Recombinant Shigella dysenteriae serotype 1 Cellulose synthesis regulatory protein (SDY_1050)

What is the molecular structure and classification of SDY_1050 protein?

SDY_1050 (UniProt ID: Q32HJ0) is a full-length protein consisting of 564 amino acids (1-564) from Shigella dysenteriae serotype 1. It functions as a cellulose synthesis regulatory protein and is classified as a probable diguanylate cyclase (DGC). The protein contains multiple transmembrane domains as evidenced by its amino acid sequence, which includes hydrophobic regions characteristic of membrane proteins . The protein is commonly referred to by several synonyms in the literature, including dgcQ, yedQ, and DGC, reflecting its functional role in cellulose synthesis regulation .

How does SDY_1050 function in the context of Shigella dysenteriae pathogenesis?

SDY_1050 (DgcQ) plays a critical role in Shigella pathogenesis through its function as a diguanylate cyclase that synthesizes cyclic di-GMP, a bacterial second messenger involved in regulating various cellular processes including virulence and biofilm formation . In the context of Shigella infection, cellulose production represents a stress response-mediated process that is modulated by pyrimidine nucleotide biosynthetic pathways . Research indicates that DgcQ activity is directly regulated by metabolites involved in these pathways, with UTP enhancing its enzymatic activity while N-carbamoyl-aspartate (an intermediate of the de novo pathway) inhibits it . This regulatory mechanism allows the bacterium to modulate cellulose production based on cellular conditions, which can impact bacterial survival within the host and contribute to pathogenesis mechanisms exhibited during Shigella infection .

What are the optimal expression systems for producing recombinant SDY_1050 protein?

The recombinant SDY_1050 protein is optimally expressed in E. coli expression systems, which provide several advantages for producing this specific bacterial protein . When expressing SDY_1050, researchers should consider using E. coli strains designed for membrane protein expression, as the protein contains transmembrane domains. The expression construct typically includes an N-terminal His-tag to facilitate purification without interfering with the protein's functional domains . E. coli expression systems are preferred because they allow for proper folding of the bacterial protein, high yield production, and compatibility with the protein's native post-translational modifications. The expression conditions should be carefully optimized regarding temperature, induction time, and inducer concentration to maximize protein yield while maintaining proper folding and activity of this regulatory protein .

What purification strategies yield the highest purity and functionality for SDY_1050 protein?

For optimal purification of SDY_1050 protein, a multi-step purification strategy is recommended. The most effective approach begins with immobilized metal affinity chromatography (IMAC) utilizing the N-terminal His-tag present on the recombinant protein . This initial purification step should be followed by additional chromatographic techniques such as ion exchange or size exclusion chromatography to achieve purity greater than 90% as determined by SDS-PAGE . Throughout the purification process, it is critical to maintain buffer conditions that stabilize the protein, typically using Tris/PBS-based buffers at pH 8.0 with the addition of stabilizing agents such as trehalose (6%) . To preserve the functional integrity of SDY_1050 during purification, all steps should be performed at controlled temperatures (usually 4°C), and protease inhibitors should be included to prevent degradation of this regulatory protein .

What reconstitution and storage protocols maximize stability and activity of SDY_1050?

For optimal reconstitution of lyophilized SDY_1050 protein, researchers should briefly centrifuge the vial before opening to ensure all content is at the bottom. The protein should be reconstituted in deionized sterile water to achieve a concentration of 0.1-1.0 mg/mL . To maximize stability during storage, a final glycerol concentration of 5-50% is recommended, with 50% being the standard recommendation for long-term storage . The reconstituted protein should be aliquoted to avoid repeated freeze-thaw cycles, which significantly reduce protein stability and function. For storage protocols, short-term working aliquots can be maintained at 4°C for up to one week, while long-term storage requires temperatures of -20°C or preferably -80°C . When using stored protein, it's essential to thaw aliquots quickly and maintain them on ice during experimental procedures to preserve enzymatic activity. These careful handling procedures are necessary because the diguanylate cyclase activity of SDY_1050 is particularly sensitive to denaturation from temperature fluctuations .

How can researchers design experiments to assess SDY_1050's diguanylate cyclase activity?

To effectively assess the diguanylate cyclase activity of SDY_1050, researchers should implement a multi-faceted experimental approach focusing on the protein's ability to synthesize cyclic di-GMP. A robust experimental design would include:

• Enzymatic activity assays: Measure the conversion of GTP to cyclic di-GMP using high-performance liquid chromatography (HPLC) or liquid chromatography-mass spectrometry (LC-MS). The reaction mixture should contain purified SDY_1050, GTP substrate, and essential cofactors such as Mg²⁺ .

• Allosteric regulation assessment: Evaluate how UTP enhances and N-carbamoyl-aspartate inhibits enzymatic activity by including these compounds at varying concentrations in the reaction mixture. This would verify the regulatory mechanisms described in the literature .

• Mutation studies: Create site-directed mutations in key catalytic domains of SDY_1050 to identify specific amino acid residues essential for enzymatic function, particularly those involved in the binding of UTP and N-carbamoyl-aspartate .

