Recombinant Protochlamydia amoebophila GTPase obg (obg)

How does the GTPase activity of Obg from Protochlamydia amoebophila compare to other bacterial Obg proteins?

Obg proteins, including that from P. amoebophila, display biochemical properties distinct from typical Ras-like GTPases:

These distinct biochemical features suggest that Obg proteins act as intracellular sensors with their nucleotide-bound state controlled primarily by relative GTP/GDP concentration rather than accessory proteins . This makes P. amoebophila Obg an interesting model for studying GTPase regulation in a minimalist bacterial system.

What are effective methods for expressing and purifying recombinant P. amoebophila Obg protein?

Based on protocols used for related Obg proteins, the following methodology is recommended:

• Gene cloning:

  • Amplify the P. amoebophila obg gene (PC0023) using PCR with specific primers

  • Clone into an expression vector (pET-based systems are commonly used)

  • Verify the construct by sequencing

• Protein expression:

  • Transform into E. coli BL21(DE3) or similar expression strain

  • Grow cultures at 37°C until OD₆₀₀ reaches 0.6-0.8

  • Induce with IPTG (0.1-1.0 mM)

  • Continue incubation at lower temperature (16-25°C) for 4-16 hours

• Protein purification:

  • Harvest cells and lyse using sonication or pressure-based methods

  • Clarify lysate by centrifugation

  • Perform affinity chromatography (His-tag purification is common)

  • Further purify by ion-exchange or size-exclusion chromatography

  • Assess purity by SDS-PAGE and protein concentration by Bradford assay

The expected molecular weight of recombinant P. amoebophila Obg is approximately 37.1 kDa, which should be visible as a distinct band on SDS-PAGE .

What assays can be used to measure the GTPase activity of recombinant Obg protein?

Several complementary approaches can be employed to measure GTPase activity:

• Colorimetric phosphate detection assay:

  • Based on quantification of inorganic phosphate released during GTP hydrolysis

  • Malachite green-based assays are widely used and can be adapted to 384-well format

  • Provides robust Z' values (average of 0.58 ± 0.02 reported for Obg GC assays)

• Fluorescence-based assays:

  • Utilize fluorescent guanine nucleotide analogs such as mant-GTP and mant-GDP

  • Measure changes in fluorescence upon nucleotide binding or hydrolysis

  • Useful for kinetic measurements and determining binding constants

• Radiometric assays:

  • Use [γ-³²P]GTP to directly measure release of radioactive phosphate

  • Highly sensitive but requires radioactive materials handling

For optimal results, reaction conditions should include:

• Buffer: 50 mM Tris-HCl (pH 7.5)

• Salt: 50-100 mM KCl or NaCl

• Magnesium: 5-10 mM MgCl₂

• GTP: 50-500 μM

• Protein: 0.5-5 μM

• Temperature: 25-37°C

• Time points: Multiple measurements over 60-120 minutes

What role does Obg play in ribosome biogenesis and translation in Chlamydia-related bacteria?

Obg serves as a critical checkpoint in ribosome biogenesis and maturation in Chlamydia-related bacteria:

• Ribosome assembly:

  • Interacts specifically with the 50S large ribosomal subunit

  • Functions as a ribosome-dependent GTPase that aids in ribosome maturation

  • May act as a molecular scaffold during ribosome assembly

• rRNA processing:

  • Involved in the maturation of ribosomal RNA

  • Affects pre-16S rRNA processing

  • Mutations in Obg can lead to accumulation of precursor rRNAs

• Ribosomal protein interactions:

  • Interacts with specific ribosomal proteins (e.g., L13 in E. coli homologs)

  • Positioned near the GTPase activating center of the ribosome

  • May function as a chaperone for ribosomal protein folding

• Translation regulation:

  • Acts as a regulatory checkpoint before 70S ribosome assembly

  • Links cellular stress responses to translation efficiency

  • May halt translation during nutrient limitation

Notably, while most bacterial Obg proteins have a C-terminal domain considered essential for ribosome binding, the Chlamydia abortus YhbZ (Obg homolog) lacks this domain but still co-fractionates with the E. coli 50S large ribosomal subunit, suggesting alternative binding mechanisms in Chlamydia-related bacteria .

How does Obg function in the bacterial stress response, particularly in obligate intracellular bacteria?

