Recombinant Burkholderia xenovorans Monofunctional biosynthetic peptidoglycan transglycosylase (mtgA)

What is mtgA and what is its primary function in Burkholderia xenovorans?

Monofunctional biosynthetic peptidoglycan transglycosylase (mtgA) in Burkholderia xenovorans is a protein involved in cell wall synthesis. It specifically catalyzes the polymerization of lipid II molecules into glycan strands of peptidoglycans, which are essential structural components of bacterial cell walls . This enzyme plays a critical role in maintaining cellular integrity, providing protection against osmotic pressure, and determining cell shape. Unlike bifunctional transglycosylases that also possess transpeptidase activity, mtgA focuses solely on the transglycosylation reaction, making it a specialized component in the peptidoglycan assembly machinery.

The specific enzymatic classification of mtgA is EC 2.4.2.-, indicating its function as a glycosyltransferase that forms glycosidic bonds . In B. xenovorans strain LB400, mtgA represents one of many specialized proteins that contribute to this organism's remarkable adaptability and metabolic versatility.

What are the optimal storage and handling conditions for recombinant B. xenovorans mtgA?

For researchers working with recombinant B. xenovorans mtgA, proper storage and handling is critical to maintain protein stability and activity. The recommended storage conditions are:

It's important to note that repeated freezing and thawing is not recommended as it can lead to protein denaturation and loss of enzymatic activity . When planning experiments, researchers should prepare appropriately sized aliquots to minimize freeze-thaw cycles.

The high glycerol content (50%) in the storage buffer serves as a cryoprotectant, preventing ice crystal formation that could damage protein structure. The Tris-based buffer maintains optimal pH for protein stability. For experimental use, researchers should consider how the storage buffer components might affect their specific assay conditions and adjust protocols accordingly.

How can researchers verify the activity of recombinant mtgA in vitro?

Verifying the enzymatic activity of recombinant mtgA requires specialized assays that measure transglycosylase function. Several methodological approaches can be employed:

• Substrate utilization assays: Using synthetic lipid II or fluorescently labeled analogs to monitor polymerization activity. The rate of substrate consumption can be measured by HPLC or other chromatographic techniques.

• Product formation analysis: Analyzing the formation of glycan strands through methods such as mass spectrometry, which can detect the polymerized products of varying lengths.

• Complementation studies: As demonstrated in research with E. coli, functional verification can be performed by complementing an mtgA deletion strain with the recombinant protein. Restoration of wild-type phenotypes (normal cell morphology, polymer accumulation patterns) confirms functional activity of the recombinant protein .

• Radioactive assays: Using radiolabeled substrates to trace the formation of peptidoglycan strands, followed by quantification of incorporated radioactivity.

For rigorous characterization, researchers should combine multiple approaches to assess both the kinetic parameters (Km, Vmax) and the specificity of the enzyme for different substrates.

What methods are effective for studying cell phenotype changes resulting from mtgA manipulation?

Research on mtgA has revealed significant phenotypic changes in bacterial cells following gene deletion. To effectively study these changes, the following methodological approaches are recommended:

• Microscopic analysis: Quantitative assessment of cell dimensions (length, width, volume) using phase contrast microscopy combined with image analysis software. This approach revealed that mtgA deletion in E. coli led to increased cell width without affecting cell length .

• Comparative morphological studies: Examining wild-type, mtgA-deleted, and complemented strains under various growth conditions. This approach has shown that the enlarged cell phenotype (termed "fat cell") is specifically associated with polymer-producing conditions .

• Biomass and polymer quantification: Measuring dry cell weight and polymer accumulation to establish correlations between morphological changes and metabolic outcomes. In E. coli, mtgA deletion resulted in a 1.6-fold increase in polymer accumulation per cell .

• Gene expression analysis: Using transcriptomics to identify compensatory mechanisms activated in response to mtgA deletion, which may explain the observed phenotypic changes.

• Cell wall composition analysis: Employing biochemical assays to characterize changes in peptidoglycan structure and cross-linking in mtgA mutants.

The experimental design should include appropriate controls, including complementation experiments where the deleted gene is reintroduced to confirm that observed phenotypes are directly attributable to mtgA function rather than secondary mutations or polar effects.

How does mtgA deletion affect polymer accumulation and cell morphology?

The deletion of mtgA has been found to significantly impact both bacterial cell morphology and polymer accumulation capacity. In E. coli, mtgA deletion resulted in an enlarged cell phenotype specifically under polymer-producing conditions, with cells becoming "fat" rather than "tall" . This morphological change was directly associated with enhanced polymer accumulation.

Quantitative analysis revealed the following impacts:

Importantly, complementation experiments confirmed that these phenotypic changes were directly attributable to mtgA deletion, as reintroduction of the mtgA gene restored normal cell morphology and polymer accumulation levels . The specificity of this effect to polymer-producing conditions suggests a metabolic interaction between cell wall synthesis and polymer accumulation pathways.

The enlarged cell phenotype is postulated to increase the volumetric capacity for accumulating intracellular material, which explains the enhanced polymer production. This finding has significant implications for biotechnological applications, particularly in the production of biopolymers.

What is the evolutionary context of mtgA within the Burkholderia genus?

Understanding mtgA in B. xenovorans requires consideration of the evolutionary dynamics within the Burkholderia genus. B. xenovorans LB400 has one of the largest known bacterial genomes (9.73 Mbp), characterized by high genomic plasticity and functional specialization across its three replicons (two chromosomes and one megaplasmid) .

