects the rate of GTP hydrolysis in vivo by disrupting interaction with regulatory proteins

  • Studies on related GTPases show that these mutations can be dominant to wild-type protein

• Nucleotide binding pocket mutations:

  • Mutations that introduce polar residues in the nucleotide binding pocket (like N116K/Y in Ras) may create proteins unable to bind GTP correctly

  • Some mutations affecting GTP binding do not alter protein stability, while others may destabilize the protein

• Surface residue mutations:

  • Mutations on the protein surface can disrupt proper localization and protein-protein interactions

  • Critical surface residues (like D52 in MglA) may be essential for interaction with cytoskeletal components or other regulatory proteins

The study of these mutations provides insights into Obg's role in controlling B. bacteriovorus predatory cycle and stress responses.

What is the relationship between Obg's GTPase activity and B. bacteriovorus' predatory behavior?

The relationship between Obg's GTPase activity and B. bacteriovorus' predatory behavior likely involves several aspects:

• Cell cycle coordination:

  • Obg likely helps coordinate the transition between the free-living attack phase and intraperiplasmic growth phase

  • Its GTPase activity may serve as a molecular switch that responds to prey encounter signals

• Stress response during predation:

  • Invading prey cells exposes B. bacteriovorus to various stresses

  • Obg's known role in bacterial stress responses suggests it may help predators adapt to conditions inside prey cells

  • This adaptation is critical during the transition from attack phase to growth phase

• Ribosome biogenesis regulation:

  • During intraperiplasmic growth, B. bacteriovorus undergoes rapid protein synthesis

  • Obg's established role in ribosome maturation may facilitate the increased translational capacity needed during this phase

  • This function would support the rapid elongation and growth observed inside the bdelloplast

• Nutrient sensing:

  • Obg may sense nucleotide levels (GTP/GDP ratios) which reflect the metabolic state

  • This sensing could help regulate predation based on available resources

  • Studies of bacterial adaptation to starvation conditions show that evolved B. bacteriovorus populations develop enhanced starvation survival rather than improved killing efficiency

Understanding these relationships requires further research, including specific studies examining Obg activity during different predatory phases.

How does B. bacteriovorus Obg compare to Obg proteins from non-predatory bacteria?

B. bacteriovorus Obg shares core functional domains with Obg proteins from non-predatory bacteria but may have evolved specialized features related to its predatory lifestyle:

• Sequence conservation:

  • The central G domain remains highly conserved across both predatory and non-predatory bacteria

  • The N-terminal glycine-rich domain (Obg fold) is also conserved

  • The C-terminal domain shows greater variability and may contain adaptations specific to predatory behavior

• Functional divergence:

  • While all Obg proteins participate in stress response and ribosome biogenesis, B. bacteriovorus Obg likely has additional functions

  • These may include regulation of the biphasic lifecycle transitions and adaptation to intraperiplasmic growth

  • The predatory nature of B. bacteriovorus suggests its Obg protein may integrate signals related to prey detection or consumption

• Structural adaptations:

  • The GTPase domain of Obg proteins contains conserved G1-G5 motifs across species

  • Switch regions may show adaptations that alter GTPase activity rates or protein-protein interactions

  • B. bacteriovorus Obg may have surface features that facilitate interactions with predation-specific partner proteins

• Regulatory network differences:

  • The regulatory networks controlling Obg expression and activity likely differ between predatory and non-predatory bacteria

  • In non-predatory bacteria like E. coli, Obg concentration correlates with growth rate

  • In B. bacteriovorus, Obg regulation may be tied to predation cycle phases rather than simple growth rate

Comparative studies between B. bacteriovorus Obg and those from well-studied model organisms would provide valuable insights into predation-specific adaptations.

What functional parallels exist between Obg and other GTPases involved in bacterial predation?

