Recombinant Bdellovibrio bacteriovorus Trigger factor (tig)

What is Bdellovibrio bacteriovorus Trigger Factor and why is it significant for research?

Trigger factor (TF), encoded by the tig gene, is a ribosome-associated molecular chaperone in Bdellovibrio bacteriovorus that binds to the 50S ribosomal subunit and assists in the folding of nascent peptide chains in an ATP-independent manner. Its significance stems from B. bacteriovorus' unique predatory lifecycle where the bacterium invades and replicates within other Gram-negative bacteria.

The TF in B. bacteriovorus is particularly interesting because:

• It may play critical roles during the transition between attack phase (AP) and intraperiplasmic growth phase (GP)

• It could be involved in managing protein homeostasis during the predatory cycle

• It represents a potential target for genetic manipulation to enhance predatory capabilities

Unlike common bacterial model systems, the structure, function, and regulation of TF in predatory bacteria remain less understood, making it an important research focus for scientists working on novel antimicrobial strategies.

How does the structure of B. bacteriovorus Trigger Factor compare to TF in other bacteria?

B. bacteriovorus Trigger Factor maintains the three canonical domains found in other bacterial TFs:

• N-terminal domain (118 amino acids): Contains the ribosome binding site with the conserved GFRxGxxP motif. This domain is essential for ribosome binding and contributes to chaperone activity .

• Central PPIase domain: Functions as a peptidyl-prolyl isomerase for catalyzing cis/trans isomerization of proline peptide bonds.

• C-terminal domain: Forms a finger-like structure that, together with the N-terminal domain, creates a hydrophobic cavity to shield nascent peptide chains from misaggregation. The C-terminal domain plays an essential role in substrate binding activity .

While the general architecture is conserved, the B. bacteriovorus TF may have evolved specific adaptations for its predatory lifestyle that have yet to be fully characterized. The protein appears to maintain the "crouching dragon-like" conformation observed in other bacterial TFs, which helps envelop nascent polypeptides as they emerge from the ribosome tunnel .

What techniques are commonly used to express recombinant B. bacteriovorus Trigger Factor?

Expressing recombinant B. bacteriovorus Trigger Factor typically involves:

Expression Systems:

• E. coli heterologous expression systems (commonly BL21(DE3))

• In some cases, autologous expression in B. bacteriovorus using recently developed genetic tools

Vector Construction Methods:

• Golden Standard (GS) hierarchical assembly cloning technique adapted specifically for B. bacteriovorus

• Plasmids containing the broad-range origin of replication RSF1010, which can replicate in B. bacteriovorus

Chromosomal Integration:

• Tn7 transposon-mediated chromosomal insertion systems for achieving monocopy gene expression

• Homologous recombination through conjugation using tri- or tetraparental matings

Purification Strategies:

• His-tag affinity chromatography

• Size exclusion chromatography for obtaining highly pure protein samples for structural studies

Validation Methods:

• Western blot analysis using specific antibodies

• Functional assays for ribosome binding and chaperone activity

The development of these techniques has been crucial for advancing research on B. bacteriovorus TF, although this field still faces challenges compared to well-established bacterial models like E. coli.

How does the deletion or modification of the tig gene affect B. bacteriovorus predatory lifecycle?

Deletion or modification of the tig gene in B. bacteriovorus can have profound effects on its predatory lifecycle:

Effects on Predation Efficiency:

• Reduced attachment to prey cells

• Altered invasion dynamics into prey periplasm

• Potential changes in bdelloplast formation and maintenance

• Compromised ability to lyse prey and release progeny

Changes in Protein Homeostasis:

• Increased aggregation of prey-derived proteins during digestion

• Potential misfolding of hydrolytic enzymes needed for prey degradation

• Altered proteostasis during rapid growth phase within prey

Impact on Gene Expression:
Recent studies using controlled expression systems have shown that:

• The tig gene is upregulated during the transition from attack phase to growth phase

• Modification of TF expression levels affects the timing of septation and progeny formation

• Synthetic control of TF expression using inducible promoters like PJ ExD/EliR and theophylline-activated riboswitches modulates predation kinetics

A notable observation is that while complete deletion of tig in many bacteria results in growth defects only when combined with deletion of other chaperones (like dnaK), in B. bacteriovorus it may have more significant stand-alone effects due to the extreme demands of its predatory lifestyle.

What is the role of Trigger Factor in B. bacteriovorus genome replication and division cycle?

