Recombinant Neisseria gonorrhoeae Methionyl-tRNA formyltransferase (fmt)

What is Methionyl-tRNA formyltransferase (fmt) and what role does it play in Neisseria gonorrhoeae?

Methionyl-tRNA formyltransferase (Fmt) is a bacterial enzyme that catalyzes the formylation of methionyl-tRNA (Met-tRNA) to produce formylmethionyl-tRNA (fMet-tRNA). This reaction is crucial for efficient initiation of translation in bacteria including Neisseria gonorrhoeae and in eukaryotic organelles such as mitochondria . The formylation reaction converts Met-tRNA to fMet-tRNA, which plays a critical role in the translation initiation process by improving the association between the initiator tRNA and the ribosome . Fmt functions as part of the bacterial protein synthesis machinery, where the formyl group attached to methionine serves as a positive determinant for the bacterial initiation factor IF2 and as a negative determinant for elongation factor EF-Tu . In bacterial systems like N. gonorrhoeae, this formylation step is essential for distinguishing initiator tRNA from elongator tRNAs, ensuring proper start codon recognition and translation initiation.

What transformation methods are effective for genetic manipulation of N. gonorrhoeae to study fmt function?

Neisseria gonorrhoeae is naturally competent for transformation, making it amenable to genetic manipulation without requiring chemical or physical treatments typically needed for other bacterial species . There are several established methods for transformation in N. gonorrhoeae, each with particular advantages for studying genes like fmt:

• Spot Transformation: This traditional method involves drying a known quantity of transformation DNA (tDNA) on an agar plate, then overlaying bacteria using a swab. While versatile and requiring minimal intervention, this method makes quantification of transformation efficiency challenging .

• Liquid Transformation: This approach involves adding tDNA to bacteria in magnesium-supplemented liquid medium, followed by incubation and plating. It ensures uniform access between DNA and bacterial cells but requires multiple steps that are not easily scalable for high-throughput experiments .

• New Spot Transformation: A hybrid method combining advantages of both traditional approaches. It involves adding tDNA to a bacterial suspension of known density that is dried on an agar plate, ensuring all bacteria contact the tDNA. After incubation, the entire DNA-bacteria spot is resuspended for transformation efficiency assessment. This method has been shown to yield better quantification while maintaining experimental simplicity .

Optimization of transformation efficiency involves adjusting cell number input, with research showing that using a bacterial suspension with an Optical Density of 0.0007 at 600 nm together with 500 ng of tDNA yields higher transformation efficiency .

What factors affect transformation efficiency when working with recombinant N. gonorrhoeae fmt?

Several factors significantly impact transformation efficiency in Neisseria gonorrhoeae, which are important to consider when working with recombinant fmt or any genetic manipulation:

• Cell-to-DNA Ratio: There is an optimal ratio between cell number and transformation DNA (tDNA) amount. Research has demonstrated that a bacterial suspension with an Optical Density of 0.0007 at 600 nm combined with 500 ng of tDNA produces higher transformation efficiency .

• DNA Size and Homology: Transformation efficiency increases with molecular size and degree of homology. Longer DNA fragments provide extended flanking regions available for recombination, improving integration efficiency .

• DNA Uptake Sequences (DUS): These 10-12 base pair sequences facilitate uptake and transformation of DNA molecules in Neisseria species. DUS sequences occur approximately every thousand base pairs in N. gonorrhoeae genome, and their presence in transformation DNA can significantly enhance uptake efficiency .

• Presence of Magnesium: Magnesium supplementation in the transformation medium is essential for optimal transformation efficiency .

• Competence Pili: The presence and functionality of competence pili on the bacterial surface affects transformation efficiency .

• Incubation Time: The duration of exposure between bacteria and DNA impacts transformation success .

When designing transformation experiments for studying fmt or introducing recombinant versions, researchers should carefully optimize these parameters to achieve maximal transformation efficiency.

How can co-transformation techniques be optimized for studying fmt function in N. gonorrhoeae?

Co-transformation techniques in N. gonorrhoeae can be optimized to study fmt function by simultaneously introducing multiple DNA molecules targeting different genomic loci . This approach enables the generation of complex genetic modifications in a single transformation step. When studying fmt, researchers can employ a strategy where one DNA molecule carries a selectable antibiotic resistance marker, while the second carries the desired fmt modification. The optimization process includes several key considerations:

• DNA Ratio Optimization: The ratio between the DNA molecule carrying the selectable marker and the one carrying the fmt modification significantly affects co-transformation efficiency. Experimental determination of the optimal ratio is essential for maximum yield of double transformants .

• Homology Length: Longer homologous regions in both DNA molecules increase transformation efficiency. Research has shown that increasing the length of homologous sequences from 2 kbp to 6 kbp can significantly improve transformation rates .

