Recombinant Bacillus subtilis Trigger factor (tig)

What is Bacillus subtilis trigger factor and what are its primary functions?

Trigger factor in B. subtilis is a 42 kDa molecular chaperone that belongs to the FK506-binding family of peptidyl-prolyl cis-trans isomerases. It serves dual functions in bacterial cells:

• As a ribosome-associated molecular chaperone that binds to the 50S subunit, assisting in the folding of nascent polypeptide chains

• As a peptidyl-prolyl cis-trans isomerase that catalyzes protein folding reactions limited by prolyl isomerization

Research has demonstrated that B. subtilis trigger factor exhibits remarkably high catalytic activity in protein folding, with a kcat/KM value of 1.4 × 10^6 M^-1 s^-1, approximately 40-fold higher than that of cyclophilin (another cytosolic peptidyl-prolyl isomerase) . This high catalytic efficiency stems from its tight binding to protein substrates, reflected in its low KM value of 0.5 μM and strong inhibition by unfolded proteins .

How conserved is trigger factor across bacterial species?

Trigger factor exhibits an interesting evolutionary pattern characterized by:

• Single-copy presence in virtually all bacteria with very few exceptions

• High conservation of functional domains, particularly the ribosome binding site (RBS) motif in the N-terminal domain

• Variable sequence homology between species despite functional conservation

What is the structural organization of B. subtilis trigger factor?

B. subtilis trigger factor consists of three distinct functional domains:

• N-terminal domain (~150 amino acids): Contains the ribosome binding site (RBS) motif and contributes to both ribosome interaction and substrate holding

• Middle domain (~150 amino acids): Possesses the peptidyl-prolyl isomerase (PPIase) activity

• C-terminal domain (~150 amino acids): Functions as the main substrate-holding region

The N-terminal domain is particularly critical as it contains the conserved ribosome binding site that allows trigger factor to associate with the 50S ribosomal subunit. This association enables trigger factor to interact with nascent polypeptides as they emerge from the ribosome .

How can recombinant B. subtilis trigger factor be expressed and purified?

Expression protocol:

• Clone the tig gene from B. subtilis into an expression vector such as pTrc99a

• Transform the construct into an expression host (typically E. coli BL21)

• Induce expression using IPTG, with optimal concentration typically around 40-80 μM to avoid toxicity issues

• Harvest cells and lyse under native conditions

Purification approach:

• For easier purification, express trigger factor with a His-tag

• Purify using Ni-NTA affinity chromatography

• Further purify using ion-exchange chromatography and/or size exclusion chromatography

• Verify purity by SDS-PAGE and activity by enzymatic assays

When expressing complete TF with its N-terminal domain intact, researchers should be cautious about expression levels, as excessive TF production can cause growth inhibition and cell filamentation in bacterial hosts .

What methods are available to assay trigger factor's peptidyl-prolyl isomerase activity?

The peptidyl-prolyl isomerase activity of trigger factor can be measured using several approaches:

1. Ribonuclease T1 refolding assay:

• This assay monitors the rate of refolding of denatured ribonuclease T1, where the rate-limiting step is prolyl cis/trans isomerization

• The refolding kinetics can be followed by changes in fluorescence properties

• The catalytic efficiency is calculated from the kcat/KM values, which for B. subtilis trigger factor is approximately 1.4 × 10^6 M^-1 s^-1

2. Tetrapeptide-based chromogenic or fluorogenic substrates:

• Synthetic peptides containing proline residues coupled to chromophores

• Changes in spectral properties upon isomerization allow direct measurement of activity

3. Protease-coupled assays:

• Utilize protease sensitivity differences between cis and trans conformations

• Activity is measured by the rate of proteolytic cleavage after isomerization

When performing these assays, it's important to include appropriate controls, such as assaying in the presence of FK506 or other inhibitors that specifically block the PPIase activity.

How does trigger factor dosage affect bacterial cell growth and protein synthesis?

Research has revealed a clear dosage constraint mechanism for trigger factor, with significant cellular consequences when TF is overexpressed:

Growth and morphological effects:

• Excessive TF production leads to cell elongation and filamentation

• The growth inhibition increases proportionally with TF expression levels

• At high IPTG concentrations (>120 μM), growth is severely inhibited

Protein synthesis impacts:

• Total protein concentration decreases to approximately 65% of normal levels when TF is overexpressed

• The formation of functional protein complexes, such as the FtsZ Z-ring, is inhibited, explaining the observed cell division defects

• TF overexpression enhances sensitivity to ribosome-binding antibiotics like tetracycline, chloramphenicol, and kanamycin

These effects can be completely rescued by deleting the N-terminal domain of TF or partially rescued by removing either the ribosome binding site motif or the C-terminal domain. This demonstrates that both ribosome binding and substrate holding activities contribute to the toxicity of excessive TF .

How do the different domains of trigger factor contribute to its function?

N-terminal domain:

• Essential for ribosome binding through its conserved ribosome binding site (RBS) motif

• Also contributes to substrate holding activity

• Deletion of this domain completely eliminates the toxic effects of TF overexpression

• The domain is critical for TF's co-translational chaperone function

Middle domain (PPIase domain):

C-terminal domain:

• Functions as the main substrate-holding region

• Forms stable intermediate complexes with unfolded proteins

• Deletion reduces but does not eliminate the toxicity of TF overexpression

The functional interplay between these domains allows trigger factor to act both as a catalyst (through its PPIase activity) and as a holdase chaperone (through its substrate binding domains).

What is the basis for trigger factor's dosage constraint mechanism?

