Recombinant Nocardia farcinica Translation initiation factor IF-2 (infB), partial

What is the role of translation initiation factor IF-2 in Nocardia farcinica?

Translation initiation factor IF-2 in N. farcinica, like in other bacteria, plays a critical role in protein synthesis by facilitating the binding of initiator tRNA (fMet-tRNA) to the ribosome. It interacts with both 30S and 50S ribosomal subunits and is responsible for positioning the ribosomal subunits in a distinct rotational orientation during the subunit-joining step of initiation . Specifically, IF-2 stabilizes an intermediate state where ribosomal subunits adopt a semirotated conformation with the 50S L1 stalk in a half-closed position .

Research methodologies to study this function include:

• Base-specific chemical probing to identify IF-2 protection sites on ribosomes

• Single-molecule FRET assays to monitor subunit orientation during IF-2 binding

• In vitro translation assays with purified components

How is the infB gene structured and expressed in Nocardia farcinica?

The infB gene in bacteria typically encodes multiple forms of IF-2. While N. farcinica-specific data is limited, studies in other bacteria show that infB codes for at least two forms of translational initiation factor IF2: a full-length form (IF2-α) and truncated forms (IF2-β/IF2-γ) .

The gene structure facilitates this through:

• Multiple translational initiation sites within the same open reading frame

• Different Shine-Dalgarno sequences that control expression of different isoforms

• Independent translation events rather than proteolytic cleavage

Experimental approaches to study infB expression include:

• Gene fusion constructs with reporter genes like lacZ

• In vitro dipeptide synthesis assays

• Deletion analysis of 5'-non-translated regions

• Edman degradation to sequence N-terminal regions of purified proteins

What expression systems are optimal for producing recombinant N. farcinica IF-2?

Based on successful expression of other N. farcinica proteins, the following methodological approach is recommended:

• Vector selection: pET expression systems (particularly pET30a) have been successfully used for other N. farcinica proteins .

• Host strain: E. coli BL21(DE3) is preferred for high-level expression of recombinant proteins.

• Induction conditions:

  • IPTG concentration: 0.2 mM has been effective for other N. farcinica proteins

  • Temperature optimization is critical: expression increases with higher induction temperatures

• Expression verification: SDS-PAGE analysis and Western blotting with anti-His antibodies

A comparative analysis of expression conditions for a N. farcinica protein (Nfa34810) showed:

For N. farcinica proteins, soluble expression is typically achieved using E. coli systems with proper optimization of induction parameters .

What purification strategies are most effective for recombinant N. farcinica IF-2?

For efficient purification of recombinant N. farcinica IF-2, a multi-step approach is recommended:

• Affinity chromatography: Ni-NTA columns for His-tagged proteins have shown excellent results with other N. farcinica recombinant proteins, achieving purity of approximately 96% .

• Additional purification steps if needed:

  • Ion exchange chromatography to separate charge variants

  • Size exclusion chromatography to eliminate aggregates and ensure homogeneity

• Optimization parameters:

  • Buffer composition (typically phosphate or Tris-based)

  • Salt concentration (typically 100-500 mM NaCl)

  • pH optimization (typically 7.0-8.0)

  • Inclusion of stabilizing agents (glycerol, reducing agents)

• Quality control methods:

  • SDS-PAGE for purity assessment

  • Western blotting for identity confirmation

  • Mass spectrometry for intact mass verification

  • Circular dichroism for secondary structure confirmation

When purifying other N. farcinica proteins, researchers have typically achieved high purity levels (>95%) using properly optimized Ni-NTA chromatography protocols .

How can the functional activity of recombinant N. farcinica IF-2 be assessed in vitro?

