Recombinant Streptomyces coelicolor Prolipoprotein diacylglyceryl transferase 2 (lgt2)

What is the function of Prolipoprotein diacylglyceryl transferase (Lgt) in bacterial cells?

Prolipoprotein diacylglyceryl transferase (Lgt) catalyzes the transfer of an sn-1,2-diacylglyceryl group from phosphatidylglycerol to prolipoproteins, representing the first step in bacterial lipoprotein biogenesis . This enzyme plays a critical role in the post-translational modification of bacterial lipoproteins, which are essential components of bacterial cell membranes. In Streptomyces coelicolor, Lgt is part of a four-step lipoprotein biogenesis pathway that includes Lgt, Lsp (lipoprotein signal peptidase), and Lnt enzymes (for N-acylation) . This pathway is crucial for proper lipoprotein localization and function in the bacterial membrane.

Why does Streptomyces coelicolor have two copies of the lgt gene?

Streptomyces coelicolor encodes two functional copies of Lgt that cannot be removed in the same strain, suggesting functional redundancy with an essential role . Research has demonstrated that while either copy can be deleted individually, attempts to delete both copies simultaneously are unsuccessful, indicating that at least one functional copy is required for cell viability . This redundancy may represent an evolutionary adaptation that ensures the essential process of lipoprotein biogenesis can proceed even if one copy is mutated or inactivated. The presence of multiple copies of essential genes is a common strategy in bacteria for maintaining critical cellular functions under different environmental conditions or developmental stages.

How is the lipoprotein biogenesis pathway in Streptomyces coelicolor different from other bacteria?

The lipoprotein biogenesis pathway in Streptomyces coelicolor has several distinctive features compared to other bacteria. First, S. coelicolor employs both the Sec and Tat secretion pathways for lipoprotein export, with approximately 20% of lipoprotein precursors being exported via the Tat pathway . Second, unlike many Gram-positive bacteria, S. coelicolor performs N-acylation of lipoproteins using two non-essential Lnt enzymes, a feature more commonly associated with Gram-negative bacteria . Third, S. coelicolor contains two functional copies of Lgt, whereas most bacteria contain only one . Fourth, while Lsp (lipoprotein signal peptidase) is essential in many bacteria, it can be deleted in S. coelicolor, though this results in growth and developmental defects .

What are the recommended methods for isolating recombinant Lgt2 from Streptomyces coelicolor?

For isolation of recombinant Lgt2 from Streptomyces coelicolor, researchers should first consider the membrane-bound nature of this protein. Based on studies of Lgt in E. coli, which demonstrated that the enzyme is embedded in the membrane by seven transmembrane segments with its N-terminus facing the periplasm and C-terminus facing the cytoplasm , similar topology might be expected for S. coelicolor Lgt2. The isolation protocol should begin with cell lysis under gentle conditions to preserve membrane integrity, followed by membrane fraction isolation through differential centrifugation. For solubilization, appropriate detergents must be selected that maintain protein structure and function while extracting Lgt2 from the membrane. Affinity chromatography using a fusion tag (His-tag or FLAG-tag) added to the recombinant protein can facilitate purification. Throughout the process, protease inhibitors should be included to prevent degradation, and all buffers should be optimized for pH and salt concentration to maintain protein stability.

What genetic tools are most effective for creating lgt2 knockout or knockdown strains in Streptomyces coelicolor?

For creating lgt2 knockout or knockdown strains in Streptomyces coelicolor, researchers should consider several genetic approaches. Traditionally, the Redirect PCR targeting method has been used for gene deletion in Streptomyces species, but this approach can lead to complications, as observed with lsp deletion . When using cosmid-based methods like Redirect, researchers should be aware that transient duplication of genes present on the cosmid (such as cell division genes) can lead to secondary mutations and phenotypic changes unrelated to the target gene deletion . A more targeted approach using suicide vectors, as demonstrated for lsp deletion, may be preferable to avoid these issues . For lgt2 specifically, since complete removal of both lgt genes appears to be lethal, researchers might consider conditional knockout systems or knockdown strategies using antisense RNA or CRISPR interference. Recent advances in CRISPR/Cas9 editing of Streptomyces genomes provide more precise tools for genetic manipulation without requiring cosmid libraries, potentially avoiding the secondary mutations observed with earlier methods .

What are the optimal expression conditions for producing active recombinant Lgt2 protein?

To produce active recombinant Lgt2 protein, expression conditions must be carefully optimized. Since Lgt is a membrane protein with multiple transmembrane segments , expression systems capable of properly inserting membrane proteins are essential. For heterologous expression, E. coli strains specifically designed for membrane protein expression (such as C41(DE3) or C43(DE3)) may be suitable. Alternatively, homologous expression in Streptomyces species might provide better yields of properly folded protein. Expression should be conducted at lower temperatures (16-20°C) to slow protein synthesis and facilitate proper folding. Induction conditions should be mild to prevent formation of inclusion bodies, with lower concentrations of inducer and longer expression times. The growth medium should be supplemented with appropriate phospholipids to ensure availability of the phosphatidylglycerol substrate. For purification, gentle detergents such as n-dodecyl-β-D-maltoside (DDM) or digitonin should be used to solubilize the membrane while maintaining protein structure and function. Activity assays using synthetic prolipoprotein substrates can confirm proper folding and function of the recombinant enzyme.