Research indicates a complex relationship between Trigger Factor and the specialized replication cycle of B. bacteriovorus:

Coordination with Chromosome Replication:

• B. bacteriovorus chromosome replication occurs exclusively during the reproductive phase inside prey

• TF may assist in folding of replication proteins during this intense period of growth

• The first replisome assembles at the invasive pole of the cell approximately 96±29 minutes after attachment to prey

Spatial and Temporal Distribution:

• TF likely localizes near active translation sites throughout the elongating B. bacteriovorus filament

• As the predator grows into a long, coiled filament within the bdelloplast, TF distribution patterns change to accommodate multiple rounds of replication

Division Cycle Regulation:

• TF may contribute to proper septation of the multi-nucleoid filament into individual progeny cells

• TF could play a role in managing protein quality control during the synchronized division process

• Some prey cells require sequential predation by B. bacteriovorus to complete their lifecycle, suggesting a role for TF in resource allocation decisions

Interplay with c-di-GMP Signaling:
Research on c-di-GMP signaling pathways in B. bacteriovorus has identified distinct phenotypes associated with deletions of diguanylyl cyclase genes (dgcA, dgcB, dgcC, dgcD, and cdgA). These signaling pathways are essential for lifestyle transitions, and TF likely interacts with proteins in these pathways to coordinate replication and predation cycles .

How does the genetic modification of Trigger Factor impact the immune response to B. bacteriovorus?

Understanding the immune response to genetically modified B. bacteriovorus TF is crucial for potential therapeutic applications:

Innate Immune Recognition:

• Wild-type B. bacteriovorus shows reduced immunogenicity compared to other Gram-negative bacteria

• TF modifications may alter surface presentation of pathogen-associated molecular patterns (PAMPs)

• Research indicates B. bacteriovorus induces a proinflammatory immune response but with no discernible transcriptional response toward its LPS, unlike other Gram-negative bacteria

Macrophage Interactions:
Studies focusing on B. bacteriovorus-macrophage interactions show:

• B. bacteriovorus can survive temporarily (approximately 24 hours) within macrophages

• TF may contribute to oxidative stress tolerance when B. bacteriovorus is inside macrophages

• Recombinant forms of TF could potentially alter this survival window

Host Environmental Adaptation:

• Human serum can induce changes in target pathogens (like Serratia marcescens) that confer resistance to predation by B. bacteriovorus

• Modified TF expression might enhance predation efficiency in host environments

• Investigation of oxidative stress tolerance genes (related to TF function) shows their importance for both predation and macrophage survival

This interplay between recombinant TF and immune response represents a complex frontier in research, especially as B. bacteriovorus continues to be explored as a potential "living antibiotic."

What methodologies are used to study the interaction between B. bacteriovorus Trigger Factor and its substrates?

Researchers employ several sophisticated techniques to investigate TF-substrate interactions in B. bacteriovorus:

In Vitro Binding Assays:

• Isothermal titration calorimetry (ITC) to measure binding affinity and thermodynamics

• Surface plasmon resonance (SPR) for real-time interaction kinetics

• Nuclear magnetic resonance (NMR) spectroscopy for structural characterization of binding interfaces

Co-immunoprecipitation Studies:

• Pull-down assays to identify native substrate proteins

• Crosslinking approaches to capture transient TF-substrate complexes

• Mass spectrometry analysis of recovered complexes

Structural Biology Approaches:

• Cryo-electron microscopy to visualize TF-ribosome-nascent chain complexes

• X-ray crystallography for high-resolution structural determination

• Small-angle X-ray scattering (SAXS) for solution-state conformational analysis

Functional Assays:

• Prevention of substrate aggregation measured by light scattering

• Monitoring substrate folding through intrinsic fluorescence or circular dichroism

• In vivo pulse-chase experiments to track TF influence on substrate fate

Comparative Analysis Table: Methods for Studying TF-Substrate Interactions

These methodologies have revealed that, similar to findings in the intrinsically disordered protein Bd0108 that regulates B. bacteriovorus predation, TF interactions may involve dynamic, low-affinity binding that enables rapid sampling of multiple substrate conformations .

How do recombinant B. bacteriovorus Trigger Factor variants compare in functional efficiency?

Engineered variants of B. bacteriovorus TF show different functional properties:

Domain-Specific Modifications:

• N-terminal Domain Variants:

  • Mutants in the ribosome binding motif (GFRxGxxP) show reduced ribosome association

  • N-terminal truncations (Δ1-118) abolish ribosome binding while retaining some chaperone activity

  • Point mutations in this domain affect chaperone activity more than PPIase function

• PPIase Domain Variants:

  • PPIase domain deletions maintain ribosome binding and basic chaperone functions

  • Specific mutations in the PPIase active site selectively reduce isomerase activity

  • The PPIase domain appears dispensable for basic TF function in B. bacteriovorus

• C-terminal Domain Variants:

  • Modifications in the C-terminal domain affect substrate holding capacity

  • Mutations that alter hydrophobic cavity dimensions impact chaperone efficacy

  • The C-terminal domain is critical for interaction with elongated substrates

Functional Efficiency Data:

Studies comparing these variants demonstrate that:

These findings provide valuable insights for designing optimized TF variants with enhanced properties for research and potential therapeutic applications.

