Recombinant Clavibacter michiganensis subsp. sepedonicus Elongation factor G (fusA), partial

What is Elongation factor G (fusA) and what is its function in Clavibacter michiganensis subsp. sepedonicus?

Elongation factor G (fusA) is a critical protein involved in bacterial protein synthesis that catalyzes the translocation step during elongation. In C. michiganensis subsp. sepedonicus, fusA functions as a GTPase that facilitates the movement of tRNA and mRNA through the ribosome after peptide bond formation. Unlike some other bacteria where fusA may have evolved additional functions, in C. michiganensis subsp. sepedonicus, the fusA gene appears to maintain its primary role in translation elongation. The protein is essential for bacterial survival and growth, making it an important target for understanding bacterial physiology and potential antimicrobial development. Comparative analysis with other actinomycetes suggests high conservation of this protein's core functional domains despite the extensive genomic rearrangements that have occurred in C. michiganensis subsp. sepedonicus .

How conserved is the fusA gene across Clavibacter species and related bacteria?

The fusA gene shows significant conservation across Clavibacter species and related actinomycetes, reflecting its essential role in protein synthesis. Alignment studies reveal that fusA in C. michiganensis subsp. sepedonicus shares high sequence identity (typically 90-95%) with orthologous genes in C. michiganensis subsp. michiganensis . This conservation is maintained despite the extensive chromosomal rearrangements that have occurred between these subspecies due to insertion sequence elements. The conservation pattern follows the taxonomic relationships within the family Microbacteriaceae in the order Actinomycetales, with decreasing similarity as taxonomic distance increases. Functional domains involved in GTP binding and ribosome interaction are the most highly conserved regions, while surface-exposed regions show higher variability. This conservation pattern makes fusA a potential marker for understanding evolutionary relationships within the Clavibacter genus .

What expression systems are most effective for producing recombinant fusA from C. michiganensis subsp. sepedonicus?

For expressing recombinant fusA from C. michiganensis subsp. sepedonicus, several expression systems have been evaluated with varying degrees of success:

The choice of expression system depends on research objectives. For structural studies requiring large quantities, E. coli BL21(DE3) with codon optimization may be preferable despite inclusion body challenges. For functional studies, Arctic Express strains grown at 16°C often produce more properly folded protein. Codon optimization based on the high G+C content (72.0%) of C. michiganensis subsp. sepedonicus is generally necessary for efficient expression .

How does the genomic context of fusA in C. michiganensis subsp. sepedonicus affect its expression and function?

The genomic context of fusA in C. michiganensis subsp. sepedonicus is significantly impacted by the extensive genomic rearrangements that have occurred within this species. The genome contains 106 insertion sequence (IS) elements, including 71 IS1121 elements, 25 ISCmi2 elements, and 9 ISCmi3 elements . These elements have mediated extensive chromosomal rearrangements compared to related subspecies. While fusA itself appears to be intact and functional, several genes in C. michiganensis subsp. sepedonicus have been disrupted by IS elements, with at least five CDSs directly interrupted .

The rearrangements may have impacted operonic structures and regulatory elements controlling fusA expression. Comparative genomics reveals that in some cases, IS element insertion has split coding sequences and moved the parts to distant chromosomal locations, which could potentially affect genes involved in fusA regulation . This genomic instability needs to be considered when designing expression constructs, as native regulatory elements may not function as expected in recombinant systems.

What structural and functional differences exist between fusA in C. michiganensis subsp. sepedonicus and fusA in other bacterial pathogens?

The fusA protein in C. michiganensis subsp. sepedonicus maintains the classical five-domain architecture common to bacterial elongation factors, but shows several notable differences from fusA in other bacterial pathogens:

• Domain III variations: Structural analyses suggest alterations in Domain III compared to other bacterial pathogens, potentially affecting interactions with ribosomal RNA.

• G' domain specificity: The G' subdomain of Domain I shows sequence signatures characteristic of Gram-positive actinomycetes with high G+C content like C. michiganensis subsp. sepedonicus.

• Functional diversity: Unlike in Pseudomonas plecoglossicida where fusA has been implicated in iron acquisition from plant ferredoxins , C. michiganensis fusA appears primarily dedicated to protein synthesis.

• Drug resistance correlations: While fusA mutations in Corynebacterium glutamicum and Brevibacterium flavum have been associated with fusidic acid resistance and increased lysine production , the relationship between fusA mutations and antibiotic resistance in C. michiganensis has not been fully characterized.

These structural and functional differences likely reflect the different evolutionary pressures experienced by C. michiganensis as an endophytic plant pathogen with limited ability to persist outside its host .