The dosage constraint of trigger factor appears to arise from its intrinsic functional properties:

• Ribosome binding interference:

  • Excessive TF proteins bind heavily to ribosomes

  • This competitive binding can impede normal translation processes

  • The evidence includes enhanced sensitivity to ribosome-binding antibiotics when TF is overexpressed

• Over-capture of substrate proteins:

  • Native-PAGE analysis reveals dispersive bands in TF-overexpressing strains

  • These bands represent stable TF-substrate complexes

  • The complexes can be pulled down with TF-His using Ni-NTA chromatography

  • This suggests that excess TF over-captures substrate proteins, maintaining them in an unfolded state

• Impact on specific cellular processes:

  • FtsZ polymerization is inhibited in TF-overexpressing cells

  • This explains the observed cell filamentation phenotype

  • The effect is abolished when the N-terminal domain is deleted

This dosage constraint may explain why trigger factor is almost universally maintained as a single-copy gene in bacterial genomes, with evolutionary mechanisms ensuring the removal or subfunctionalization of any duplicated copies .

How does B. subtilis trigger factor compare with other peptidyl-prolyl isomerases?

In B. subtilis, two cytosolic peptidyl-prolyl cis-trans isomerases have been identified: cyclophilin (ppiB gene product) and trigger factor (tig gene product). Comparative analysis reveals significant differences:

Catalytic efficiency:

Trigger factor exhibits approximately 40-fold higher specific activity than cyclophilin in catalyzing the refolding of ribonuclease T1. This high catalytic efficiency results from tight binding to protein substrates, reflected in the low KM value of 0.5 μM .

What evolutionary patterns are observed in trigger factor gene duplication events?

Genomic analysis has revealed fascinating evolutionary patterns regarding trigger factor gene duplication:

• Rarity of duplication:

  • Trigger factor is singly copied in virtually all bacteria

  • Only a very few bacterial species possess multiple TF homologs

• Domain retention pattern:

  • In species with multiple TF homologs, only one complete TF copy exists

  • Other homologs typically lack the N-terminal domain containing the ribosome binding site motif

• Subfunctionalization:

  • N-terminal deficient TF homologs in bacteria with multiple TF genes often partition the function of TF through subfunctionalization

  • This allows retention of useful enzymatic functions while avoiding the toxicity associated with multiple complete TF copies

This pattern strongly suggests that dosage constraint drives the evolutionary fate of duplicated trigger factor genes, with mutations leading either to gene loss or to subfunctionalization through domain deletion .

How do mutations in trigger factor affect its function across different bacterial species?

Cross-species analysis of trigger factor mutations reveals consistent functional patterns:

An interesting example is the expression of Myxococcus xanthus TF homologs in E. coli. While expression of N-terminal deficient homologs (MXAN_6153 or MXAN_1178) did not affect growth, chimeric proteins where their N-terminal domains were replaced with that of the complete TF (MXAN_2013) significantly inhibited growth .

How does trigger factor coordinate with other molecular chaperones?

Trigger factor functions as part of a complex protein quality control network in bacteria, interacting with other chaperone systems:

• Sequential action with Hsp70 system:

  • Trigger factor acts as the first chaperone to interact with nascent chains emerging from the ribosome

  • Subsequently, DnaK (bacterial Hsp70) can engage with the partially folded protein

  • This sequential action ensures efficient folding of complex proteins

• Functional overlap:

  • Experiments show that while individual deletion of trigger factor or DnaK has minimal impact on cell viability

  • The double deletion creates severe growth defects, particularly at elevated temperatures

  • This indicates partial functional redundancy between these chaperone systems

• Cooperative substrate handling:

  • Trigger factor can maintain substrates in a folding-competent state

  • This allows subsequent engagement by downstream chaperones

  • The handoff mechanism between different chaperones ensures efficient folding pathways

This coordination between different molecular chaperones forms a functional network that minimizes protein misfolding and aggregation during and after translation.

What role does trigger factor play in bacterial stress response?

While trigger factor is primarily known for its role in co-translational protein folding, research has identified important contributions to stress response:

Starvation conditions:

• In B. subtilis, the simultaneous disruption of trigger factor and cyclophilin genes shows pronounced growth defects specifically under amino acid starvation

• This suggests an essential role for these prolyl isomerases under nutrient limitation conditions

Temperature stress:

• Trigger factor's holdase activity becomes particularly important during heat stress

• At elevated temperatures, it can prevent aggregation of thermally destabilized proteins

Oxidative stress protection:

• Trigger factor may shield sensitive residues from oxidative damage during translation

• This protective role could be particularly important during oxidative stress conditions

The stress-protective functions of trigger factor may explain why it is universally conserved across bacterial species, despite not being essential under optimal growth conditions.

How can trigger factor be utilized in recombinant protein production systems?

Trigger factor's natural role in protein folding can be leveraged for biotechnological applications:

Co-expression strategies:

• Co-express trigger factor with difficult-to-fold recombinant proteins

• Optimize the ratio of trigger factor to target protein to enhance solubility

• Consider using N-terminal domain variants to avoid growth inhibition effects

Fusion protein approaches:

• Generate trigger factor fusion constructs with recombinant proteins

• The PPIase and holdase functions can enhance solubility and folding

• Include protease cleavage sites for subsequent separation if needed

In vitro applications:

• Add purified trigger factor to in vitro translation systems

• Enhance refolding yields of denatured proteins

• Use as an additive in protein crystallization trials to improve protein stability

When implementing these strategies, researchers should be mindful of the potential negative effects of excessive trigger factor expression, particularly when using the complete N-terminal containing form .