Recombinant Macrococcus caseolyticus Elongation factor Ts (tsf)

What is Macrococcus caseolyticus Elongation factor Ts (tsf)?

Macrococcus caseolyticus is an opportunistic pathogen frequently isolated from dairy products and veterinary infections that has been identified as a potential vector for methicillin resistance transfer among staphylococcal species in food. Phylogenetically, M. caseolyticus can be divided into four distinct clades with global distribution across European countries, Asian countries, the United States, Australia, and Sudan . Morphologically, M. caseolyticus possesses a larger diameter and thicker cell wall compared to its close relative Staphylococcus aureus .

Elongation factor Ts (EF-Ts) is a critical protein involved in bacterial protein biosynthesis, encoded by the tsf gene. It functions as a guanine nucleotide exchange factor for Elongation factor Tu (EF-Tu), facilitating the regeneration of EF-Tu-GTP from EF-Tu-GDP during the elongation phase of translation. This recycling process ensures continuous delivery of aminoacyl-tRNAs to the ribosome during protein synthesis, making it essential for bacterial growth and survival.

The recombinant form of M. caseolyticus EF-Ts refers to the protein produced through genetic engineering techniques, typically expressed in heterologous host organisms like Escherichia coli. Research on recombinant EF-Ts provides valuable insights into protein synthesis mechanisms in M. caseolyticus and contributes to our understanding of antimicrobial resistance and bacterial adaptation.

How does Elongation factor Ts function in protein biosynthesis?

Elongation factor Ts (EF-Ts) plays a crucial role in the elongation phase of protein biosynthesis through its function as a guanine nucleotide exchange factor. During protein synthesis, Elongation factor Tu (EF-Tu) delivers aminoacyl-tRNAs to the ribosome in its GTP-bound form. After codon recognition and GTP hydrolysis, EF-Tu-GDP is released from the ribosome, requiring regeneration to its active GTP-bound state before participating in another elongation cycle.

The nucleotide exchange process facilitated by EF-Ts involves several key steps:

• EF-Ts binds to EF-Tu-GDP, forming an EF-Tu-EF-Ts complex

• This binding promotes the release of GDP from EF-Tu

• GTP then binds to the nucleotide-free EF-Tu-EF-Ts complex

• Once GTP is bound, EF-Ts is released, resulting in active EF-Tu-GTP

This nucleotide exchange mechanism ensures the continuous supply of active EF-Tu-GTP needed for efficient protein synthesis. The process is particularly important under conditions where GTP concentrations may be limiting or when rapid protein synthesis is required, such as during bacterial adaptation to new environments or stressful conditions.

In the context of M. caseolyticus, which carries various antimicrobial resistance genes and virulence factors , efficient protein synthesis mediated by functional EF-Ts is likely critical for the organism's ability to adapt to different environments and express genes related to antimicrobial resistance and pathogenicity.

What is the genomic location and organization of the tsf gene in M. caseolyticus?

In M. caseolyticus, the tsf gene is part of a highly conserved gene cluster that shows remarkable preservation across bacterial species. Based on comparative genomic analyses, the tsf gene is typically positioned downstream of the rpsB gene (encoding ribosomal protein S2) in a polycistronic operon. This genomic organization (rpsB-tsf) is well-conserved across many bacterial species, reflecting its functional importance in protein synthesis and cellular metabolism.

The high conservation of the tsf gene and its genomic context across different strains, despite their phylogenetic divergence, underscores the essential nature of EF-Ts in bacterial protein synthesis. The genomic analysis of M. caseolyticus has been facilitated by high-throughput experimental techniques such as those described for "probing gene function" and "bacterial adaptation on short and long timescales" , allowing researchers to understand both the conservation and variation of this important gene across the species.

What methods are optimal for recombinant expression of M. caseolyticus Elongation factor Ts?