How might fusA mutations affect virulence and host specificity in C. michiganensis subsp. sepedonicus?

The relationship between fusA mutations and virulence in C. michiganensis subsp. sepedonicus remains largely unexplored, but several mechanisms can be proposed based on research in related bacteria:

• Growth rate effects: Since fusA is essential for protein synthesis, mutations affecting its efficiency would impact bacterial growth rates. In P. plecoglossicida, fusA knockout significantly affected growth capabilities . Even subtle changes in growth rate could affect the ability of C. michiganensis subsp. sepedonicus to establish infection within potato tissues.

• Stress response modification: Evidence from other bacteria suggests fusA's involvement in stress response. In P. plecoglossicida, expression of fusA was significantly reduced under temperature stress (4°C, 12°C, and 37°C) . Similar temperature sensitivity might affect C. michiganensis' ability to adapt to environmental conditions.

• Host interaction proteins: Efficient translation of virulence factors is critical for pathogenicity. Subtle alterations in fusA function could differentially affect the translation of specific virulence proteins, particularly those with rare codons or complex secondary structures in their mRNAs.

• Evolutionary implications: The estimated divergence time between C. michiganensis subsp. sepedonicus and C. michiganensis subsp. michiganensis (53,000 to 1,120,000 years ago) occurred after the speciation of their respective hosts (potato and tomato) , suggesting fusA evolution may have contributed to host adaptation.

What techniques are most effective for studying the ribosomal interactions of recombinant fusA from C. michiganensis subsp. sepedonicus?

For investigating ribosomal interactions of recombinant fusA from C. michiganensis subsp. sepedonicus, researchers should consider this methodological hierarchy:

A comprehensive approach might begin with filter binding to establish basic parameters, progress to FRET for understanding dynamics, and culminate in cryo-EM studies for structural detail. For C. michiganensis subsp. sepedonicus specifically, the high G+C content (72.0%) presents challenges for heterologous ribosome production, so hybrid systems using purified components might be necessary.

How can comparative genomics of fusA across Clavibacter subspecies inform our understanding of bacterial adaptation and pathogenicity?

Comparative genomics of fusA across Clavibacter subspecies provides valuable insights into bacterial adaptation and pathogenicity through several analytical approaches:

These approaches can collectively illuminate how changes in fundamental cellular machinery like fusA may contribute to host specificity and virulence in plant pathogens.

What are the optimal conditions for expressing and purifying recombinant C. michiganensis subsp. sepedonicus fusA?

The optimal conditions for expressing and purifying recombinant C. michiganensis subsp. sepedonicus fusA are outlined below:

Expression Protocol:

• Vector selection: pET-28a(+) with N-terminal His-tag provides good results due to the placement of the tag away from critical functional domains.

• Host strain: E. coli BL21(DE3) pLysS helps control leaky expression of potentially toxic fusA protein.

• Growth conditions: Initial growth at 37°C to OD600 = 0.6-0.8, followed by temperature reduction to 18°C before induction.

• Induction parameters: 0.2-0.5 mM IPTG, 16-18 hours at 18°C.

• Media composition: TB (Terrific Broth) supplemented with 1% glucose to suppress basal expression and 5-10 μM ZnCl2 to stabilize zinc-finger domains.

Purification Strategy:

• Initial capture: Ni-NTA affinity chromatography with gradual imidazole gradient (10-250 mM) to separate full-length protein from truncated products.

• Intermediate purification: Heparin affinity chromatography exploits fusA's nucleic acid binding properties.

• Polishing step: Size exclusion chromatography in buffer containing 20 mM HEPES pH 7.5, 100 mM KCl, 10 mM MgCl2, 5% glycerol, and 1 mM DTT.

This methodological approach addresses the challenges posed by the high G+C content (72.0%) of C. michiganensis subsp. sepedonicus and the potential toxicity of overexpressed translation factors.

What mutagenesis approaches are most effective for studying functional domains of fusA in C. michiganensis subsp. sepedonicus?

For studying the functional domains of fusA in C. michiganensis subsp. sepedonicus, several mutagenesis approaches can be employed:

For C. michiganensis subsp. sepedonicus specifically, the high G+C content (72.0%) can complicate PCR-based methods, requiring specialized polymerases and buffer conditions. When designing primers for site-directed mutagenesis, researchers should avoid regions containing repetitive sequences associated with the numerous IS elements found in the genome . In designing domain swapping experiments, the extensive genomic rearrangements observed in C. michiganensis subsp. sepedonicus compared to related species suggest a high tolerance for structural changes, potentially facilitating chimeric protein construction.

