Recombinant Bifidobacterium adolescentis Translation initiation factor IF-2 (infB), partial

What is Translation Initiation Factor IF-2 and what role does it play in protein synthesis within Bifidobacterium adolescentis?

Translation Initiation Factor 2 (IF2) is a guanine nucleotide-binding protein that plays an essential role throughout the entire translation initiation pathway in bacteria. It remains ribosome-bound from the initial formation of the 30S initiation complex (30SIC) through the assembly of the 70S initiation complex (70SIC), ultimately facilitating the formation of the first peptide bond. The protein undergoes significant conformational changes during these steps, which are influenced by its interactions with various ligands including the 30S and 50S ribosomal subunits, fMet-tRNA, GTP, GDP·Pi, and GDP .

In Bifidobacterium species, IF2 serves as a metabolic sensor that oscillates between an active GTP-bound form under favorable growth conditions and an inactive ppGpp-bound form under stress conditions. This regulatory mechanism becomes particularly important when nutrient shortages occur, as the inactive ppGpp-bound form helps reduce protein synthesis when it would be detrimental to cellular survival .

How is the infB gene expressed in Bifidobacterium, and what forms of IF-2 are produced?

The infB gene encodes two distinct forms of translational initiation factor IF2: IF2 alpha (97,300 daltons) and IF2 beta (79,700 daltons). These two forms differ at their N-terminus as demonstrated through Edman degradation analysis. The N-terminal amino acid sequences of the two forms match perfectly with the DNA sequences at the beginning of the infB open reading frame and an in-phase region 471 bp downstream .

Expression studies using gene fusions between the proximal half of the infB gene and the lacZ gene have confirmed that the two proteins differ at their N-terminus. Importantly, deletion experiments involving the 5'-non-translated region of the fused gene (including the Shine/Dalgarno ribosomal binding site) resulted in expression of IF2 beta-beta-galactosidase but not IF2 alpha-beta-galactosidase. This finding strongly suggests that IF2 beta results from independent translation rather than from proteolytic cleavage of IF2 alpha .

What experimental approaches are effective for isolating and purifying recombinant IF-2 from Bifidobacterium adolescentis?

Effective purification of recombinant IF-2 from Bifidobacterium typically requires a multi-step approach. One successful strategy involves C-terminal His6-tag fusion proteins purified by Ni-NTA affinity chromatography, as demonstrated with other Bifidobacterium proteins . This approach allows for selective isolation of the target protein while maintaining its functional properties.

For expression systems, researchers have developed specialized vectors for Bifidobacterium. The Bifidobacteria Expression SysTem (BEST) represents an advanced approach that allows controlled expression of recombinant proteins in Bifidobacterium species . This system can be adapted for IF-2 production by replacing the target gene in the expression vector with the infB gene sequence.

The purification process should account for the two different forms of IF-2 (alpha and beta). When designing expression constructs, researchers should consider whether to express both forms or focus on one specific form depending on their research objectives.

How does IF-2 function as a metabolic sensor during stress responses in Bifidobacterium, and what methodologies can be used to study this function?

Research has demonstrated that IF-2 binds ppGpp at the same nucleotide-binding site and with similar affinity as GTP. This makes GTP and ppGpp alternative physiologically relevant IF-2 ligands. The ppGpp binding has significant functional consequences: it interferes with IF-2-dependent initiation complex formation, severely inhibits initiation dipeptide formation, and effectively blocks the initiation step of translation .

To study this function, researchers can employ several methodologies:

• Nucleotide binding assays: Using purified IF-2 to measure binding affinities for GTP and ppGpp under various conditions.

• Translation initiation complex formation assays: Measuring the formation of 30S and 70S initiation complexes in the presence of GTP versus ppGpp.

• Dipeptide formation assays: Quantifying fMet-puromycin formation as a measure of IF-2 activity in the presence of different nucleotides.

• Structural studies: Employing techniques such as X-ray crystallography or cryo-EM to visualize conformational changes in IF-2 upon binding different nucleotides.

What are the functional differences between IF-2 alpha and IF-2 beta in Bifidobacterium, and how can these be experimentally characterized?

The two forms of IF-2 (alpha and beta) differ at their N-terminus, suggesting potential functional distinctions. To characterize these differences experimentally, researchers have employed several approaches:

• Gene fusion studies: Constructing fusions between the proximal half of the infB gene and reporter genes such as lacZ can help confirm the existence of two translation products with different N-termini .

• Deletion analysis: Removing the 5'-non-translated region, including the Shine/Dalgarno ribosomal binding site, prevents expression of IF-2 alpha while still allowing expression of IF-2 beta, suggesting independent translational initiation for each form .

• In vitro dipeptide synthesis assays: These can be used to assess the functional activity of each IF-2 form in initiating protein synthesis. The assay typically uses fMet-tRNA and various labeled aminoacyl-tRNAs to measure the formation of initiation dipeptides .

• Ribosome binding studies: Comparing the interaction of each IF-2 form with ribosomes can reveal potential differences in their roles during translation initiation.

A comparative analysis of functional parameters between IF-2 alpha and IF-2 beta might show:

What expression systems are most effective for producing recombinant Bifidobacterium IF-2, and what challenges need to be addressed?

Several expression systems have been developed for producing recombinant proteins in Bifidobacterium, with the BEST (Bifidobacteria Expression SysTem) being a notable recent advancement. This system allows for the expression of heterologous proteins in Bifidobacterium under different stress conditions .