Several functional parallels exist between Obg and other GTPases involved in bacterial predation or motility:

• Comparison with MglA GTPase:

  • MglA, a 22kDa Ras-related GTPase, controls motility in Myxococcus xanthus, another predatory bacterium

  • Like Obg, MglA functions as a molecular switch cycling between GTP-bound "on" and GDP-bound "off" states

  • Both proteins likely coordinate complex cellular behaviors in response to environmental cues

  • Mutations in conserved residues of MglA affect localization and function, providing a model for studying Obg

• Role in coordinating cellular processes:

  • Both Obg and predatory-specific GTPases coordinate multiple cellular systems

  • While Obg primarily regulates ribosome biogenesis and stress responses, it may also influence cytoskeletal dynamics during predation

  • The spatial and temporal regulation seen in MglA may have parallels in Obg function during B. bacteriovorus predation cycle

• Interaction with regulatory proteins:

  • MglA interacts with MglB, which functions as a GAP (GTPase Activating Protein)

  • Similar regulatory proteins likely modulate Obg activity during different phases of predation

  • Understanding these interactions is critical for deciphering Obg's role in predation

• Response to starvation conditions:

  • Both Obg and predatory movement-associated GTPases help bacteria respond to nutrient limitation

  • In evolved B. bacteriovorus populations, improved starvation survival was observed rather than enhanced predation efficiency

  • This suggests GTPases like Obg may prioritize survival under stress over predation efficiency

Studying these parallels provides a framework for understanding the specialized functions of GTPases in predatory bacteria.

How might Obg be targeted to modify B. bacteriovorus predation efficiency for biocontrol applications?

Targeting Obg to modify B. bacteriovorus predation efficiency for biocontrol applications could be approached through several strategies:

• Genetic engineering of obg expression levels:

  • Overexpression of wild-type obg may enhance metabolic rates and stress tolerance

  • Controlled expression using inducible promoters could create "tunable" predators

  • Expression timing could be optimized for different target bacteria or environments

• Introduction of activity-enhancing mutations:

  • Mutations analogous to activating mutations in Ras-like GTPases (G21V, L22V, Q82A/R)

  • These mutations could create constitutively active forms with altered GTP hydrolysis rates

  • Such modifications might accelerate the predation cycle or improve stress tolerance

• Creation of specialized Obg variants:

  • Chimeric proteins combining domains from different bacterial Obg proteins

  • Domain swapping experiments to identify regions responsible for specific functions

  • Directed evolution approaches to select for enhanced predation under specific conditions

• System-level modifications:

  • Co-engineering of Obg and its interaction partners

  • Modification of regulatory pathways controlling Obg activity

  • Integration with other predation enhancement strategies

• Application-specific optimization:

  • For biofilm targeting: Engineer Obg variants that enhance B. bacteriovorus survival in biofilm environments

  • For clinical applications: Develop strains with enhanced predation against specific pathogens

  • For environmental applications: Create variants optimized for different temperature, pH, or oxygen conditions

The natural resistance of B. bacteriovorus to β-lactam antibiotics also allows for potential combination therapies where both the predatory bacterium and conventional antibiotics are used together .

What role might Obg play in the evolution of B. bacteriovorus predation strategies?

Obg likely plays significant roles in the evolution of B. bacteriovorus predation strategies:

• Adaptation to selective pressures:

  • Studies on parallel evolution in B. bacteriovorus during long-term coculture showed mutations in several genes, potentially including those in regulatory networks involving Obg

  • Instead of evolving improved killing efficiency, B. bacteriovorus adapted to better withstand nutrient limitation

  • This suggests Obg's stress response function may be more critical for evolutionary fitness than its role in predation optimization

• Balancing predation efficiency with survival:

  • Obg's dual role in stress response and cellular processes creates evolutionary trade-offs

  • Enhanced predation might come at the cost of reduced stress tolerance

  • Evolutionary pressures likely shape Obg function to balance these competing needs

• Host range determination:

  • Obg's regulatory functions may influence which prey bacteria can be successfully attacked

  • Evolution of Obg and its partners could contribute to host range expansion or specialization

  • This has implications for the therapeutic potential of B. bacteriovorus against different pathogens

• Resistance to counter-predation mechanisms:

  • As prey bacteria evolve resistance, B. bacteriovorus must adapt its predation strategies

  • Obg's role in regulating cellular processes may facilitate rapid adaptation to changing prey defenses

  • Understanding this co-evolutionary process is essential for applications in biocontrol

• Lifecycle variations:

  • Some B. bacteriovorus strains can grow host-independently

  • Obg likely plays a role in regulating the transition between predatory and non-predatory growth

  • Evolution of this regulatory capacity may represent adaptation to environments with variable prey availability

Research on experimental evolution of B. bacteriovorus in different conditions could reveal how Obg functions have been shaped by selective pressures.