• In vivo reporter systems: Develop bacterial reporter systems where cyclic di-GMP production activates a fluorescent or colorimetric output, allowing for real-time monitoring of SDY_1050 activity in living bacterial cells under various conditions .

These experimental approaches would provide comprehensive insights into both the basic enzymatic function and the complex regulatory mechanisms of SDY_1050 as a diguanylate cyclase.

What methodologies can be used to study the relationship between SDY_1050 and cellulose production in Shigella?

To study the relationship between SDY_1050 (DgcQ) and cellulose production in Shigella, researchers should employ multiple complementary methodologies:

• Gene knockout and complementation studies: Generate SDY_1050 deletion mutants and complemented strains to directly assess the protein's effect on cellulose production. This genetic approach establishes causality between the protein and the phenotype .

• Cellulose detection assays: Implement quantitative and qualitative methods to measure cellulose production, including:

  • Congo Red binding assays (quantitative spectrophotometric measurements)

  • Calcofluor staining with fluorescence microscopy

  • Biochemical quantification of cellulose using enzymatic hydrolysis followed by glucose measurement

• Metabolic pathway analysis: Investigate the interplay between pyrimidine biosynthetic pathways and cellulose production by manipulating growth conditions or introducing mutations in key metabolic genes. This would validate the connection between pyrimidine salvage pathway activation and cellulose production via SDY_1050 .

• Cyclic di-GMP measurement: Employ LC-MS/MS to quantify intracellular cyclic di-GMP levels in wild-type versus mutant strains under various conditions, establishing the link between SDY_1050 activity and second messenger production that ultimately regulates cellulose synthesis .

• Protein-protein interaction studies: Use co-immunoprecipitation or bacterial two-hybrid systems to identify interactions between SDY_1050 and other proteins involved in cellulose biosynthesis machinery .

This multi-faceted approach provides mechanistic insights into how SDY_1050 regulates cellulose production in response to metabolic cues in Shigella.

What techniques can be used to study the localization and membrane dynamics of SDY_1050?

To comprehensively investigate the localization and membrane dynamics of SDY_1050, researchers should employ several complementary techniques:

• Fluorescent protein fusion imaging: Create SDY_1050-GFP (or other fluorescent protein) fusions to visualize the protein's subcellular localization using confocal microscopy. This approach allows for real-time tracking of protein movement within living bacterial cells while maintaining physiological conditions .

• Immunofluorescence microscopy: Develop specific antibodies against SDY_1050 for immunostaining to detect the native protein without the potential artifacts associated with protein fusions. This technique provides high specificity when combined with appropriate controls .

• Membrane fractionation and Western blotting: Perform subcellular fractionation to physically separate inner membrane, outer membrane, periplasmic, and cytoplasmic fractions, followed by immunoblotting to determine the distribution of SDY_1050 across these compartments. This biochemical approach provides quantitative data on protein localization .

• FRAP (Fluorescence Recovery After Photobleaching): Apply this technique to SDY_1050-fluorescent protein fusions to measure the mobility of the protein within membranes, providing insights into its dynamics and potential interactions with other membrane components .

• Cryo-electron microscopy: Utilize high-resolution imaging to visualize the organization of SDY_1050 within the membrane context, potentially revealing structural information about its integration and clustering behavior .

These methodologies collectively provide a comprehensive understanding of how SDY_1050 is positioned and functions within the bacterial membrane architecture, which is crucial for interpreting its regulatory role in cellulose synthesis.

How does allosteric regulation by pyrimidine pathway metabolites affect SDY_1050 function?

Allosteric regulation of SDY_1050 (DgcQ) by pyrimidine pathway metabolites represents a sophisticated control mechanism that directly links cellular metabolism to cellulose production in Shigella. Research indicates that DgcQ enzymatic activity is enhanced by UTP while being inhibited by N-carbamoyl-aspartate, an intermediate of the de novo pyrimidine biosynthesis pathway . This dual regulatory mechanism creates a metabolic sensing system that allows the bacterium to modulate cellulose production based on the predominant pyrimidine nucleotide biosynthetic pathway being utilized.

The molecular mechanism behind this allosteric regulation likely involves conformational changes in the protein structure upon binding of these metabolites. When cells predominantly use the salvage pathway for pyrimidine synthesis, elevated UTP levels stimulate DgcQ activity, leading to increased production of the second messenger c-di-GMP and consequently enhanced cellulose synthesis . Conversely, active de novo pyrimidine synthesis results in accumulation of N-carbamoyl-aspartate, which inhibits DgcQ activity.

This regulatory system provides Shigella with the ability to adjust cellulose production based on environmental conditions, as the salvage pathway depends on the environmental availability of pyrimidine bases. Understanding these allosteric mechanisms is crucial for researchers developing strategies to target bacterial cellulose production or studying bacterial adaptation mechanisms during infection processes .

What roles does SDY_1050 play in Shigella biofilm formation and host-pathogen interactions?