Obg serves as a central regulatory hub in stress response pathways in obligate intracellular bacteria like Protochlamydia and Chlamydia:

• Stringent response:

  • Potentially interacts with alarmone (p)ppGpp, a key stress signaling molecule

  • May compensate for the absence of typical stringent response proteins in Chlamydia

  • Connects stress sensing to cellular growth control

• Amino acid starvation response:

  • Enables cell cycle interruption and entry into persistence/stasis

  • Critical for survival during nutrient limitation inside host cells

  • YhbZ (Chlamydial Obg) is one of the few stringent response-related proteins retained in Chlamydia

• Growth rate regulation:

  • Bacterial growth rate depends on cellular Obg concentration

  • Heterologous expression of Chlamydial YhbZ in E. coli leads to growth defects and cell elongation

  • Functions as a growth rate regulator during environmental challenges

• Developmental transitions:

  • May play a role in developmental transitions between reticulate bodies (RBs) and elementary bodies (EBs) in Chlamydia

  • Could coordinate metabolic adjustments during different life cycle stages

  • Potentially involved in cellular differentiation processes

This multifunctional role makes Obg particularly important in obligate intracellular bacteria with reduced genomes that have lost many conventional stress response mechanisms.

How can Obg structure-function relationships be investigated to identify potential antibiotic targets?

Investigating structure-function relationships of Obg presents several approaches for antibiotic development:

• Structural analysis:

  • Crystallographic studies of P. amoebophila Obg in different nucleotide-bound states

  • Comparison with Obg structures from other bacteria to identify conserved pockets

  • Molecular dynamics simulations to identify flexible regions and allosteric sites

• Functional domains investigation:

  • Mutagenesis of key residues in the GTP-binding domain to identify critical catalytic residues

  • Creation of domain-deletion constructs to determine domain-specific functions

  • Analysis of the unique G-domains that prevent nucleotide binding

• Nucleotide-binding studies:

  • Characterization of interactions with fluorescent guanine nucleotide analogs (mant-GTP/GDP)

  • Determination of binding constants and dissociation rates

  • Investigation of factors affecting nucleotide exchange and hydrolysis

• Protein-protein interactions:

  • Identification of novel interactors using techniques like pull-down assays and mass spectrometry

  • Characterization of interactions with biphasic binding patterns

  • Analysis of the role of the intrinsically disordered C-terminal domain in protein interactions

A validated high-throughput screening assay for Obg inhibitors has been developed with excellent performance (Z' value of 0.58 ± 0.02), enabling the identification of lead compounds that could be developed into broad-spectrum antibiotics .

What is the evolutionary significance of Obg conservation across diverse bacterial phyla, including Chlamydia-related organisms?

The evolutionary conservation of Obg across bacteria provides valuable insights:

• Phylogenetic analysis:

  • Obg is universally conserved from bacteria to humans, indicating fundamental cellular roles

  • Protochlamydia amoebophila Obg represents an evolutionary intermediate between free-living bacterial and host-adapted Chlamydial homologs

  • Analysis of sequence conservation reveals domains under selective pressure

• Structural adaptations:

  • Chlamydial Obg homologs (YhbZ) have evolved structurally, lacking the C-terminal domain present in most bacteria

  • This structural adaption may reflect specialization for the intracellular lifestyle

  • Despite these changes, core functions in ribosome association are maintained

• Functional conservation in organelles:

  • Obg homologs exist in chloroplasts (e.g., Oryza sativa ObgC1), reflecting bacterial origin

  • Chloroplast Obg proteins maintain ancestral roles in DNA replication and ribosome biogenesis

  • This supports endosymbiotic theory and functional conservation across evolutionary time

• Genomic context:

  • In some bacteria, Obg is encoded near sporulation genes (e.g., downstream of Spo0B in B. subtilis)

  • In Chlamydia-related organisms, genomic context differs but functional roles remain similar

  • Analysis of genomic location provides insights into co-evolution with other cellular systems

The remarkable conservation of Obg across phylogenetically diverse bacteria, including obligate intracellular Chlamydia-related organisms with highly reduced genomes, underscores its fundamental importance in bacterial physiology and adaptation .

What is the relationship between Obg GTPase and toxin-antitoxin systems in Chlamydia-related bacteria?

Recent research has revealed potential connections between Obg GTPase and toxin-antitoxin (TA) systems in Chlamydia-related bacteria:

• Genomic context:

  • Several Chlamydia-related organisms encode type II TA modules as shown in the table below:

• Functional relationship:

  • Obg GTPases can interact with the RelA/SpoT homolog proteins that regulate (p)ppGpp levels

  • Many of the TA modules in Chlamydia-related bacteria belong to the RelEB family

  • This suggests potential regulatory crosstalk between Obg and TA systems during stress responses

• Persistence regulation:

  • Both Obg and TA systems are implicated in bacterial persistence mechanisms

  • Obg enables entry into persistence during amino acid starvation in Chlamydia

  • TA modules contribute to persistence in various bacteria, potentially complementing Obg function

• Research approaches:

  • Co-immunoprecipitation experiments to identify direct interactions between Obg and TA components

  • Expression analysis to determine co-regulation during stress conditions

  • Functional studies using recombinant proteins to test biochemical interactions

This emerging field presents interesting research opportunities for understanding how these ancient bacterial regulatory systems cooperate in the unique context of obligate intracellular bacteria.

