Recombinant Leuconostoc citreum Ribosome-recycling factor (frr)

What is Leuconostoc citreum and why is it important for recombinant protein research?

Leuconostoc citreum is a gram-positive, non-sporulating hetero-fermentative lactic acid bacterium (LAB) that plays a significant role in the fermentation industry. It has gained importance in recombinant protein research due to several advantageous characteristics:

• Food-grade status, making it suitable for production of proteins intended for food or pharmaceutical applications

• Fermentation capabilities that produce aromatic compounds and useful byproducts like dextran

• Genomic plasticity, demonstrated by specialized metabolic adaptations in strains like the pseudofructophilic F192-5

• Potential as an expression host with promising results using bicistronic design expression systems

These characteristics make L. citreum a valuable alternative to traditional expression hosts, particularly where food-grade production is required. Recent development of expression systems specifically optimized for L. citreum has further enhanced its utility in recombinant protein research.

What is the function of ribosome-recycling factor (frr) in bacterial translation?

The ribosome recycling factor (RRF), encoded by the frr gene, performs an essential role in the final stage of protein translation in bacteria:

• It dissociates ribosomes from mRNA after translation termination, effectively "recycling" ribosomes for new rounds of protein synthesis

• It works synergistically with elongation factor G (EF-G) and initiation factor 3 (IF3) to split post-termination ribosomal complexes into their subunits

• It facilitates efficient transition between termination and initiation phases of translation

Research has conclusively demonstrated that frr is essential for bacterial cell growth. Studies with Escherichia coli showed that strains carrying frame-shifted frr in the chromosome were non-viable unless complemented with a functional frr gene, confirming that ribosome recycling factor is indispensable for cellular viability and protein synthesis in bacteria . This essential nature must be considered when designing recombinant expression systems involving frr modification.

What are the structural characteristics of the frr gene in bacterial systems?

Computer-based secondary structure analysis has revealed that the ribosome recycling factor protein has a structure consisting of three primary domains:

• Domain A: Contains the N-terminal helix

• Domain B: Includes coil, alpha-helix, and beta-strand structures

• Domain C: Comprises the C-terminal helix

The functional importance of these domains has been revealed through mutation studies:

What expression systems are currently used for recombinant protein production in Leuconostoc citreum?

Several expression systems have been developed for recombinant protein production in L. citreum, with significant recent advances:

• Bicistronic Design (BCD) Expression System:

  • Structure: Includes a short leader peptide (1st cistron) followed by the target gene (2nd cistron) under control of a single promoter

  • Validation: Functionality verified using superfolder green fluorescent protein (sfGFP) as a reporter

  • Enhancements: Improved through engineering of the Shine-Dalgarno sequence (SD2) for the 2nd cistron and stronger promoter (P710V4) isolated through FACS screening

• Monocistronic Design (MCD) Expression System:

  • Conventional approach used as comparison for evaluating BCD performance

  • Significantly lower expression levels compared to optimized BCD systems

• Dextran-free Expression Host Systems:

  • Developed through homologous recombination of the dextransucrase gene (dsrC)

  • Provides hosts for production of recombinant proteins without viscosity issues caused by dextran

Comparative performance data for these systems using model proteins shows the engineered BCD system significantly outperforms conventional approaches:

These systems have been successfully validated with various model proteins, including glutathione-s-transferase, human growth hormone, and α-amylase .

How can bicistronic design (BCD) expression systems be optimized for frr expression in Leuconostoc citreum?

Optimizing a bicistronic design expression system for frr expression in L. citreum requires several strategic approaches:

• Engineering the Shine-Dalgarno Sequence (SD2):

  • Optimize the SD2 sequence for the 2nd cistron (containing frr) through FACS screening of random libraries

  • Implement the enhanced SD2 (eSD2) previously isolated for general recombinant protein expression

• Promoter Engineering:

  • The P710V4 promoter, identified through random library screening, shows strong activity in L. citreum

  • For frr expression, consider inducible promoters to control expression level, as overexpression of translation factors can be detrimental

• Leader Peptide Optimization:

  • Test multiple leader peptide variants to identify sequences that enhance frr expression

  • Optimize length and sequence of the 1st cistron for efficient translation initiation of the downstream frr gene

• Host Strain Selection:

  • Consider dextran-free mutant strains as expression hosts to facilitate downstream purification

  • Evaluate pseudofructophilic strains (like F192-5) for expression in fructose-rich media

Implementation should include quantitative validation using appropriate reporter systems. Previous success with model proteins suggests this approach has high potential for optimized frr expression in L. citreum .