What controls should be included when studying B. bacteriovorus Obg function in predation assays?

When studying B. bacteriovorus Obg function in predation assays, comprehensive controls should include:

• Strain controls:

  • Wild-type B. bacteriovorus (positive control for normal predation)

  • Complemented Obg mutant strains (to verify phenotype rescue)

  • Non-predatory bacteria (negative predation control)

  • Host bacteria without predator (prey survival control)

• Obg protein variant controls:

  • Catalytically inactive Obg mutant (e.g., mutations in G1 domain)

  • Constitutively active Obg variant (e.g., G21V equivalent)

  • Tagged Obg protein control (to verify tag doesn't affect function)

• Environmental condition controls:

  • Aerobic vs. microaerobic conditions (B. bacteriovorus requires oxygen for optimal predation)

  • pH controls (optimal pH range 7.0-8.0)

  • Temperature series (optimal temperature 30-35°C)

  • Ion concentration controls (Ca²⁺ 15-25 mM, Na⁺ concentration 0%)

• Predator-prey ratio controls:

  • Different ratios should be tested (optimal ratio approximately 1:10,000)

  • Time-course sampling to capture complete predation dynamics

• Technical controls:

  • Microscopy: Fixed cells for comparison with live imaging

  • Plaque assays: Phage contamination controls

  • Gene expression: No reverse transcriptase controls for RT-PCR

  • Protein purification: Tag-only protein expression control

• Specificity controls:

  • Other GTPase mutants to verify effects are Obg-specific

  • Multiple prey species to assess host range effects

  • Different B. bacteriovorus strains to confirm strain-independent effects

The experimental data from predation assays should be analyzed using appropriate statistical methods, with multiple biological and technical replicates to ensure reliability.

What physiological parameters should be optimized when working with recombinant B. bacteriovorus Obg protein?

When working with recombinant B. bacteriovorus Obg protein, several physiological parameters should be optimized:

• Buffer composition:

  • pH: Typically 7.0-8.0 to match the optimal pH range for B. bacteriovorus

  • Salt concentration: 150-300 mM NaCl is common for GTPases

  • Divalent cations: 5-10 mM MgCl₂ is essential for GTPase activity

  • Reducing agents: 1-5 mM DTT or β-mercaptoethanol to maintain cysteine residues

  • Stabilizers: 5-10% glycerol to improve protein stability

• Nucleotide binding conditions:

  • GTP/GDP concentration: Typically 0.1-1 mM

  • Incubation time: Allow sufficient time for nucleotide binding (15-30 minutes)

  • Temperature: 25-30°C is optimal for most GTPase binding assays

• GTPase activity measurement conditions:

  • Reaction time: Time course experiments to determine linear range

  • Temperature: Assay at physiologically relevant temperatures (30-35°C)

  • Enzyme concentration: Determine appropriate concentration for linear kinetics

  • Detection method: Colorimetric phosphate detection, HPLC, or coupled enzymatic assays

• Protein stability factors:

  • Storage temperature: Typically -80°C for long-term, -20°C with glycerol for short-term

  • Freeze-thaw cycles: Minimize these as they can affect activity

  • Protein concentration: Higher concentrations often improve stability

• Interaction studies parameters:

  • Partner proteins: Consider co-purification with known binding partners

  • Detergents: Low concentrations may be needed if membrane interactions are involved

  • Crowding agents: PEG or BSA can mimic cellular conditions

• Structural analysis conditions:

  • For circular dichroism: Low salt buffers and appropriate protein concentration

  • For crystallization: Screen various precipitants, pH values, and temperatures

  • For NMR studies: Isotopic labeling and appropriate buffer conditions

These parameters should be systematically optimized for each specific application of the recombinant protein.