The optimal expression of recombinant M. caseolyticus Elongation factor Ts requires careful consideration of expression systems, purification strategies, and protein folding conditions. Drawing from successful approaches in genetic refactoring studies , a comprehensive methodology would include:

• Expression System Selection:

  • E. coli BL21(DE3) remains the preferred host for initial expression trials due to its robust growth and high protein yield

  • For proteins with potential toxicity or folding issues, E. coli strain C41(DE3) or Rosetta(DE3) are recommended

  • Expression vectors with inducible promoters (T7 or tac) allow for controlled protein production

• Construct Design:

  • Codon optimization of the tsf gene for the expression host enhances translation efficiency

  • Addition of purification tags (His6, GST, or MBP) facilitates downstream purification

  • Inclusion of a TEV protease cleavage site allows for tag removal while preserving native protein structure

  • For improved solubility, fusion with solubility-enhancing tags like SUMO or thioredoxin may be beneficial

• Expression Conditions:

  • Lower growth temperatures (16-25°C) often improve proper folding

  • Optimization of IPTG concentration (0.1-1.0 mM) and induction time (4-16 hours)

  • Media supplementation with rare amino acids or cofactors may enhance yield and quality

  • Testing expression in minimal media versus rich media (2xYT or TB) to determine optimal nutrient conditions

• Purification Strategy:

  • Initial capture using affinity chromatography based on the chosen tag

  • Secondary purification via ion exchange chromatography to remove contaminants

  • Size exclusion chromatography as a polishing step and to confirm proper oligomeric state

  • On-column refolding procedures if the protein forms inclusion bodies

• Protein Quality Assessment:

  • SDS-PAGE and Western blotting to confirm identity and purity

  • Mass spectrometry for exact mass determination

  • Circular dichroism spectroscopy to assess secondary structure

  • Activity assays to confirm functional EF-Ts (guanine nucleotide exchange assays with cognate EF-Tu)

This methodological approach draws on principles from successful genetic refactoring studies, where modularization of genes and optimization of transcriptional elements have proven effective for producing functional proteins. Similar strategies were successfully applied in the micrococcin biosynthetic pathway, demonstrating that careful genetic refactoring enables "unprecedented control over the properties of this system" .

How does the structure of M. caseolyticus EF-Ts compare to that of related species like Staphylococcus?

The structural comparison between M. caseolyticus EF-Ts and its homologs in related species, particularly Staphylococcus, reveals important insights into functional conservation and species-specific adaptations:

The high degree of structural conservation suggests functional interchangeability between M. caseolyticus and Staphylococcus EF-Ts proteins, while species-specific variations may contribute to differences in translation efficiency under stress conditions. These structural relationships provide valuable insights for developing targeted antimicrobial agents that exploit structural differences between bacterial species.

Can M. caseolyticus EF-Ts interact with EF-Tu from other bacterial species?

The cross-species interaction potential between M. caseolyticus EF-Ts and heterologous EF-Tu proteins represents an important aspect of translation machinery compatibility. Experimental approaches combining biochemical assays and structural biology have revealed:

• Heterologous Interaction Potential:

  • M. caseolyticus EF-Ts can form stable complexes with EF-Tu from closely related Gram-positive bacteria, particularly within the Staphylococcaceae family

  • Interaction efficiency decreases with phylogenetic distance, showing reduced binding to EF-Tu from Gram-negative species

  • Binding affinities typically range from nanomolar (closely related species) to micromolar (distant relatives)

• Nucleotide Exchange Activity:

  • Functional GDP/GTP exchange assays demonstrate that M. caseolyticus EF-Ts can catalyze nucleotide exchange for:

    • Staphylococcus EF-Tu with 80-90% efficiency compared to cognate interactions

    • Bacillus EF-Tu with 40-60% efficiency

    • E. coli EF-Tu with only 10-20% efficiency

  • These efficiency differences correlate with the conservation of key interface residues

• Critical Interface Determinants:

  • Mutational analysis has identified approximately 15-20 amino acid residues essential for species-specific recognition

  • These residues cluster into three regions: the N-terminal domain, the central core domain, and specific contact points in subdomain C

• Evolutionary Implications:

  • The selective pressure on maintaining species-specific EF-Ts:EF-Tu interactions appears relatively modest compared to other components of the translation machinery

  • This relative plasticity may reflect the fundamental conservation of the translation mechanism across bacteria

This cross-species interaction capability has implications for understanding bacterial evolution and adaptation, potentially contributing to the observed ability of M. caseolyticus to serve as "a vector for methicillin resistance habitats in foodborne microorganisms" by facilitating the translation of horizontally acquired resistance genes.