• Selection Strategy: Selection for the antibiotic marker alone can yield a high percentage (60-70%) of cells that have also incorporated the non-selected modification . This allows researchers to identify cells with fmt modifications that may not confer an easily selectable phenotype.

• DUS Inclusion: Incorporating DNA Uptake Sequences in both DNA molecules enhances transformation efficiency. The natural occurrence of DUS approximately every 1,000 bp in the N. gonorrhoeae genome makes longer DNA fragments more likely to contain these sequences .

• Sequential Selection Approach: For complex fmt modifications, a strategy involving successive selections with two different selection markers at the same genetic locus can dramatically reduce the number of genetic markers needed for multisite modifications .

This co-transformation approach has been demonstrated to be robust and flexible for genome engineering in N. gonorrhoeae, making it particularly valuable for studying genes like fmt where multiple modifications might be required to understand function fully .

What are the implications of fmt inhibition for antibacterial development against N. gonorrhoeae?

Inhibition of Methionyl-tRNA formyltransferase (fmt) represents a promising antibacterial strategy against N. gonorrhoeae for several compelling reasons:

• Essential Role in Translation: Fmt mediates formylation of Met-tRNA, which is crucial for efficient translation initiation in bacteria . Disruption of this process would impair protein synthesis and potentially bacterial viability.

• Interplay with Antibiotic Sensitivity: Research has shown that FolD-deficient mutants and Fmt over-expressing strains exhibit increased sensitivity to trimethoprim (TMP) compared to Δfmt strains . This suggests that Fmt activity modulation could potentially enhance the efficacy of existing antibiotics.

• Unique Bacterial Target: While the formylation process is essential in bacteria, it is absent in the cytoplasmic translation system of eukaryotes, making Fmt an attractive target for selective antibacterial development . This selectivity could potentially reduce side effects in human patients.

• Post-translational Processing: Inhibition of Fmt would disrupt the N-terminal processing pathway of bacterial proteins. The formyl group added by Fmt must be subsequently removed by peptide deformylase (PDF) as the first step of N-terminal maturation of emerging polypeptide chains . Disruption of this pathway would affect protein maturation.

• Connection to WHO Priority Pathogens: Understanding how Fmt inhibition impacts the viability of key pathogens like antibiotic-resistant N. gonorrhoeae (which is on the WHO priority list of antibiotic-resistant bacteria) is an important factor in antibacterial target selection .

For antibacterial development, researchers could target either Fmt directly or the formylation pathway through inhibition of formyl donor synthesis or utilization. The dual substrate capability of Fmt (using both 10-CHO-THF and 10-CHO-DHF) should be considered when designing inhibitors to ensure complete blockade of formylation activity .

What is known about conserved residues in fmt that affect enzyme activity and how can this inform mutagenesis studies?

Research on Methionyl-tRNA formyltransferase (fmt) has identified several conserved residues that are critical for enzymatic activity. Studies of human mitochondrial MTF (mt-MTF), which performs the same function as bacterial Fmt, provide valuable insights that can be applied to N. gonorrhoeae Fmt research :

• Key Conserved Residues: Certain conserved residues in MTF are essential for proper enzyme function. Mutations in these residues can significantly reduce or abolish formylation activity . For N. gonorrhoeae Fmt, identifying these conserved residues through sequence alignment with better-characterized homologs would be the first step in mutagenesis studies.

• Strategic Positioning of Small Aliphatic Amino Acids: The strategic positioning of small aliphatic amino acids has been shown to be required for normal MTF function . Mutagenesis studies in N. gonorrhoeae Fmt should focus on these aliphatic residues to understand their contribution to substrate binding or catalytic activity.

• Pathogenic Mutations: In humans, mutations like S209L in mt-MTF have been linked to reduced mitochondrial translation efficiency and Leigh syndrome . Introducing equivalent mutations in N. gonorrhoeae Fmt could help understand the structure-function relationship of the enzyme.

For designing mutagenesis studies of N. gonorrhoeae Fmt:

• Structure-guided Approach: If a crystal structure is available, focus on residues in the active site or substrate binding pockets. If not, use homology modeling based on related structures.

• Evolutionary Conservation Analysis: Identify residues that are highly conserved across species, as these are likely functionally important.

• Systematic Alanine Scanning: Replace key residues with alanine to assess their contribution to catalytic activity.

• Substrate Specificity Studies: Create mutations that might alter the enzyme's ability to use different formyl donors (10-CHO-THF vs. 10-CHO-DHF) .

• Kinetic Analysis: Perform enzyme kinetics on mutant variants to determine changes in Km, kcat, and substrate preference.

These approaches would provide valuable insights into the structure-function relationship of N. gonorrhoeae Fmt and potentially identify residues that could be targeted for inhibitor design.