SDY_1050 (DgcQ) serves as a critical regulatory nexus in Shigella biofilm formation and host-pathogen interactions through its control of cellulose production and cyclic di-GMP synthesis. As a diguanylate cyclase, SDY_1050 produces cyclic di-GMP, which is a universal bacterial second messenger that orchestrates the transition between motile and sessile lifestyles . In the context of biofilm formation, SDY_1050-mediated cellulose production contributes significantly to the extracellular matrix component of biofilms, enhancing bacterial aggregation, surface adhesion, and resistance to environmental stresses including antimicrobials and host immune defenses .

During host-pathogen interactions, SDY_1050's regulatory activity influences multiple aspects of Shigella pathogenesis. While Shigella is primarily known for its invasive capabilities within host epithelial cells, the timing and extent of cellulose production can modulate bacterial adhesion properties and affect the efficacy of the Type III Secretion System (T3SS) . Research indicates that the T3SS interacts with adhesion functions in Shigella, suggesting a potential regulatory cross-talk between virulence mechanisms and cellulose production pathways .

The metabolic sensing function of SDY_1050, particularly its responsiveness to pyrimidine pathway metabolites, likely allows Shigella to adapt its virulence expression and biofilm formation capabilities based on the specific metabolic environment encountered within different host niches . This adaptability represents a sophisticated survival strategy during infection, potentially influencing the progression from initial colonization to established infection and persistence.

What is the relationship between SDY_1050 activity and the Type III Secretion System in Shigella virulence?

Mechanistically, the connection may involve several pathways:

• Coordinated regulation: Cyclic di-GMP produced by SDY_1050 may influence the expression or assembly of T3SS components through transcriptional or post-translational mechanisms.

• Metabolic integration: Both systems respond to environmental and metabolic cues, suggesting that SDY_1050's sensitivity to pyrimidine metabolites might indirectly affect T3SS function through global metabolic changes .

• Temporal expression patterns: The activities of SDY_1050 and T3SS may be coordinated during different stages of infection to optimize virulence. For example, reduced cellulose production might be necessary during periods of active T3SS utilization for invasion .

• Structural interactions: The membrane localization of both SDY_1050 and T3SS components may allow for direct or indirect physical interactions that affect their respective functions .

Understanding this relationship has significant implications for developing novel anti-virulence strategies that could target multiple aspects of Shigella pathogenesis simultaneously.

How does SDY_1050 compare functionally and structurally to homologous proteins in other bacterial species?

The distinctive feature of SDY_1050 compared to homologs in other species is its specific allosteric regulation by pyrimidine pathway metabolites—enhanced by UTP and inhibited by N-carbamoyl-aspartate . This metabolic sensing capability appears to be specialized in Shigella and closely related enterobacteria, suggesting evolutionary adaptation to their particular ecological niches and pathogenic lifestyles.

These comparative differences highlight the evolutionary specialization of SDY_1050 for Shigella's particular pathogenic strategy and environmental adaptation requirements.

What methodological approaches can resolve contradicting data about SDY_1050 function across different experimental systems?

To resolve contradicting data about SDY_1050 function across different experimental systems, researchers should implement a comprehensive methodological framework that addresses potential sources of variability:

This systematic approach would help reconcile contradictory findings and establish a more robust understanding of SDY_1050's true biological function and regulation.

How can systems biology approaches integrate SDY_1050 function into broader cellular networks?

Systems biology approaches can effectively integrate SDY_1050 function into broader cellular networks by applying multi-omics strategies and computational modeling techniques. This comprehensive integration would reveal how this regulatory protein influences and is influenced by various cellular processes in Shigella:

• Multi-omics data integration: Combine transcriptomics, proteomics, and metabolomics data from wild-type and SDY_1050 mutant strains to map the global impact of this protein on cellular networks. This approach would identify genes, proteins, and metabolites whose levels change in response to SDY_1050 activity, creating a functional network centered on this regulatory protein .

• Protein-protein interaction mapping: Employ techniques such as bacterial two-hybrid screens, co-immunoprecipitation coupled with mass spectrometry, or proximity labeling methods to identify direct interaction partners of SDY_1050. These interaction networks would place the protein within specific cellular complexes and pathways .

• Metabolic flux analysis: Implement isotope-labeled metabolite tracing to quantify how SDY_1050 activity affects carbon flow through central metabolism, particularly focusing on connections to pyrimidine biosynthesis pathways that allosterically regulate the protein .

• Mathematical modeling of regulatory circuits: Develop computational models that incorporate SDY_1050's function as a cyclic di-GMP producer, its allosteric regulation by metabolites, and downstream effects on cellulose production and virulence mechanisms. These models would predict system-level behaviors under various conditions .

• Network perturbation analysis: Systematically perturb connected pathways (e.g., through genetic mutations or chemical inhibitors) and measure the impact on SDY_1050-dependent processes to validate predicted network connections and identify regulatory feedback loops .

By applying these complementary systems biology approaches, researchers would develop an integrated understanding of how SDY_1050 functions within the complex cellular network of Shigella, providing insights into potential intervention strategies targeting this key regulatory node.