How does Obg GTPase contribute to pathogenesis in Chlamydia-related infections?

Obg GTPase plays several key roles that contribute to the pathogenesis of Chlamydia-related infections:

• Developmental cycle regulation:

  • May regulate transitions between infectious elementary bodies (EBs) and replicative reticulate bodies (RBs)

  • Could influence the timing of host cell exit and new infections

  • Potentially coordinates cellular processes during developmental transitions

• Persistence and chronic infection:

  • Enables entry into persistence during stress conditions

  • Contributes to long-term survival within host cells

  • May play a role in treatment failures and chronic disease states

• Reproductive tract pathology:

  • Chlamydia infections are associated with reproductive tract complications

  • Undiagnosed chlamydial infections can lead to pelvic inflammatory disease, ectopic pregnancy, and tubal factor infertility

  • Obg's role in persistence may contribute to these long-term sequelae

• Impact on host cellular processes:

  • Interactions with host cell machinery could alter normal cellular functions

  • May contribute to inflammatory responses and tissue damage

  • Potential involvement in cell cycle disruption of infected cells

Understanding Obg's role in pathogenesis has implications for developing new diagnostic and therapeutic approaches for Chlamydia-related infections, which remain significant global health concerns .

What methodological approaches can be used to study Obg function in the context of host-pathogen interactions?

Studying Obg in host-pathogen contexts requires specialized techniques:

• Cell infection models:

  • Infection of relevant cell lines with wild-type and modified Chlamydia

  • Temporal analysis of Obg expression during different infection stages

  • Correlating Obg levels with bacterial developmental transitions

• Molecular manipulation approaches:

  • Given that Chlamydia are resistant to genetic manipulation, heterologous expression in E. coli can provide insights

  • Gradient centrifugation experiments to study ribosome association

  • Complementation studies to test functional conservation

• Microscopy techniques:

  • Immunofluorescence localization of Obg during infection

  • Co-localization with bacterial nucleoids or host cell structures

  • Live-cell imaging using fluorescently tagged proteins

• Proteomics and interactomics:

  • Identification of host proteins that interact with bacterial Obg

  • Temporal changes in protein-protein interactions during infection

  • Analysis of post-translational modifications of Obg during infection

• Animal models:

  • Testing the effects of Obg-targeting compounds in animal infection models

  • Analysis of bacterial persistence in various tissues

  • Evaluation of host inflammatory responses

These approaches can help elucidate how Obg functions during infection and identify potential intervention points for treating Chlamydia-related diseases.

What are the most promising approaches for developing Obg-targeting antimicrobials?

Several strategic approaches show promise for developing Obg-targeting antimicrobials:

• High-throughput screening:

  • The 384-well GTPase assay developed for Obg has demonstrated robust performance (Z' value of 0.58 ± 0.02)

  • This platform enables screening of large compound libraries

  • Secondary assays using fluorescent nucleotide analogs (mant-GTP/GDP) provide confirmation of direct targeting

• Structure-based drug design:

  • Crystal structures of Obg in different nucleotide-bound states can guide rational design

  • Virtual screening and molecular docking can identify compounds with high binding probability

  • Fragment-based approaches may identify building blocks for novel inhibitors

• Allosteric inhibition:

  • Targeting sites outside the GTP-binding pocket may offer higher specificity

  • The interaction between Obg and novel protein partners presents alternative targeting strategies

  • The intrinsically disordered C-terminal domain offers unique targeting opportunities

• Broad-spectrum potential assessment:

  • Testing candidate compounds against Obg proteins from diverse pathogens

  • Cloning and purification of Obg from organisms like Klebsiella pneumoniae and MRSA for comparison studies

  • Assessment using colorimetric and fluorescence-based activity assays

• Delivery system development:

  • For intracellular pathogens like Chlamydia, effective delivery systems are crucial

  • Nanoparticle-based approaches may enhance intracellular delivery

  • Cell-penetrating peptides could facilitate entry into infected cells

The essential nature of Obg and its conservation across bacterial species makes it a promising target for novel broad-spectrum antibiotics that could address the growing challenge of multidrug-resistant infections .

How can advanced structural biology techniques advance our understanding of Obg function?

Advanced structural biology techniques offer powerful approaches to elucidate Obg function: