Recombinant Bacillus subtilis Uncharacterized glycosyltransferase ykcC (ykcC)

What is the predicted function of YkcC in Bacillus subtilis?

YkcC is predicted to function as a glycosyltransferase that converts UDP-glucose and undecaprenyl phosphate (C55-P) to C55-P-glucose on the cytoplasmic side of the plasma membrane. This enzyme acts cooperatively with YkcB, another glycosyltransferase, and YngA, a flippase, in a multicomponent glycosylation system. After YkcC produces C55-P-glucose, YngA flips it to the outer surface of the plasma membrane, where YkcB transfers glucose from C55-P-glucose to various cell surface components . This glycosylation pathway contributes to the structural integrity of the cell surface and affects antimicrobial susceptibility profiles.

How is ykcC genetically organized in the B. subtilis genome?

The ykcC gene is located in the same operon as ykcB, suggesting coordinated expression and related functional roles. Studies have indicated the existence of a feedback loop that senses the enzymatic products of YkcB and upregulates the ykcBC promoter. This was demonstrated when FLAG-tagged ykcC was more highly expressed in a ΔykcB strain than in the parent strain . This operon structure and regulatory mechanism are important considerations when designing genetic manipulation experiments targeting ykcC.

What cellular processes are affected by YkcC activity?

YkcC activity influences several critical cellular processes:

• Cell wall integrity and composition

• Antimicrobial susceptibility, particularly to vancomycin

• Phage resistance

• Lipoteichoic acid production

• Biofilm formation capability

• Undecaprenyl phosphate (C55-P) recycling

These processes collectively contribute to bacterial survival, virulence, and interaction with the environment .

What are effective approaches for creating and validating ykcC knockout mutants?

To create ykcC knockout mutants, researchers should consider the following methodology:

• Design primers flanking the ykcC gene with appropriate restriction sites

• Amplify upstream and downstream regions of ykcC

• Insert an antibiotic resistance cassette between these regions

• Transform the construct into B. subtilis using natural competence or electroporation

• Select transformants on appropriate antibiotic media

• Verify knockout by PCR, sequencing, and complementation studies

For validation, researchers should confirm the absence of ykcC expression using RT-PCR or Western blotting. Complementation experiments, where the ykcC gene is reintroduced on a plasmid, are essential to verify that observed phenotypes are specifically due to ykcC deletion rather than polar effects . Additionally, researchers should examine the expression of neighboring genes, particularly ykcB, to ensure the knockout doesn't disrupt the entire operon's function.

What methods can be used to measure YkcC enzymatic activity in vitro?

For in vitro assessment of YkcC enzymatic activity, researchers can employ these methodologies:

• Membrane preparation: Isolate membrane fractions from B. subtilis expressing recombinant YkcC with an affinity tag.

• Substrate preparation: Synthesize radiolabeled UDP-glucose (commonly using UDP-[14C]glucose) and purified C55-P.

• Enzymatic assay: Incubate membrane preparations containing YkcC with substrates in appropriate buffer conditions (typically containing divalent cations like Mg2+).

• Product detection: Extract lipids using chloroform-methanol extraction, separate by thin-layer chromatography, and quantify C55-P-glucose formation through autoradiography or scintillation counting.

• Kinetic analysis: Determine enzyme kinetics by varying substrate concentrations and measuring initial reaction rates.

Control experiments should include heat-inactivated enzyme preparations and assays with membranes from ykcC knockout strains .

How can researchers effectively overexpress and purify recombinant YkcC?

For optimal expression and purification of recombinant YkcC, consider the following protocol:

• Expression system selection: Given that YkcC is a membrane protein with multiple predicted transmembrane domains, expression systems like E. coli C41(DE3) or C43(DE3) strains specifically designed for membrane proteins are recommended.

• Construct design: Create a fusion construct with an N-terminal or C-terminal affinity tag (His-tag or FLAG-tag), considering that tag placement may affect protein folding and activity.

• Expression conditions: Optimize temperature (typically 16-25°C), inducer concentration, and expression duration to maximize protein yield while minimizing inclusion body formation.

• Membrane extraction: Harvest cells and disrupt using sonication or French press, followed by differential centrifugation to isolate membrane fractions.

• Detergent solubilization: Solubilize membrane proteins using detergents such as n-dodecyl-β-D-maltoside (DDM) or lauryldimethylamine oxide (LDAO).