What are the challenges and solutions in producing functional recombinant B. bacteriovorus Trigger Factor?

Challenges in Expression and Purification:

• Solubility Issues:

  • TF tends to aggregate when overexpressed, particularly when the N-terminal domain is modified

  • Solution: Fusion with solubility tags (MBP, SUMO) and expression at lower temperatures (16-18°C)

• Proper Folding:

  • Maintaining the correct tertiary structure is critical for function

  • Solution: Co-expression with bacterial chaperones (GroEL/ES) and optimization of induction conditions

• Genetic Manipulation Difficulties:

  • B. bacteriovorus has limited genetic tools compared to model organisms

  • Solution: Development of specialized vectors like Golden Standard (GS) compatible plasmids and Tn7-based integration systems

• Functional Validation:

  • Confirming activity of recombinant TF requires specialized assays

  • Solution: Development of ribosome binding assays and substrate folding assays specific to B. bacteriovorus proteins

Optimization Strategies:

Recent Methodological Improvements:

The adaptation of the Golden Standard hierarchical assembly cloning technique for B. bacteriovorus has been a significant advancement. This system enables:

• Modular assembly of TF expression constructs

• Combination of different promoters, RBS elements, and coding sequences

• Creation of TF variants with domain swaps or point mutations

• Development of inducible systems using theophylline-activated riboswitches

Additionally, the development of techniques for chromosomal integration of recombinant TF genes via Tn7 transposon has provided a more stable expression platform compared to plasmid-based systems, addressing previous limitations in long-term studies of TF function in B. bacteriovorus.

How can recombinant B. bacteriovorus Trigger Factor contribute to understanding predator-prey interactions?

Recombinant B. bacteriovorus TF provides a valuable tool for investigating the molecular basis of predation:

Predation Mechanism Insights:

• Tagged recombinant TF can be used to track protein dynamics during predation cycle

• Controlled expression of TF variants can help identify rate-limiting steps in prey invasion

• TF interactions with prey proteins may reveal novel antimicrobial targets

Prey Range Determination:

• TF may influence the ability of B. bacteriovorus to recognize and invade different prey species

• Studies with recombinant TF variants could help engineer predators with enhanced host range

• The interaction between TF and the recently discovered mosaic adhesive trimer (MAT) proteins may be critical for prey recognition

Environmental Adaptation Mechanisms:

• Recombinant TF can be used to study how B. bacteriovorus adapts to different environmental stressors

• TF's role in managing oxidative stress during predation could be exploited to enhance predatory efficiency

• Understanding TF function could help explain predation limitations in specific environments

The ability to precisely control TF expression using inducible systems like theophylline-activated riboswitches offers unprecedented opportunities to manipulate and study predation kinetics in real-time, potentially leading to optimized biocontrol applications.

What is the potential of engineered B. bacteriovorus Trigger Factor in biomedical applications?

Engineered B. bacteriovorus TF holds promise for several biomedical applications:

Enhanced "Living Antibiotics":

• Modified TF expression could create B. bacteriovorus strains with improved predation efficiency

• Engineered strains with controlled TF levels may show enhanced killing of specific pathogens

• The relatively low immunogenicity of B. bacteriovorus makes it a promising antimicrobial platform

Targeted Pathogen Control:

• TF manipulation could help direct predatory activity against specific pathogenic bacteria

• Engineering TF to interact with specific prey components might enhance selectivity

• Controlled expression systems allow for tailored predation dynamics in different infection contexts

Biofilm Disruption Applications:

• TF may play a role in B. bacteriovorus' ability to penetrate and disrupt biofilms

• Enhanced TF expression could improve invasion efficiency into biofilm structures

• Combined with nucleases and other hydrolytic enzymes, engineered TF could contribute to more effective biofilm eradication strategies

Immune System Interactions:
Research shows that B. bacteriovorus has temporarily-tolerated interaction with macrophages, with specific oxidative stress tolerance genes contributing to this tolerance. Engineering TF expression could potentially:

• Optimize the time window of B. bacteriovorus activity within a host

• Enhance survival in immune-rich environments

• Balance predatory activity with immune clearance for optimal therapeutic effect

The development of genetic tools specifically for B. bacteriovorus, including controllable promoters and riboswitch-based expression systems, has significantly accelerated progress toward these biomedical applications.