The Burkholderia genus exhibits remarkable genomic diversity, with only 44% gene conservation between B. xenovorans and Burkholderia cepacia complex strain 383 . Even among four B. xenovorans strains, genome size varies from 7.4 to 9.73 Mbp, with over 20% of the LB400 sequence being recently acquired through lateral gene transfer .

This evolutionary context suggests that genes like mtgA may have undergone selection pressure specific to the ecological niche of B. xenovorans. The significant finding that 17.6% of proteins in B. xenovorans have a better paralog within the same genome than an ortholog in different genomes highlights the importance of gene duplication and divergence in this species . This raises questions about potential functional redundancy or specialization of mtgA in B. xenovorans compared to homologous proteins in other bacterial species.

The genomic analysis also revealed differential selective pressure across the three replicons of B. xenovorans LB400, with more relaxed selection on the two smaller replicons compared to the largest chromosome . This pattern suggests that genes involved in core cellular functions might be subject to different evolutionary constraints than those involved in niche adaptation.

What is the relationship between mtgA function and antibiotic resistance mechanisms?

While the search results don't directly address the relationship between mtgA and antibiotic resistance, the function of mtgA in peptidoglycan synthesis places it at a critical intersection with antibiotic resistance mechanisms. Several research-driven considerations emerge:

• Cell wall-targeting antibiotics: As mtgA is involved in peptidoglycan synthesis, alterations in its activity could affect susceptibility to antibiotics targeting cell wall assembly, such as β-lactams, glycopeptides, and bacitracin.

• Permeability barriers: The enlarged cell phenotype observed with mtgA deletion might alter membrane permeability and diffusion characteristics, potentially affecting antibiotic penetration into the cell.

• Stress responses: Changes in cell wall integrity resulting from mtgA dysfunction could trigger stress responses that upregulate resistance mechanisms such as efflux pumps or alternative metabolic pathways.

• Biofilm formation: Cell morphology changes associated with mtgA deletion might impact biofilm formation capacity, which is often linked to increased antibiotic tolerance.

Methodological approaches to investigate these relationships would include:

• Minimum inhibitory concentration (MIC) testing of various antibiotic classes against wild-type and mtgA-mutant strains

• Transcriptomic analysis to identify changes in expression of resistance genes in response to mtgA manipulation

• Time-kill kinetics to assess the dynamics of antibiotic action against mtgA mutants

• Biofilm formation assays to evaluate structural and resistance properties

These investigations would contribute to understanding the broader role of cell wall synthesis enzymes in antibiotic resistance phenotypes.

How can mtgA manipulation be leveraged for biotechnological applications?

The discovery that mtgA deletion leads to enlarged cells with enhanced polymer accumulation capacity presents significant opportunities for biotechnological applications. Research has demonstrated that mtgA-deleted E. coli strains produced 35% more P(LA-co-3HB) (7.0 g/l) compared to control strains (5.2 g/l) . This finding suggests several potential applications:

• Enhanced biopolymer production: The "fat cell" phenotype created by mtgA deletion increases volumetric capacity for accumulating intracellular biopolymers, making it a promising strategy for improving production yields of commercially valuable compounds.

• Metabolic engineering platform: mtgA deletion could serve as a novel genetic background for metabolic engineering approaches aimed at producing various intracellular compounds, potentially improving yields through increased cellular storage capacity.

• Combinatorial approaches: Researchers have suggested that mtgA deletion should be combined with conventional engineering approaches to maximize benefits . This combinatorial strategy could involve:

  • Coupling mtgA deletion with pathway optimization for target compounds

  • Combining with other cell morphology modifications

  • Integrating with fermentation process improvements

The methodology for implementing these applications would involve:

• Introducing mtgA deletion into production strains using precise genome editing techniques

• Characterizing the resulting strains for growth, stability, and production characteristics

• Optimizing fermentation conditions specifically for the altered cell morphology

• Developing extraction and recovery processes tailored to the enlarged cell phenotype

This approach represents a novel strategy that moves beyond traditional metabolic engineering by manipulating cellular morphology to create improved "cellular factories" for biopolymer production.

What are the future research directions for understanding mtgA function in B. xenovorans?

Given the current knowledge about mtgA and the broader context of B. xenovorans biology, several promising research directions emerge:

• Comparative genomics and evolution: Further investigation of mtgA across Burkholderia species could reveal evolutionary patterns in peptidoglycan synthesis enzymes. With only 44% gene conservation between B. xenovorans and related species , understanding the specific adaptations in mtgA could provide insights into niche specialization.

• Regulatory networks: Exploring how mtgA expression is regulated in response to environmental conditions, particularly in the context of B. xenovorans' remarkable metabolic versatility and environmental adaptability.

• Structural biology: Determining the crystal structure of B. xenovorans mtgA would enable detailed understanding of its catalytic mechanism and potential for rational enzyme engineering.

• Environmental stress responses: Investigating how mtgA function changes under various environmental stresses, particularly in relation to B. xenovorans' ability to degrade polychlorinated biphenyls and other aromatic compounds .

• Systems biology approach: Integrating mtgA function into broader metabolic models of B. xenovorans to understand its role in the context of the organism's complex metabolism, which includes at least eleven "central aromatic" and twenty "peripheral aromatic" pathways .

• Synthetic biology applications: Exploring the potential to engineer mtgA variants with enhanced or modified activities for biotechnological applications, particularly in bioremediation contexts where B. xenovorans is already valuable.

These research directions would benefit from integrated methodological approaches combining genomics, transcriptomics, proteomics, and metabolomics to build a comprehensive understanding of mtgA's role in B. xenovorans biology.