Recombinant Yersinia pseudotuberculosis serotype IB Galactokinase (galK)
Description
Recombinant Protein Production in Y. pseudotuberculosis
While GalK (galactokinase) is not discussed in the provided sources, other recombinant proteins from Y. pseudotuberculosis serotype IB have been studied. For example:
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Tetraacyldisaccharide 4'-kinase (LpxK): A recombinant form of this enzyme (UniProt: B2KA30) from Y. pseudotuberculosis serotype IB (strain PB1/+) has been produced. This protein is involved in lipid A biosynthesis and is critical for lipopolysaccharide (LPS) assembly .
Genetic Engineering of Y. pseudotuberculosis
Several studies describe recombinant Y. pseudotuberculosis strains engineered for vaccine development or antigen delivery:
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Strain χ10068: A recombinant attenuated strain modified to express the Yersinia pestis F1 antigen. This strain induced 70–90% protection against plague in mice via oral immunization .
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Strain χ10069: Engineered with ΔyopK, ΔyopJ, and Δasd mutations to deliver a Y. pestis YopENt138-LcrV fusion protein, providing cross-protection against pneumonic plague .
Antigen Delivery Mechanisms
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Recombinant Y. pseudotuberculosis strains utilize the type III secretion system (T3SS) to secrete fusion proteins (e.g., YopE-LcrV) under calcium-deprived conditions at 37°C .
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These systems enable efficient mucosal and systemic immune responses in murine models .
LPS and Immune Evasion
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The LPS core of Y. pseudotuberculosis interacts with host CD209 receptors, facilitating bacterial invasion of dendritic cells and macrophages .
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Rough LPS mutants (lacking O-antigen) show enhanced dissemination to mesenteric lymph nodes and spleen compared to smooth LPS variants .
O-Antigen Biosynthesis Gene Clusters
Though unrelated to GalK, Y. pseudotuberculosis O-antigen gene clusters (located between hemH and gsk) are critical for LPS diversity and pathogenicity. These clusters exhibit horizontal gene transfer, enabling rapid evolution of antigenic variants .
Critical Challenges and Gaps
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Absence of GalK-Specific Data: None of the provided sources mention galactokinase (GalK) in Y. pseudotuberculosis.
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Potential Misidentification: The query may conflate GalK with other kinases (e.g., LpxK) or metabolic enzymes described in these studies.
Recommendations for Further Research
To address the lack of GalK-specific data:
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Explore genomic databases (e.g., UniProt, NCBI) for Y. pseudotuberculosis GalK sequences.
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Investigate metabolic pathways in Yersinia species to identify GalK’s role in carbohydrate utilization.
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Use structural homology modeling to predict GalK’s function based on related bacterial enzymes.
Product Specs
Target Background
KEGG: ypb:YPTS_1247
Q&A
Experimental Design for Studying Recombinant Galactokinase in Yersinia pseudotuberculosis
Q: How can I design an experiment to study the role of recombinant galactokinase in Yersinia pseudotuberculosis serotype IB? A: To study the role of recombinant galactokinase, you can use a combination of molecular biology techniques such as PCR, gene cloning, and transformation into Y. pseudotuberculosis. Use a plasmid system to express the galK gene, and compare the metabolic and virulence capabilities of the recombinant strain with a wild-type strain. Analyze growth rates, sugar metabolism, and virulence factor expression using techniques like RT-qPCR and Western blotting.
Data Analysis and Contradiction Resolution
Q: How do I resolve contradictory data regarding the role of galactokinase in carbohydrate metabolism in Y. pseudotuberculosis? A: Contradictions can arise from differences in experimental conditions or strain backgrounds. To resolve these, ensure consistency in experimental design, including growth conditions (e.g., temperature, media) and strain selection. Use statistical analysis to compare results across different studies, and consider factors like gene regulation and environmental cues that might influence galactokinase activity.
Advanced Research Questions: Gene Regulation and Virulence
Q: What is the relationship between galactokinase expression and virulence gene regulation in Y. pseudotuberculosis? A: Investigate how galactokinase affects the expression of virulence genes by analyzing regulatory networks. Use techniques like RNA sequencing to identify changes in gene expression profiles when galK is overexpressed or knocked out. Examine the role of regulatory proteins like YmoA, which influences virulence gene expression in Yersinia species .
Methodological Considerations for Recombinant Protein Expression
Q: What are the best methods for expressing and purifying recombinant galactokinase from Y. pseudotuberculosis serotype IB? A: For expression, use a suitable host like E. coli with a plasmid containing the galK gene under a strong promoter (e.g., T7). Optimize growth conditions for high protein yield. For purification, use affinity chromatography (e.g., His-tag) followed by size exclusion chromatography to ensure purity.
Serotype-Specific Variations and Their Implications
Q: How do different serotypes of Y. pseudotuberculosis, such as IB, vary in their O-antigen structures and how might this impact galactokinase function? A: Different serotypes have distinct O-antigen structures due to variations in gene clusters . This diversity can affect the bacterium's interaction with the host and potentially influence metabolic pathways like galactose metabolism. Investigate how these structural differences impact the role of galactokinase in carbohydrate metabolism and virulence.
Genomic Analysis and Virulence Factors
Q: How can whole-genome sequencing help in understanding the virulence factors and genetic diversity of Y. pseudotuberculosis isolates? A: Whole-genome sequencing allows for detailed analysis of virulence genes, plasmids, and genetic elements like prophages that contribute to pathogenicity . Use bioinformatics tools to identify variations in gene content and expression that correlate with virulence and metabolic capabilities, including those related to galactokinase.
Epidemiological Studies and Outbreak Investigations
Q: How can molecular typing methods like core-genome multilocus sequence typing (cgMLST) aid in investigating outbreaks of Y. pseudotuberculosis? A: cgMLST is useful for identifying clonal outbreaks by analyzing genetic similarity among isolates . This method helps trace the source of outbreaks and understand the spread of specific strains, which can inform public health interventions and studies on virulence factors like galactokinase.
Biochemical Assays for Galactokinase Activity
Q: What biochemical assays can be used to measure the activity of recombinant galactokinase from Y. pseudotuberculosis? A: Use spectrophotometric assays that measure the conversion of galactose to galactose-1-phosphate, typically coupled with a secondary reaction to detect NADH or ATP production. Alternatively, radioisotope assays can provide sensitive detection of galactose phosphorylation.
Environmental Cues and Gene Expression
Q: How do environmental cues influence the expression of galactokinase in Y. pseudotuberculosis, and what are the implications for pathogenesis? A: Environmental factors such as temperature and nutrient availability can significantly affect gene expression in Yersinia species . Investigate how these cues regulate galK expression and how this impacts the bacterium's ability to metabolize carbohydrates during infection.