• Affinity purification: Purify using immobilized metal affinity chromatography (IMAC) for His-tagged proteins or anti-FLAG affinity chromatography for FLAG-tagged proteins.

• Quality assessment: Evaluate protein purity by SDS-PAGE and Western blotting, and confirm activity using functional assays .

This approach has been successfully used to generate His-tagged YkcC for structural and functional studies, though care must be taken to maintain proper protein folding in the detergent-solubilized state.

How does YkcC activity influence vancomycin susceptibility in B. subtilis?

The relationship between YkcC activity and vancomycin susceptibility involves several interconnected mechanisms:

• C55-P-glucose accumulation: In strains with ykcB knockout but functional ykcC, C55-P-glucose accumulates due to continued synthesis by YkcC without consumption by YkcB.

• C55-P recycling disruption: The accumulation of C55-P-glucose likely disrupts the recycling of the lipid carrier C55-P, which is essential for cell wall synthesis.

• Cell wall composition alteration: Depletion of available C55-P affects the synthesis of various cell wall components, including peptidoglycan, wall teichoic acids, and lipoteichoic acids.

• Vancomycin target modification: These alterations in peptidoglycan structure reduce the binding affinity of vancomycin to its target (D-Ala-D-Ala termini), conferring tolerance.

Importantly, overexpression of ykcC enhances vancomycin tolerance in both wild-type and ΔykcB strains, while ykcC knockout abolishes the vancomycin tolerance observed in ΔykcB mutants . This indicates that YkcC activity is a critical determinant of vancomycin susceptibility, likely through its effects on cell wall composition and structure.

What is the relationship between YkcC function and bacteriophage resistance?

YkcC plays a crucial role in bacteriophage resistance through its involvement in cell surface glycosylation:

• O-antigen biosynthesis: YkcC functions within the O-antigen biosynthetic cluster OBC3, contributing to the synthesis of long O-polysaccharides (O-PS).

• Phage receptor modification: The long O-PS serves as the primary receptor for phage attachment, particularly for phage ΦGP100 infection.

• Structural mutations: A single amino acid substitution (P229T) in YkcC confers resistance to phage ΦGP100. This proline at position 229 is highly conserved among orthologs of this glycosyltransferase, suggesting its importance in enzyme function .

• Altered O-PS synthesis: Both the P229T mutation and complete deletion of ykcC result in resistance to phage ΦGP100, likely by preventing the synthesis of the O-PS structures that serve as phage receptors.

This demonstrates how subtle changes in glycosyltransferase activity can dramatically alter bacterial susceptibility to bacteriophage infection, providing insight into bacterial-phage co-evolution mechanisms.

How do the functions of YkcC and YkcB interrelate and influence each other?

The functional relationship between YkcC and YkcB reveals a sophisticated interdependence:

• Sequential enzymatic action: YkcC synthesizes C55-P-glucose, which serves as the substrate for YkcB. YkcB transfers glucose from C55-P-glucose to cell surface components.

• Regulatory feedback: YkcC expression appears to be regulated by a feedback mechanism that senses YkcB products. In ykcB knockout strains, YkcC expression from the native ykcBC promoter is increased .

• Phenotypic dependence: Many phenotypes observed in ykcB knockout mutants (vancomycin tolerance, decreased lipoteichoic acid, reduced biofilm formation) are dependent on functional ykcC, as they are abolished in double ykcB/ykcC knockout strains .

• C55-P recycling coordination: Both enzymes appear to influence the recycling of the lipid carrier C55-P, with disruption of this balance leading to various phenotypic changes.

This interrelationship suggests that YkcC and YkcB function as a coordinated system rather than independent enzymes, with the balance of their activities being critical for normal cell surface glycosylation and antimicrobial susceptibility.

What specific mutations in ykcC confer phage resistance, and what is their mechanistic basis?

Research has identified specific mutations in ykcC that confer phage resistance:

These findings demonstrate how single nucleotide polymorphisms can dramatically alter bacterial surface structures and host-phage interactions, providing insight into potential mechanisms of evolved phage resistance.

How does overexpression of ykcC affect antimicrobial susceptibility profiles?