For IF-2 expression specifically, researchers should consider:

• Vector selection: The BEST system utilizes specialized plasmids that function effectively in Bifidobacterium species and can be regulated by various stressors such as heat-shock, pH changes, and bile salts .

• Signal peptide optimization: Different signal peptides can significantly affect protein secretion efficiency. For example, replacing a signal peptide from L. lactis (SP Exp4) with one from B. longum (SP BL1181) resulted in a sevenfold increase in secretion of the target protein in one study .

• Induction conditions: The BEST system can function both constitutively and under induction by various stresses. Researchers should optimize induction conditions for their specific experimental needs .

Challenges that need to be addressed include:

• Protein stability: Ensuring the stability of recombinant IF-2 in the Bifidobacterium cellular environment and during purification.

• Functional assessment: Verifying that recombinant IF-2 retains its nucleotide binding properties and ability to participate in translation initiation.

• Yield optimization: Enhancing expression levels without compromising bacterial viability.

How does IF-2 contribute to the stress response and survival mechanisms of Bifidobacterium adolescentis in the gastrointestinal environment?

IF-2 plays a crucial role in bacterial adaptation to stress, particularly in the challenging environment of the gastrointestinal tract. In Bifidobacterium species, which are important members of the intestinal microbiota, IF-2 functions as a metabolic sensor that helps regulate protein synthesis in response to changing environmental conditions .

Under stress conditions such as nutrient limitation, pH changes, or exposure to bile salts—all common in the gastrointestinal tract—cellular ppGpp levels increase. IF-2 binds ppGpp with affinity similar to GTP, leading to inhibition of translation initiation . This mechanism allows Bifidobacterium to rapidly down-regulate protein synthesis when faced with unfavorable conditions, conserving energy and resources until conditions improve.

Experimental approaches to study this aspect include:

• Growth and survival assays: Comparing wild-type Bifidobacterium with strains expressing modified IF-2 under various stress conditions.

• Proteomics analysis: Identifying changes in the bacterial proteome under stress conditions with normal versus modified IF-2 function.

• In vivo colonization studies: Assessing the ability of different IF-2 variants to support bacterial persistence in animal models.

What role might IF-2 play in the adhesion of Bifidobacterium adolescentis to intestinal epithelial cells, and how could this be investigated?

While IF-2 is primarily known for its role in translation initiation, proteins with moonlighting functions are common in bacteria. The potential role of IF-2 in adhesion to intestinal epithelial cells (IECs) could be investigated by drawing parallels with other Bifidobacterium adhesion proteins such as BopA .

BopA has been identified as a B. bifidum-specific protein involved in adhesion to IECs. Strains expressing BopA show enhanced adhesion capabilities . Similar studies could be designed to investigate whether IF-2, particularly its different forms, might contribute to adhesion:

• Comparative adhesion assays: Using wild-type Bifidobacterium adolescentis versus strains with modified IF-2 expression to compare adhesion to intestinal epithelial cell lines such as Caco-2 and T84 .

• Inhibition studies: Purified recombinant IF-2 could be tested for its ability to inhibit adhesion of B. adolescentis to IECs, similar to how purified BopA was shown to have an inhibitory effect on adhesion of B. bifidum S17 .

• Molecular interaction studies: Techniques such as surface plasmon resonance or pull-down assays could identify potential interactions between IF-2 and intestinal epithelial cell surface components.

• In vivo colonization studies: Animal models could be used to assess whether modifications to IF-2 affect intestinal colonization by B. adolescentis.

How can recombinant B. adolescentis IF-2 be employed as a tool for studying bacterial translation regulation under various environmental conditions?

Recombinant B. adolescentis IF-2 can serve as a valuable tool for understanding bacterial translation regulation, particularly in response to environmental stressors. Several experimental approaches can be employed:

• In vitro translation systems: Purified recombinant IF-2 can be incorporated into cell-free translation systems to study its effects on protein synthesis under different conditions (varying nucleotide concentrations, pH, temperature, etc.).

• Structure-function studies: Creating point mutations or truncations in recombinant IF-2 can help identify specific domains responsible for nucleotide binding, ribosome interaction, and response to stress conditions.

• Crosslinking experiments: Chemical crosslinking followed by mass spectrometry can identify interaction partners of IF-2 under different environmental conditions.

• Ribosome profiling: This technique can reveal how modifications to IF-2 affect ribosome positioning and translation efficiency across the bacterial transcriptome.

A comparative analysis of translation efficiency under different conditions might show:

What comparative differences exist in IF-2 structure and function across different Bifidobacterium species, and what methodologies are best suited for such comparisons?

Comparative analysis of IF-2 across different Bifidobacterium species can provide insights into evolutionary adaptations and species-specific functions. Methodological approaches for such comparisons include:

• Sequence analysis: Bioinformatic comparison of infB genes and predicted protein sequences across species can identify conserved domains and species-specific variations.

• Recombinant expression: Expressing IF-2 from different Bifidobacterium species in a common host system allows direct functional comparisons under identical conditions.

• Structural studies: Techniques such as X-ray crystallography, NMR, or cryo-EM can reveal species-specific structural features of IF-2.

• Functional assays: Comparing nucleotide binding properties, interaction with ribosomes, and activity in translation initiation across species.

• Stress response analysis: Evaluating how IF-2 from different species responds to various stressors, particularly those relevant to their ecological niches.

For example, B. adolescentis is predominantly found in the adult gut, while other species like B. longum subsp. infantis are more abundant in the infant gut. Comparative studies might reveal adaptations in IF-2 that reflect these different environmental niches and their associated stressors.