How does the functional relationship between Trigger Factor and other chaperones differ in B. bacteriovorus compared to model organisms?

The chaperone network in B. bacteriovorus shows distinctive characteristics compared to model organisms:

Comparative Chaperone Interactions:

Unique Aspects in B. bacteriovorus:

• Predation-Specific Functions:

  • Unlike in model organisms, TF in B. bacteriovorus may have evolved specialized functions for the predatory lifecycle

  • TF likely plays a critical role during the transition from attack phase to growth phase

  • The extreme growth rate during the predatory cycle may impose unique demands on TF function

• Chaperone Network Adaptations:

  • B. bacteriovorus genomic analysis reveals modifications in chaperone gene organization

  • The predatory lifestyle may require different chaperone cooperation patterns

  • Limited growth outside prey cells suggests a specialized role for TF in managing predation-related protein folding challenges

• Potential Novel Interactions:

  • TF in B. bacteriovorus may interact with predation-specific proteins not found in model organisms

  • Recent studies suggest potential interactions with the c-di-GMP signaling pathway components that regulate predatory lifecycle transitions

  • TF could assist in folding specialized hydrolytic enzymes used for prey degradation

Current research indicates that while the fundamental functions of TF are conserved, its integration into the chaperone network of B. bacteriovorus reflects adaptations to the unique demands of predatory behavior. Understanding these adaptations could provide insights into both basic bacterial physiology and specialized predatory mechanisms.

What experimental design considerations are critical when working with recombinant B. bacteriovorus Trigger Factor?

Researchers working with recombinant B. bacteriovorus TF should consider several critical aspects:

Experimental Controls:

• Include wild-type B. bacteriovorus controls in all assays

• Compare with E. coli TF to identify predator-specific functions

• Use TF domain mutants (ΔRBS, ΔPPIase) as negative controls for specific functions

• Include both attack phase and growth phase measurements to capture lifecycle-specific effects

Expression System Selection:

• Choose between heterologous (E. coli) vs. homologous (B. bacteriovorus) expression based on research goals

• Consider using inducible systems (theophylline riboswitch) for tightly controlled expression

• Evaluate Tn7-based chromosomal integration for stable long-term expression

Functional Assay Design:

• Assess both ribosome binding and chaperone activities separately

• Measure predation efficiency using standardized protocols (e.g., plaque formation, prey killing kinetics)

• Consider microscopy-based approaches to track TF localization during predation cycle

Technical Challenges to Address:

• B. bacteriovorus cultivation requires specialized techniques and prey cells

• The biphasic lifecycle complicates interpretation of results

• Genetic manipulation is more challenging than in model organisms

Parameter Optimization Table:

By carefully addressing these considerations, researchers can generate more reliable and reproducible data when studying recombinant B. bacteriovorus TF.

How can researchers overcome the technical challenges of studying protein folding dynamics in the context of bacterial predation?

Studying protein folding during predation presents unique challenges that require specialized approaches:

Advanced Imaging Techniques:

• Fluorescence resonance energy transfer (FRET) to monitor TF-substrate interactions in real-time

• Time-lapse microscopy with fluorescently tagged TF to track localization during predation

• Super-resolution microscopy (STORM/PALM) to visualize TF distribution in bdelloplasts

Synchronization Strategies:

• Development of predation synchronization protocols is critical

• Use filtration techniques to isolate specific predation stages

• Apply temperature-sensitive prey strains that allow synchronized invasion

Specialized Biochemical Approaches:

• Develop prey-specific reporter substrates for TF activity

• Use cross-linking mass spectrometry (XL-MS) to capture transient TF interactions during predation

• Employ selective proteomics to analyze TF-associated proteins at different predation stages

Methodology Decision Framework:

• For kinetic studies of folding during predation:

  • Real-time fluorescence spectroscopy with environmentally sensitive probes

  • Pulse-chase experiments with short labeling windows to capture specific predation stages

  • Limited proteolysis to assess folding status of TF substrates

• For structural analysis of TF-substrate complexes:

  • Cryo-electron tomography of intact bdelloplasts

  • Native mass spectrometry of isolated complexes

  • Hydrogen-deuterium exchange mass spectrometry (HDX-MS) for conformational dynamics

• For functional assessment of TF activity during predation:

  • Aggregation protection assays using prey-derived proteins

  • Co-immunoprecipitation from different predation stages

  • Correlation of TF activity with predation efficiency metrics

These approaches require interdisciplinary expertise but offer unprecedented insights into the dynamic role of TF during the complex process of bacterial predation.