Multiple complementary assays can be employed to assess the functional activity of recombinant N. farcinica IF-2:

• GTPase activity assay:

  • Measure GTP hydrolysis rates using colorimetric phosphate detection or radioactive GTP

  • Compare kinetic parameters (Km, kcat) to IF-2 from other bacterial species

  • Assess the effect of ribosomes on GTPase activity

• Ribosome binding assays:

  • Measure binding to 30S and 70S ribosomes using filter binding or surface plasmon resonance

  • Assess protection of specific rRNA residues using chemical probing methods

  • Map ribosome interaction sites, particularly in domains V and VI of 23S rRNA (positions A2476, A2478, and the sarcin-ricin loop)

• Initiator tRNA binding assays:

  • Measure binding affinity to fMet-tRNA using fluorescence-based methods

  • Assess the effect of nucleotide binding (GTP vs. GDP) on tRNA binding

• Translation initiation complex formation:

  • Reconstitute 30S initiation complexes with purified components

  • Monitor 70S complex formation using light scattering or sucrose gradient centrifugation

  • Assess the rotational state of the ribosome using FRET-based methods

• Protein folding/chaperone activity:

  • Assess ability to promote refolding of denatured GFP or other model proteins

  • Compare chaperone activity to other bacterial IF-2 proteins

What structural domains are present in N. farcinica IF-2 and how do they contribute to function?

While the specific structure of N. farcinica IF-2 has not been fully characterized, bacterial IF-2 proteins typically contain several functional domains that likely exist in the N. farcinica homolog:

• N-terminal domain (NTD):

  • Highly variable in length and sequence among bacteria

  • Important for cold adaptation in some bacteria

  • May be involved in ribosome assembly/maturation

  • Can be completely deleted while maintaining basic translational function in vitro

• G-domain (G2):

  • Contains GTP binding and hydrolysis machinery

  • Undergoes significant conformational changes upon nucleotide binding

  • Interacts with the 50S ribosomal subunit

  • Specifically protects rRNA nucleotides in domain V (A2476, A2478) and domain VI (sarcin-ricin loop)

• Domain G3:

  • Functions in coordination with the G2 domain

  • Involved in relaying conformational changes

• Domain C1:

  • Connects G3 to the C-terminal domain

  • Shows independence of mobility from C2

• C-terminal domain (C2):

  • Responsible for binding initiator tRNA

  • Essential for translation initiation

  • In bacterial IF-2, shows independent mobility from C1

Experimental approaches to study domain functions include:

• Domain deletion analysis

• Site-directed mutagenesis of key residues

• Domain-specific antibody binding

• NMR structural analysis of isolated domains

How does N. farcinica IF-2 compare to IF-2 from other bacterial species?

A comparative analysis of N. farcinica IF-2 with other bacterial IF-2 proteins would reveal important similarities and differences:

• Sequence homology:

  • The G-domain and C-terminal domains are likely highly conserved

  • The N-terminal domain would show higher variability, as is typical among bacterial IF-2 proteins

• Functional conservation:

  • Core functions in translation initiation (fMet-tRNA binding, ribosome interactions) are expected to be conserved

  • Specialized functions may differ based on the ecological niche of N. farcinica as a soil-dwelling and potentially pathogenic organism

• Isoform expression:

  • Like E. coli, N. farcinica likely expresses multiple IF-2 isoforms (full-length and truncated forms)

  • The ratio of these isoforms might be regulated differently in response to environmental stressors

• Structural features:

  • Comparison to B. stearothermophilus IF-2 would reveal differences in domain organization and mobility

  • Unlike archaeal IF2 homologs, bacterial IF-2 typically shows independent mobility between the C1 and C2 domains

Methodological approaches for comparative analysis:

• Sequence alignment and phylogenetic analysis

• Heterologous complementation studies

• Structural modeling based on solved IF-2 structures

• Comparative biochemical assays under varying conditions

What role might N. farcinica IF-2 play in the bacterium's pathogenicity?

While the direct role of IF-2 in N. farcinica pathogenicity has not been established, several lines of evidence suggest potential contributions:

• Translation regulation during infection:

  • IF-2 could facilitate selective translation of virulence factors

  • Differential expression of IF-2 isoforms might occur during transition from environmental to host conditions

• Stress adaptation mechanisms:

  • IF-2 has been implicated in bacterial adaptation to stress conditions, which is critical during host infection

  • Full-length IF-2 has been shown to influence cellular recovery following DNA damage in bacteria, which could enhance survival during host immune responses

• Ribosome assembly function:

  • IF-2's role in ribosome assembly/maturation could be crucial for adaptation to the host environment

  • Proper ribosome biogenesis is essential for bacterial survival during infection

• Comparison with known N. farcinica virulence factors:

  • Known virulence factors like NFA49590 and Nfa34810 activate similar signaling pathways (MAPK, NF-κB)

  • These pathways are critical for modulating host immune responses

Research approaches to investigate this connection:

• Conditional expression systems to modulate IF-2 levels during infection

• Comparison of IF-2 expression in virulent versus attenuated strains

• Mutation studies targeting specific IF-2 domains

• Host-pathogen interaction studies using macrophage infection models

How can site-directed mutagenesis be applied to study specific domains of N. farcinica IF-2?