How can contradictory results in Obg functional studies be reconciled?

When facing contradictory results in Obg functional studies, researchers should implement the following reconciliation strategy:

• Methodological differences analysis:

  • Compare experimental conditions between studies (buffer composition, temperature, pH)

  • Assess differences in protein preparation methods (expression system, purification protocol, tag position)

  • Evaluate assay methodologies (in vitro vs. in vivo, detection methods, time scales)

• Strain-specific variations consideration:

  • Different B. bacteriovorus strains may show varied Obg functionality

  • HD100 is the most commonly studied strain but others may have different properties

  • Genome sequence comparison between strains used in different studies can identify potential modifiers

• Experimental design evaluation:

  • Sample size and statistical power analysis

  • Biological vs. technical replication strategies

  • Controls used and their appropriateness

  • Blinding and randomization procedures

• Multi-method confirmation approach:

  • Validate key findings using orthogonal methods

  • For example, complement biochemical assays with genetic approaches

  • Consider in vivo, in vitro, and in silico methods to build a complete picture

• Data integration framework:

  • Create models that can accommodate seemingly contradictory data

  • Consider that Obg may function differently under various conditions

  • Use mathematical modeling to test whether contradictions can be explained by different parameters

• Common confounding factors:

  • Protein aggregation affecting activity measurements

  • Host-independent mutants arising during cultivation

  • Prey variation affecting predation assays

  • Contamination with other bacteria or bacteriophages

• Collaborative resolution:

  • Direct collaboration between labs with contradictory results

  • Exchange of materials (strains, proteins, reagents) to identify source of variation

  • Joint design of experiments to resolve discrepancies

By systematically addressing these aspects, apparently contradictory results can often be reconciled within a more comprehensive understanding of Obg function.

What are common pitfalls in analyzing B. bacteriovorus Obg mutant phenotypes and how can they be avoided?

Common pitfalls in analyzing B. bacteriovorus Obg mutant phenotypes and their solutions include:

• Suppressor mutations development:

  • Pitfall: Obg is essential, so mutants may develop compensatory mutations

  • Solution: Sequence verify strains before and after experiments; use freshly prepared cultures; implement conditional expression systems rather than complete knockouts

• Pleiotropic effects misinterpretation:

  • Pitfall: Attributing all phenotypic changes directly to Obg when they may be downstream effects

  • Solution: Use multiple mutant types (point mutations vs. expression level changes); perform complementation with wild-type and mutant variants; examine effects on known Obg interaction partners

• Growth phase variability:

  • Pitfall: B. bacteriovorus exhibits different behaviors in attack vs. growth phase

  • Solution: Synchronize cultures; clearly define which phase is being studied; use time-course experiments to capture phase transitions

• Host-independent (HI) variant interference:

  • Pitfall: HI variants can arise spontaneously and confound predation assays

  • Solution: Regular checking for HI growth; plaque purification; prey-dependent cultivation methods

• Predator-prey ratio inconsistencies:

  • Pitfall: Variable results due to inconsistent predator-prey ratios

  • Solution: Standardize and report ratios (optimal ratio is 1:10,000); measure both predator and prey concentrations accurately

• Inadequate phenotypic assessment:

  • Pitfall: Focusing on a single aspect of predation while missing others

  • Solution: Comprehensive phenotyping including: predation efficiency, motility, prey range, bdelloplast formation, progeny release timing, and stress survival

• Environmental condition variations:

  • Pitfall: Results varying due to unstandardized conditions

  • Solution: Control and report temperature (30-35°C optimal), pH (7.0-8.0 optimal), oxygen levels (strictly aerobic), and ion concentrations (Ca²⁺ 15-25 mM)

• Technical artifact misinterpretation:

  • Pitfall: Assay-specific artifacts being attributed to biological differences

  • Solution: Use multiple assay methods to confirm findings; include appropriate technical controls; blind analysis when possible

By anticipating these pitfalls and implementing the suggested solutions, researchers can generate more reliable and interpretable data from B. bacteriovorus Obg studies.

What emerging technologies might advance our understanding of Obg's role in bacterial predation?

Several emerging technologies hold promise for advancing our understanding of Obg's role in bacterial predation:

• Advanced microscopy techniques:

  • Super-resolution microscopy to track Obg localization during predation with nanometer precision

  • Correlative light and electron microscopy (CLEM) to connect Obg localization with ultrastructural features

  • Light sheet microscopy for long-term live imaging of predation with minimal phototoxicity

• Single-cell technologies:

  • Single-cell RNA-seq to profile transcriptional changes in individual predatory cells

  • Single-cell proteomics to measure protein-level changes during predation

  • Microfluidic devices for tracking individual predator-prey interactions

• Protein interaction mapping:

  • Proximity labeling techniques (BioID, APEX) to identify proteins near Obg during different predation phases

  • Hydrogen-deuterium exchange mass spectrometry to map conformational changes upon nucleotide binding

  • Single-molecule FRET to measure Obg conformational dynamics in real-time

• Genetic engineering advances:

  • CRISPR-Cas9 genome editing for precise manipulation of obg and related genes

  • Optogenetic control of Obg activity to precisely time activation/inactivation during predation

  • Synthetic biology approaches to create B. bacteriovorus with engineered Obg variants for enhanced function

• Computational approaches:

  • Molecular dynamics simulations of Obg-nucleotide interactions

  • Machine learning analysis of predation phenotypes to identify subtle effects of mutations

  • Systems biology modeling of the predation cycle including Obg regulation networks

• Structural biology techniques:

  • Cryo-electron microscopy to solve structures of Obg in different nucleotide-bound states

  • In-cell NMR to observe Obg conformational changes inside living predatory cells

  • AlphaFold2 and other AI-based structure prediction to model Obg interactions with partners

These technologies, used in combination, could provide unprecedented insights into the dynamic functions of Obg during the complex process of bacterial predation.

How might understanding B. bacteriovorus Obg function contribute to developing novel antimicrobial strategies?

Understanding B. bacteriovorus Obg function could contribute to developing novel antimicrobial strategies in several ways:

• Enhanced predatory bacteria engineering:

  • Optimizing Obg function could create more efficient predatory bacteria for therapeutic applications

  • Engineered B. bacteriovorus with modified Obg could show enhanced predation against specific pathogens

  • Stress-tolerant variants could survive better in clinical settings or biofilms

• B. bacteriovorus-antibiotic combination therapies:

  • Understanding how Obg mediates stress responses could inform optimal antibiotic combinations

  • B. bacteriovorus naturally resists β-lactam antibiotics, enabling combination therapies

  • Strategic targeting of different cellular processes in pathogens could reduce resistance development

• Novel drug target identification:

  • Comparative analysis between predator and prey Obg could reveal unique vulnerabilities

  • Inhibitors targeting pathogen Obg but sparing predator Obg could create selective antimicrobials

  • The essential nature of Obg in bacteria makes it an attractive target

• Biofilm eradication strategies:

  • B. bacteriovorus can penetrate biofilms that resist conventional antibiotics

  • Understanding how Obg regulates this ability could inform new approaches to biofilm treatment

  • Engineering enhanced biofilm penetration through Obg modification could improve therapeutic efficacy

• Host-microbiome interaction optimization:

  • B. bacteriovorus may contribute to human gut microbiota health

  • Understanding Obg's role in regulating predation in this environment could allow targeted interventions

  • Predator-based approaches could selectively remove pathogens while sparing beneficial bacteria

• Resistance management strategies:

  • Prey can develop temporary resistance to predation

  • Insights into how Obg facilitates adaptation to prey resistance could inform counter-resistance strategies

  • Multi-predator approaches targeting different pathways could minimize resistance development

This research direction represents a promising alternative to conventional antibiotics, particularly for addressing multidrug-resistant infections.

What are the key experimental methodologies for studying different aspects of Obg function?

What analytical tools are most appropriate for interpreting Obg functional data?