Overexpression of ykcC has significant effects on antimicrobial susceptibility:

• Vancomycin tolerance: Transforming wild-type B. subtilis with a multicopy plasmid encoding FLAG-tagged ykcC under the ykcBC native promoter decreases vancomycin susceptibility .

• Enhanced effect in ΔykcB background: The vancomycin tolerance effect of ykcC overexpression is even more pronounced in a ykcB knockout background .

• Expression-dependent mechanism: The degree of vancomycin tolerance correlates with ykcC expression levels, suggesting a dose-dependent effect.

• Cell wall modifications: Increased YkcC activity likely alters cell wall composition through changes in glycolipid distribution and C55-P availability.

• Potential clinical relevance: This mechanism suggests that ykcC upregulation could be a potential resistance mechanism in clinical bacterial isolates.

These findings highlight how altered expression of a single glycosyltransferase can reconfigure antimicrobial susceptibility, underscoring the importance of gene expression regulation in bacterial adaptation to antimicrobial threats.

What effect does ykcC deletion have on bacterial fitness and virulence?

The impact of ykcC deletion on bacterial fitness and virulence involves multiple aspects:

• Growth characteristics: Unlike the ykcB knockout, which maintains normal growth, the phenotypes of ykcC knockout strains regarding growth rate and generation time have not been fully characterized.

• Biofilm formation: The ykcC gene appears to be essential for the decreased biofilm formation observed in ykcB knockout strains, as double ykcB/ykcC knockout mutants do not show decreased biofilm formation .

• Lipoteichoic acid content: Similarly, ykcC is required for the decreased lipoteichoic acid phenotype in ykcB mutants, suggesting its role in lipoteichoic acid synthesis or modification .

• Antibiotic susceptibility balance: While ykcC activity contributes to vancomycin tolerance, its role in susceptibility to other antibiotics, particularly β-lactams, requires further investigation.

• Virulence implications: The ykcB knockout showed increased pathogenicity to silkworms in a ykcC-dependent manner, suggesting that ykcC activity influences virulence factor expression or bacterial interaction with host immune responses .

These observations indicate that YkcC plays a multifaceted role in bacterial fitness and virulence, likely through its effects on cell surface structure and composition.

What are the key structural domains of YkcC and how do they contribute to its function?

YkcC structural organization includes several key domains that contribute to its glycosyltransferase function:

• Catalytic domain: YkcC belongs to the glycosyltransferase family, which typically contains a nucleotide-binding domain that recognizes UDP-glucose as the donor substrate.

• Membrane association: The protein likely contains membrane-associated regions that facilitate interaction with the lipid substrate C55-P at the cytoplasmic membrane interface.

• Conserved residues: Position P229 is highly conserved among YkcC orthologs, suggesting its importance in maintaining proper protein folding or substrate recognition .

• Structural homology: YkcC shares structural features with other glycosyltransferases involved in bacterial cell envelope biosynthesis.

• Active site architecture: Though detailed crystallographic data is not yet available, the active site likely contains residues for coordinating metal ions (typically Mg2+) and binding both the nucleotide sugar donor and lipid acceptor.

Understanding these structural elements is crucial for designing inhibitors targeting YkcC or engineering the enzyme for biotechnological applications.

How does the proline to threonine substitution at position 229 alter YkcC structure and function?

The P229T substitution in YkcC has significant structural and functional implications:

• Secondary structure disruption: Proline is unique among amino acids in its ability to introduce kinks in protein structure due to its rigid cyclic side chain and inability to participate in regular hydrogen bonding patterns. Its substitution with threonine likely alters local secondary structure.

• Conformational changes: Even though position 229 is distant from the predicted catalytic site, the substitution could induce long-range conformational changes that affect active site geometry or substrate binding .

• Protein stability: The mutation may alter protein stability or membrane association, affecting enzyme availability or localization.

• Substrate recognition: The structural changes could modify substrate recognition, particularly for the lipid carrier C55-P, altering enzyme specificity or catalytic efficiency.

• Functional consequences: These structural alterations result in impaired O-polysaccharide synthesis, conferring phage resistance by eliminating the phage receptor on the bacterial surface .

This example illustrates how single amino acid substitutions can have profound effects on enzyme function through structural perturbations, even when located away from the active site.

What experimental approaches can elucidate the structure-function relationship of YkcC?

To investigate YkcC structure-function relationships, researchers can employ these methodologies:

• Site-directed mutagenesis: Systematically mutate conserved residues and assess their impact on enzyme activity, substrate binding, and phenotypic outcomes.