Site-directed mutagenesis provides a powerful approach to dissect the structure-function relationships of N. farcinica IF-2:

• Strategic mutation target selection:

  • GTP-binding motifs (G1-G4) in the G-domain to disrupt GTPase activity

  • Conserved residues in the C2 domain involved in fMet-tRNA binding

  • Residues at domain interfaces to investigate interdomain communication

  • N-terminal region residues to study isoform-specific functions

• Mutagenesis methodology:

  • PCR-based methods (QuikChange or overlap extension PCR)

  • Gibson Assembly for larger modifications

  • CRISPR-Cas9 for genomic modifications in N. farcinica

• Functional characterization of mutants:

  • GTPase activity assays to measure the impact on nucleotide hydrolysis

  • Ribosome binding assays to assess interaction with 30S and 50S subunits

  • In vitro translation assays to determine effects on protein synthesis

  • Thermal stability assays to evaluate structural integrity

• Domain-specific mutation strategies:

What experimental approaches can be used to study the interaction of N. farcinica IF-2 with the ribosome?

Several complementary techniques can be employed to characterize the interaction between N. farcinica IF-2 and ribosomes:

• Biochemical approaches:

  • Chemical cross-linking followed by mass spectrometry to identify interaction points

  • Filter-binding assays to measure binding kinetics

  • Competition assays with other initiation factors to assess binding sites

• Structural approaches:

  • Cryo-electron microscopy of IF-2-bound ribosomal complexes

  • Chemical probing of rRNA in the presence and absence of IF-2

  • NMR studies of isolated domains interacting with ribosomal components

• Biophysical approaches:

  • Surface plasmon resonance to measure binding kinetics

  • Isothermal titration calorimetry to determine thermodynamic parameters

  • Fluorescence-based assays to monitor conformational changes

• In vivo approaches:

  • Chromatin immunoprecipitation (ChIP) to detect IF-2 association with translational machinery in cells

  • Fluorescence microscopy to visualize IF-2-ribosome co-localization

  • Genetic complementation studies with IF-2 mutants

Based on studies of bacterial IF-2, key experimental considerations include:

• IF-2 protects specific sites in domain V (A2476, A2478) and domain VI (sarcin-ricin loop) of 23S rRNA

• IF-2 binding has a tightening effect on the association of ribosomal subunits

• IF-2 stabilizes ribosomes in a semi-rotated conformation during initiation

How does the expression of N. farcinica IF-2 respond to environmental stress conditions?

While specific data on N. farcinica IF-2 stress response is limited, bacterial IF-2 typically shows regulated expression under various stress conditions. Research methodologies to investigate this include:

• Transcriptional analysis:

  • qRT-PCR to measure infB mRNA levels under different stressors

  • Promoter-reporter fusion constructs to monitor transcriptional activity

  • RNA-seq to analyze global transcriptional changes including infB

• Translational regulation:

  • Western blotting to monitor IF-2 protein levels and isoform ratios

  • Ribosome profiling to assess translation efficiency of infB

  • Analysis of 5' UTR structural changes under stress conditions

• Environmental stressors to test:

  • Temperature shifts (both cold and heat shock)

  • Nutrient limitation

  • Oxidative stress

  • pH changes

  • Exposure to antibiotics

• Isoform regulation:

  • Monitor changes in the ratio of full-length (IF-2α) vs. truncated (IF-2β/γ) forms

  • Analyze differential activities of isoforms under stress conditions

In other bacteria, cold shock activates promoters in the nusA-infB operon and stabilizes infB transcripts, increasing IF-2 levels approximately 3-fold . This cold-induced increase in IF-2 appears to be involved in ribosome assembly/maturation rather than translational bias .

How can structural biology techniques be applied to study N. farcinica IF-2?