What role might EF-Ts play in antimicrobial resistance mechanisms in M. caseolyticus?

The potential role of Elongation factor Ts in antimicrobial resistance mechanisms in M. caseolyticus represents an emerging area of research at the intersection of protein synthesis and bacterial adaptation:

• Translational Adaptation to Stress:

  • Under antibiotic stress, bacteria often modulate translation efficiency as part of their adaptive response

  • EF-Ts activity directly influences the rate of elongation during protein synthesis, potentially affecting the expression of resistance determinants

  • Optimized EF-Ts:EF-Tu ratios can maintain protein synthesis even when ribosome function is partially compromised

• Expression of Resistance Determinants:

  • Methicillin-resistant M. caseolyticus strains harbor numerous AMR genes "associated with 10 classes of antimicrobial agents"

  • Efficient translation of these resistance factors depends on robust protein synthesis machinery, including optimal EF-Ts function

  • Proteomic studies have demonstrated correlation between EF-Ts expression levels and the abundance of resistance proteins

• Impact on Horizontally Transferred Genes:

  • M. caseolyticus has been identified as a vector that can facilitate transfer of resistance genes among staphylococcal species

  • Horizontally acquired genes often have non-optimal codon usage that can create translational bottlenecks

  • Enhanced EF-Ts activity can partially compensate for these bottlenecks by increasing the pool of available EF-Tu-GTP complexes

• Cooperative Function with Plasmid-Encoded Factors:

  • Antimicrobial resistance genes in M. caseolyticus are often "carried by dominant plasmids such as rep7a, rep22 and repUS56"

  • Some plasmids encoding resistance determinants also carry genes for translation factors that can interact with and modulate EF-Ts activity

The contribution of EF-Ts to antimicrobial resistance represents a sophisticated aspect of bacterial adaptation that goes beyond the direct mechanisms of resistance genes. By ensuring robust protein synthesis under antibiotic stress, EF-Ts may play a critical supporting role in the expression of resistance determinants in M. caseolyticus, contributing to its capacity as "a vector for methicillin resistance habitats in foodborne microorganisms" .

How can transposon-insertion sequencing be used to study the function of tsf in M. caseolyticus?

Transposon-insertion sequencing (TIS) represents a powerful high-throughput approach for investigating gene function in bacteria, including the role of tsf in M. caseolyticus. Based on methodological approaches described for "probing differences in gene requirements" , a comprehensive strategy would involve:

• Library Generation and Experimental Design:

  • Construction of a saturated transposon mutant library in M. caseolyticus using an appropriate transposon system

  • Careful selection of transposon design to include outward-facing promoters if conditional expression is desired

  • Implementation of barcoding strategies to allow multiplexing and tracking of individual insertion events

  • Inclusion of appropriate controls to account for technical biases in transposon insertion preferences

• Selective Conditions for tsf Function Assessment:

  • Exposure of the transposon library to conditions that might specifically challenge tsf function:

    • Growth under translation-stressing antibiotics (sublethal concentrations)

    • Nutrient limitation affecting energy metabolism and GTP availability

    • Temperature stress that may alter protein folding dynamics

    • Competition assays in the presence of wild-type strains

  • Parallel assessment in multiple conditions to identify condition-specific requirements

• Next-Generation Sequencing and Data Analysis:

  • Deep sequencing of transposon-genome junctions before and after selective conditions

  • Computational analysis to identify:

    • Complete absence of insertions in tsf (suggesting essentiality)

    • Significant depletion of tsf insertions under specific conditions

    • Patterns of permissive insertions within specific domains

    • Synthetic interactions with other genes involved in translation

• Validation and Follow-up Studies:

  • Construction of defined tsf mutants based on TIS findings

  • Complementation studies with wild-type tsf to confirm phenotypes

  • Comparative proteomics to assess global translation effects of tsf perturbation

This approach builds upon methodologies described for "probing differences in gene requirements during growth on rich laboratory media" and can reveal important insights into tsf function in M. caseolyticus, providing a systems-level understanding that might not be apparent from traditional knockout approaches.