• Protein crystallography: Express, purify, and crystallize YkcC (potentially using detergent solubilization or lipidic cubic phase methods suitable for membrane proteins) to determine its three-dimensional structure.

• Cryo-electron microscopy: Use cryo-EM as an alternative structural approach, particularly valuable for membrane proteins that are difficult to crystallize.

• Molecular dynamics simulations: Perform computational simulations to predict protein dynamics, substrate binding modes, and the effects of mutations.

• Chimeric protein analysis: Create chimeric proteins with related glycosyltransferases to identify domains responsible for specific functions or substrate specificities.

• Cross-linking studies: Use chemical cross-linking combined with mass spectrometry to identify protein-protein interactions, particularly with YkcB and YngA.

• Enzyme kinetics with substrate analogs: Measure enzyme activity with modified substrates to probe the structural requirements for catalysis .

These complementary approaches would provide a comprehensive understanding of how YkcC structure determines its function in glycolipid synthesis and cell surface modification.

How might targeting YkcC function be exploited for antimicrobial development?

YkcC presents a promising target for novel antimicrobial strategies for several reasons:

• Vancomycin resensitization: Inhibitors of YkcC could potentially restore vancomycin sensitivity in resistant strains where C55-P-glucose accumulation contributes to tolerance.

• Biofilm disruption: Given the role of YkcC in biofilm formation, inhibitors might reduce biofilm development, enhancing the efficacy of conventional antibiotics against biofilm-associated infections.

• Combination therapies: YkcC inhibitors could be developed as adjuvants to be used in combination with vancomycin or β-lactams for synergistic antimicrobial effects.

• Narrow-spectrum activity: Since YkcC has specific functions in certain Gram-positive bacteria, inhibitors could potentially have narrow-spectrum activity, reducing disruption of beneficial microbiota.

• Reduced resistance potential: Targeting non-essential functions like glycosylation may impose less selective pressure for resistance development compared to targeting essential cellular processes.

Challenges include developing inhibitors that can penetrate the bacterial cell envelope and achieving sufficient specificity to avoid off-target effects on human glycosyltransferases .

What tools and methodologies are needed to fully characterize the YkcC glycosylation pathway?

Complete characterization of the YkcC glycosylation pathway requires development and application of several advanced methodologies:

• Metabolic labeling techniques: Develop methods to specifically label and track C55-P-glucose in living cells to monitor its synthesis and utilization.

• Mass spectrometry approaches: Implement advanced glycomics approaches to fully characterize the structures of cell surface glycolipids and identify YkcC-dependent modifications.

• Single-molecule enzymology: Apply techniques like FRET or single-molecule tracking to understand the dynamics of YkcC activity in membrane environments.

• Reconstitution systems: Develop in vitro reconstitution systems with purified components (YkcC, YkcB, YngA) in artificial membranes to recapitulate the complete glycosylation pathway.

• Temporal regulation analysis: Develop methods to monitor the temporal regulation of the ykcBC operon under different growth conditions and stresses.

• Synthetic biology approaches: Create synthetic genetic circuits to control and monitor YkcC activity to understand its impact on cellular physiology .

These tools would enable comprehensive mapping of the glycosylation pathway, its regulation, and its integration with other cellular processes.

How does YkcC function compare across different bacterial species?

Comparative analysis of YkcC homologs across bacterial species reveals important evolutionary and functional insights:

• Conservation patterns: The proline at position 229 is highly conserved among YkcC orthologs, suggesting evolutionary pressure to maintain this structural feature .

• Functional divergence: Despite sequence conservation, YkcC homologs may serve different roles in different bacterial species, potentially glycosylating different target molecules.

• Operon organization: The organization of ykcC in an operon with ykcB appears in multiple species, but variations in operon structure may reflect adaptations to different ecological niches.

• Phage resistance mechanisms: The role of YkcC in phage resistance may vary across species, reflecting co-evolution with species-specific bacteriophages.

• Antimicrobial resistance contributions: The contribution of YkcC to antimicrobial resistance may differ between species based on their cell wall composition and antimicrobial susceptibility profiles.

Systematic comparative genomics and functional studies across diverse bacterial species would provide valuable insights into the evolution and functional diversification of this glycosylation pathway.