How can functional assays be designed to evaluate the impact of fusA mutations on C. michiganensis subsp. sepedonicus virulence?

Designing functional assays to evaluate the impact of fusA mutations on C. michiganensis subsp. sepedonicus virulence requires a multi-tiered approach:

In Vitro Assays:

• Translation efficiency: Measuring poly(U)-dependent poly(Phe) synthesis rates using purified components with wild-type versus mutant fusA proteins.

• GTPase activity assays: Quantifying inorganic phosphate release to assess the catalytic efficiency of different fusA variants.

• Ribosome binding studies: Using surface plasmon resonance to measure binding kinetics between fusA variants and purified ribosomes.

Cellular Assays:

• Growth rate determination: Comparing growth curves of C. michiganensis strains expressing different fusA variants, similar to methods used for P. plecoglossicida .

• Stress response evaluation: Assessing survival under various stressors (temperature extremes, oxidative stress, pH shifts) to determine if fusA mutations affect stress adaptation.

• Biofilm formation: Quantifying biofilm development using crystal violet staining protocols similar to those used for P. plecoglossicida .

Plant Infection Assays:

• Potato microtuber infection: Inoculating microtubers with bacterial strains carrying different fusA variants and quantifying disease progression.

• Stem injection assays: Direct injection of bacteria into potato stems followed by monitoring of symptom development and bacterial spread.

• Competitive index studies: Co-inoculating wild-type and mutant strains to assess relative fitness in planta.

These assays should be designed with appropriate controls and statistical analyses, considering the temperature-dependent nature of bacterial pathogenicity as observed in other systems .

What bioinformatic approaches are most valuable for analyzing fusA sequences across the Clavibacter genus?

For comprehensive analysis of fusA sequences across the Clavibacter genus, researchers should implement these bioinformatic approaches:

Sequence-Based Analysis:

• Multiple sequence alignment: Using MAFFT or T-Coffee algorithms optimized for high G+C content sequences to properly align fusA homologs.

• Phylogenetic reconstruction: Employing maximum likelihood methods with appropriate substitution models for high G+C sequences to construct robust evolutionary trees.

• Selection pressure analysis: Calculating site-specific dN/dS ratios to identify regions under positive, neutral, or purifying selection.

• Recombination detection: Using programs like RDP4 to identify potential recombination events, particularly important given the high number of IS elements in C. michiganensis subsp. sepedonicus .

Structural Bioinformatics:

• Homology modeling: Building structural models based on existing bacterial EF-G crystal structures to predict the impact of sequence variations.

• Molecular dynamics simulations: Simulating fusA protein dynamics to assess how subspecies-specific variations might affect function.

• Coevolution analysis: Identifying co-evolving residues that might indicate functional interactions within the protein or with binding partners.

Genomic Context Analysis:

• Synteny mapping: Analyzing the genomic neighborhood of fusA across species, considering the extensive rearrangements observed in C. michiganensis subsp. sepedonicus .

• Promoter analysis: Identifying regulatory elements that might differ between subspecies.

• IS element mapping: Documenting proximity of IS elements to fusA in different species, given their abundance in C. michiganensis subsp. sepedonicus (106 IS elements) .

These approaches can reveal patterns of fusA evolution within the Clavibacter genus and provide context for experimental studies.

What are the most promising research avenues for understanding fusA's role in C. michiganensis subsp. sepedonicus pathogenicity?

Based on current knowledge, several research directions stand out as particularly promising for understanding fusA's role in C. michiganensis subsp. sepedonicus pathogenicity:

• Comparative ribosome profiling: Applying ribosome profiling to compare translational landscapes between wild-type and fusA mutant strains during infection could reveal how fusA activity influences the expression of virulence factors.

• Investigation of potential moonlighting functions: While primarily a translation factor, fusA may have secondary roles similar to those observed in other bacteria. The findings that fusA in Pectobacterium species is involved in iron acquisition from plant ferredoxins suggests potential additional functions worth exploring in C. michiganensis subsp. sepedonicus.

• Host-pathogen protein interaction studies: Investigating whether fusA directly interacts with any host proteins could reveal unexpected roles in virulence, similar to how other bacterial translation factors have been shown to moonlight as virulence factors.

• Impact of genomic instability on fusA function: Given the extensive genomic rearrangements observed in C. michiganensis subsp. sepedonicus , studying how this genomic instability affects fusA expression and function could provide insights into bacterial adaptation mechanisms.