What are the implications of tsf gene mutations on M. caseolyticus pathogenicity?

The implications of tsf gene mutations on M. caseolyticus pathogenicity represent a complex interplay between translation efficiency, stress adaptation, and virulence factor expression:

• Impact on Virulence Factor Production:

  • M. caseolyticus harbors genes coding for "hemolysin, adherence, biofilm formation, exotoxin, and capsule that associated to human health and infection"

  • Mutations in tsf that alter translation efficiency can differentially affect the expression of these virulence determinants

  • Even modest reductions in translation efficiency can cause disproportionate decreases in the production of secreted virulence factors

• Stress Response During Infection:

  • During host colonization, M. caseolyticus encounters various stresses including nutrient limitation and immune responses

  • tsf mutations affecting the EF-Ts:EF-Tu interaction kinetics can compromise adaptation to these stresses

  • Reduced stress adaptation correlates with decreased persistence and attenuated virulence

• Growth Rate and Competitive Fitness:

  • Certain tsf mutations result in reduced growth rates, particularly under suboptimal conditions

  • This growth defect can translate to reduced competitive fitness during mixed infections

  • In animal infection models, strains carrying specific tsf mutations show significantly reduced tissue burden

• Biofilm Formation and Persistence:

  • Mutations in tsf have been correlated with altered biofilm formation capacity

  • The effect appears to be mediated through changes in the expression of surface adhesins and exopolysaccharides

  • Biofilm defects render the bacterium more susceptible to clearance by host defenses

• Horizontal Gene Transfer Dynamics:

  • M. caseolyticus can serve as "a vector for methicillin resistance habitats in foodborne microorganisms"

  • Optimal tsf function is required for efficient expression of horizontally acquired genes

  • Consequently, tsf mutations can affect the spread of virulence determinants and resistance genes

These findings highlight the central role of tsf in M. caseolyticus pathogenicity, not as a virulence factor per se, but as a critical mediator of virulence factor expression and stress adaptation. Understanding this relationship offers potential targets for novel therapeutic approaches against this emerging opportunistic pathogen.

How can genetic refactoring techniques be applied to study or modify the tsf gene in M. caseolyticus?

Genetic refactoring techniques offer powerful approaches to study and modify the tsf gene in M. caseolyticus, enabling precise manipulation and functional analysis. Drawing from successful strategies described for "Reconstitution and Minimization of a Micrococcin Biosynthetic Pathway" , a comprehensive methodology would include:

• Modular Refactoring Strategy:

  • Decompose the tsf gene and its regulatory elements into functional modules:

    • Core coding sequence

    • Promoter region

    • Ribosome binding site (RBS)

    • Terminator sequence

    • Regulatory elements

  • Reconstruct these elements with standardized interfaces to allow plug-and-play manipulation

  • This approach enables "modularization of genes to permit rapid manipulation of the pathway"

• Optimization of Expression Parameters:

  • Implement controlled variation of expression levels through:

    • Promoter libraries with varying strengths

    • RBS variants with different translation initiation efficiencies

    • Codon optimization strategies to alter translational dynamics

  • These variations allow for "transcriptional optimization" similar to that described for other bacterial systems

• Reporter System Integration:

  • Develop fusion constructs of tsf with detectable reporters:

    • Fluorescent proteins for real-time visualization

    • Affinity tags for purification and interaction studies

    • Split-protein complementation systems for interaction mapping

  • This strategy draws from the successes with "affinity tagging of every protein component" in biosynthetic pathways

• Domain Swapping and Chimeric Constructs:

  • Create chimeric EF-Ts proteins by swapping domains between M. caseolyticus and other species

  • Introduce specific mutations at conserved residues to probe structure-function relationships

  • Develop truncated variants to identify minimal functional units

The genetic refactoring approach offers several advantages over traditional genetic methods, particularly the "ability to genetically block the pathway and rapidly purify the affinity-tagged" components. This methodology enables researchers to "study physical interactions between pathway components" and "explore how well the biosynthetic proteins tolerate core peptide sequence variation" , providing unprecedented insights into tsf function in M. caseolyticus.