A multi-technique structural biology approach would provide comprehensive insights into N. farcinica IF-2:

Previous structural studies of bacterial IF-2 have revealed large structural rearrangements in the G2 subdomain upon nucleotide binding and considerable flexibility within domains , information that would guide similar studies with N. farcinica IF-2.

What is the potential of N. farcinica IF-2 as a target for anti-Nocardia therapeutics?

N. farcinica IF-2 presents several characteristics that make it a potential target for antibiotic development:

• Essential function:

  • IF-2 is essential for bacterial translation initiation

  • Inhibition would block protein synthesis and bacterial growth

• Structural divergence from host factors:

  • Bacterial IF-2 differs significantly from eukaryotic initiation factors

  • These differences could be exploited for selective targeting

• Treatment challenges in nocardiosis:

  • N. farcinica shows intrinsic resistance to multiple antibiotics

  • Current treatment relies heavily on TMP-SMX, but resistance is emerging

  • New therapeutic targets are needed, especially for disseminated infections with high mortality rates (39-85%)

• Drug development approaches:

  • Structure-based design targeting GTP-binding pocket

  • Peptide inhibitors disrupting IF-2/ribosome interaction

  • Small molecules blocking IF-2/fMet-tRNA binding

  • Compounds targeting the interface between domains

• Experimental validation methods:

  • In vitro translation assays to measure inhibition of protein synthesis

  • GTPase activity assays to assess functional inhibition

  • Growth inhibition assays with N. farcinica clinical isolates

  • Cell-based infection models to evaluate efficacy

• Considerations for targeting IF-2:

  • Need to achieve selectivity over host translation factors

  • Ability to penetrate the complex cell wall of Nocardia

  • Potential for resistance development through mutations

How can genome editing techniques be applied to study N. farcinica IF-2 function in vivo?

Genome editing approaches offer powerful tools to investigate IF-2 function directly in N. farcinica:

• CRISPR-Cas9 system adaptation:

  • Design sgRNAs targeting the infB gene

  • Optimize transformation protocols for N. farcinica

  • Include appropriate selection markers

  • Consider using a temperature-sensitive plasmid system similar to that used in other studies

• Strategic genetic modifications:

  • Create conditional knockdowns using inducible promoters

  • Generate domain-specific deletions

  • Introduce point mutations in functional motifs

  • Create reporter gene fusions for localization studies

• Phenotypic analysis of mutants:

  • Growth curve analysis under various conditions

  • Stress response profiling

  • Ribosome profiling to assess translation changes

  • Virulence assessment using infection models

• Complementation strategies:

  • Express wild-type or mutant variants from plasmids

  • Use heterologous IF-2 from other bacteria to assess functional conservation

  • Create chimeric IF-2 proteins to map domain functions

• Methodological considerations:

  • N. farcinica has a high GC content that may require codon optimization

  • The complex cell wall may require specialized transformation protocols

  • Homologous recombination efficiency may be lower than in model organisms

Successful gene deletion studies have been performed with other N. farcinica genes, such as nfa34810, demonstrating the feasibility of genetic manipulation in this organism .

How does N. farcinica IF-2 contribute to ribosome assembly and maturation?

Recent research suggests bacterial IF-2 has an unexpected role in ribosome assembly and maturation that may be important in N. farcinica:

• Evidence for ribosome assembly function:

  • Cold-stress induces increased IF-2 expression in bacteria

  • IF-2 associates with immature ribosomal subunits along with other assembly factors

  • Cold-sensitive IF-2 mutations cause accumulation of immature ribosomal particles

• Molecular mechanism:

  • IF-2 exhibits GTPase-associated chaperone activity

  • This chaperone function promotes refolding of denatured proteins

  • The activity appears particularly important at low temperatures

  • May facilitate proper folding of ribosomal proteins or rRNA during assembly

• Experimental approaches to study this function:

  • Ribosome profile analysis of cells with IF-2 mutations

  • Pull-down assays to identify IF-2-associated ribosome assembly factors

  • In vitro ribosome assembly assays with purified components

  • Chaperone activity assays using model substrates like GFP

• Physiological significance:

  • This function may be critical for N. farcinica adaptation to environmental stresses

  • Could contribute to survival during temperature fluctuations in soil

  • May play a role during host infection when bacteria face stress conditions