What are the critical domains in the frr protein that affect its functionality?

Based on structural and functional studies, several critical domains and specific amino acid residues significantly impact RRF functionality:

• Domain Structure and Functional Significance:

• Key Amino Acid Residues:

  • Arginine 132 (Arg132) in domain C: Substitution observed in five independently isolated null mutants, strongly suggesting it is part of an active site

  • Other conserved residues likely involved in ribosome binding interface and EF-G interaction

Understanding these critical domains is essential for designing effective mutagenesis studies, interpreting functional impacts of mutations, and engineering variants with enhanced properties. When working with recombinant L. citreum frr, researchers should be particularly cautious about modifications to domain B for structural stability and to the Arg132 region for maintaining functional activity .

What are the effects of site-directed mutagenesis on frr function?

Site-directed mutagenesis studies on frr reveal several categories of mutations with distinct functional consequences:

• Null Mutations:

  • 52 identified null mutations classified into six categories

  • Most common in domain B (>50% of all null mutations)

  • Completely abolish RRF function, resulting in non-viable cells unless complemented

• Temperature-Sensitive (ts) Mutations:

  • 12 identified ts mutations, predominantly in domains A and C

  • Function at permissive temperatures but lose function at non-permissive (higher) temperatures

  • Valuable tools for studying RRF function through conditional inactivation

• Reversion Mutations:

  • Six documented reversions restoring function to non-functional variants

  • Provide insights into structure-function relationships

• Silent Mutations:

  • Five identified silent mutations that don't significantly affect function

  • Typically fall outside domain B, consistent with this domain's structural importance

• Specific Residue Effects:

  • Substitution of Arg132 in domain C observed in multiple independent null mutants

  • Indicates this residue's critical importance for function

When performing site-directed mutagenesis on L. citreum frr, researchers should consider potential lethality of mutations in critical domains, complementation strategies for studying essential functions, and the particular importance of domain B for structural integrity and domain C for function.

How do temperature-sensitive mutations in the frr gene affect protein synthesis?

Temperature-sensitive (ts) mutations in the frr gene have pronounced effects on protein synthesis due to RRF's essential role in translation:

• Conditional Translation Defects:

  • At permissive temperatures: Protein synthesis proceeds normally as ts-RRF retains functionality

  • At non-permissive temperatures: Ribosome recycling becomes impaired, leading to:

    • Accumulation of stalled ribosomes on mRNAs

    • Reduced availability of free ribosomes for new translation

    • Global decrease in protein synthesis rates

• Growth Arrest Phenotype:

  • ts-RRF mutants exhibit growth arrest at non-permissive temperatures

  • This arrest directly links to impaired protein synthesis due to inadequate ribosome recycling

• Structural Basis:

  • ts mutations map primarily to domains A and C, not domain B

  • Suggests domains A and C are involved in temperature-dependent functional interactions rather than core stability

• Suppressor Interactions:

  • Intergenic suppressor strains partially restore growth at non-permissive temperatures

  • Classified into four categories based on which ts alleles they suppress

For L. citreum researchers, temperature-sensitive frr mutants offer valuable tools for studying RRF's role under controlled conditions, investigating translation hierarchy when capacity is limited, and identifying species-specific interactions in the translation machinery.

What are the challenges in purifying recombinant frr protein from Leuconostoc citreum?

Purifying recombinant ribosome recycling factor from L. citreum presents several challenges that researchers must address:

These strategies should be combined with gentle purification conditions and appropriate functional validation to ensure the isolated recombinant frr retains its native activity.