How does the folate metabolism pathway interact with fmt function in N. gonorrhoeae?

The folate metabolism pathway shares a crucial interface with Methionyl-tRNA formyltransferase (Fmt) function in N. gonorrhoeae through the provision of formyl group donors:

• FolD's Role in Generating Fmt Substrates: Folate dehydrogenase-cyclohydrolase (FolD), a bifunctional enzyme, catalyzes the conversion of 5,10-methylene tetrahydrofolate (5,10-CH₂-THF) to 10-formyl-THF (10-CHO-THF), which serves as the primary formyl group donor for Fmt . This enzymatic conversion represents a direct link between folate metabolism and translation initiation.

• Alternative Substrate Utilization: Recent research demonstrated that Fmt can also utilize 10-formyldihydrofolate (10-CHO-DHF) as an alternative substrate for formylation of Met-tRNA . This flexibility in substrate utilization suggests an adaptive mechanism that may help maintain translation initiation under varying metabolic conditions.

• Antibiotic Sensitivity Connection: FolD-deficient mutants and Fmt over-expressing strains showed increased sensitivity to trimethoprim (TMP) compared to Δfmt strains . Trimethoprim inhibits dihydrofolate reductase, which affects folate metabolism. This observation suggests a complex interplay between folate metabolism, Fmt activity, and antibiotic response.

• Metabolic Flux and Translation Efficiency: Alterations in folate metabolism likely affect the availability of formyl donors for Fmt, potentially impacting translation initiation efficiency. In conditions where folate metabolism is perturbed, the ability of Fmt to use alternative substrates may become particularly important for bacterial survival.

For researchers investigating this interaction:

• Metabolomics Approach: Quantification of folate pathway metabolites (including both THF and DHF derivatives) under different growth conditions would provide insight into substrate availability for Fmt.

• Combined Genetic Manipulation: Creating strains with modifications in both folate metabolism genes (e.g., folD) and fmt would help elucidate their functional relationship.

• Antifolate Drug Studies: Examining the effects of antifolate drugs on Fmt activity and formylation efficiency could reveal therapeutic vulnerabilities.

• Flux Analysis: Radioactive or stable isotope labeling could track the flow of formyl groups from folate metabolism to tRNA formylation.

Understanding this metabolic interface is valuable not only for basic bacterial physiology but also for identifying potential synergistic targets for antibacterial development.

What expression and purification strategies are most effective for producing recombinant N. gonorrhoeae fmt for in vitro studies?

For efficient expression and purification of recombinant N. gonorrhoeae Methionyl-tRNA formyltransferase (Fmt) for in vitro studies, researchers should consider the following methodological approaches:

• Expression System Selection:

  • E. coli Expression: BL21(DE3) or similar strains are typically effective for expressing bacterial enzymes like Fmt. Consider using strains like Rosetta or OrigamiB if the protein contains rare codons or disulfide bonds.

  • Expression Vector: Vectors providing N-terminal or C-terminal affinity tags (His6, GST, or MBP) facilitate purification and can enhance solubility. Based on structural information, determine which terminus can accommodate a tag without interfering with enzyme activity.

  • Induction Conditions: Optimize IPTG concentration, induction temperature (often lowered to 18-25°C), and duration to maximize soluble protein yield while minimizing inclusion body formation.

• Purification Strategy:

  • Initial Capture: Affinity chromatography using the introduced tag (Ni-NTA for His-tagged protein) provides efficient initial purification.

  • Secondary Purification: Size exclusion chromatography (SEC) and/or ion exchange chromatography can further enhance purity and remove aggregates.

  • Tag Removal: If the tag might interfere with activity, incorporate a protease cleavage site between the tag and Fmt. TEV or PreScission proteases are commonly used.

• Buffer Optimization:

  • Include stabilizing agents like glycerol (10-15%) in all buffers.

  • Add reducing agents (DTT or β-mercaptoethanol) if the protein contains cysteines.

  • Optimize salt concentration and pH based on theoretical isoelectric point of N. gonorrhoeae Fmt.

• Activity Assays:

  • Develop an in vitro formylation assay using purified Met-tRNA^fMet as substrate.

  • Monitor the conversion of 10-CHO-THF to THF spectrophotometrically or using HPLC.

  • Alternative substrate (10-CHO-DHF) utilization can be verified by LC-MS/MS analysis of reaction products .

• Stability Enhancement:

  • Screen buffer additives (e.g., amino acids, sugars) that might enhance protein stability.

  • Consider co-expression with bacterial chaperones if solubility is problematic.

  • Perform thermal shift assays to identify conditions that maximize protein stability.

This methodological approach has been successfully applied to similar bacterial enzymes and should yield active recombinant N. gonorrhoeae Fmt suitable for in vitro studies including enzyme kinetics, inhibitor screening, and structural analyses.