What are the mechanisms of antibiotic resistance involving Lgt2, particularly regarding globomycin and antibiotic TA?

While the search results don't specifically address resistance mechanisms involving Lgt2 in S. coelicolor, they do provide insights about related antibiotics that target the lipoprotein biogenesis pathway. Globomycin, produced by Streptomyces globisporus, and antibiotic TA, produced by Myxococcus xanthus, both inhibit Lsp (lipoprotein signal peptidase) activity and are lethal to Escherichia coli . Resistance to antibiotic TA in E. coli can arise through spontaneous IS3 insertion into the lpp gene, which encodes an abundant lipoprotein that attaches the E. coli outer membrane to the peptidoglycan cell wall . Additionally, over-expression of lsp confers TA resistance in both E. coli and M. xanthus, and the latter encodes additional Lsp homologues within the TA biosynthetic gene cluster . By analogy, potential resistance mechanisms involving Lgt2 in S. coelicolor might include: (1) Mutations in the lgt2 gene that alter the binding site for inhibitors while maintaining enzymatic function; (2) Upregulation of lgt2 expression to overcome inhibition through increased enzyme concentration; (3) Compensatory upregulation of the paralogous lgt1 when lgt2 is inhibited; (4) Modifications in the structure or expression of target prolipoproteins. Future research should focus on identifying specific inhibitors of Lgt enzymes and characterizing the resulting resistance mechanisms through evolution experiments and structural studies.

What are the best techniques for studying the interaction between Lgt2 and its prolipoprotein substrates?

To study the interaction between Lgt2 and its prolipoprotein substrates, several complementary techniques can be employed. Surface plasmon resonance (SPR) or bio-layer interferometry (BLI) allows real-time monitoring of binding kinetics between purified Lgt2 (immobilized on a sensor chip) and various synthetic prolipoprotein substrates. Isothermal titration calorimetry (ITC) provides thermodynamic parameters of binding, offering insights into the energetics of the interaction. Cross-linking coupled with mass spectrometry can identify specific amino acid residues involved in the enzyme-substrate interaction. For structural studies, X-ray crystallography or cryo-electron microscopy of Lgt2 in complex with substrate analogs or inhibitors would provide detailed information about binding sites and catalytic mechanisms. Drawing from studies of E. coli Lgt, which identified critical residues Y26, N146, G154, R143, E151, R239, and E243 for enzyme function , researchers should focus on the corresponding residues in S. coelicolor Lgt2 through site-directed mutagenesis followed by activity assays. Fluorescence resonance energy transfer (FRET) assays using labeled enzyme and substrate can monitor the interaction in real-time and provide spatial information. Additionally, computational approaches such as molecular docking and molecular dynamics simulations can predict binding modes and conformational changes during catalysis.

How can researchers distinguish between the functions of the two Lgt enzymes in vivo?

To distinguish between the functions of the two Lgt enzymes in Streptomyces coelicolor in vivo, researchers should employ a multi-faceted approach. Single gene knockout strains (Δlgt1 and Δlgt2) should be created and characterized through comparative phenotypic analysis, examining growth rates, morphological development, and antibiotic production under various conditions. Complementation studies with controlled expression of each lgt gene can confirm phenotype specificity. To identify the specific substrates of each enzyme, quantitative proteomics should be performed on membrane fractions from wild-type, Δlgt1, and Δlgt2 strains, looking for lipoproteins that are specifically unprocessed in each mutant. Metabolic labeling with azide-modified fatty acids followed by click chemistry can enable visualization and identification of newly lipidated proteins specific to each Lgt enzyme. Temporally controlled gene expression using inducible promoters for each lgt gene can reveal whether they function at different developmental stages. Fluorescent protein fusions can determine whether the two enzymes localize differently within the cell membrane. Chimeric proteins, where domains are swapped between Lgt1 and Lgt2, can help identify which protein regions determine substrate specificity. Finally, transcriptomics under various growth conditions and stresses can reveal differential expression patterns of the two lgt genes, providing insights into their specialized roles.

What computational approaches can predict the structure and substrate binding sites of Lgt2?

For predicting the structure and substrate binding sites of Lgt2 from Streptomyces coelicolor, researchers should implement a comprehensive computational pipeline. Homology modeling represents the starting point, using the structure of E. coli Lgt as a template if available, or structures of related enzymes. Since Lgt is a membrane protein with seven transmembrane segments in E. coli , specialized membrane protein modeling tools like MEMOIR or ROSETTA-MP should be employed. The model should be refined through molecular dynamics simulations in a lipid bilayer environment to accurately represent the membrane-embedded state of the protein. Conservation analysis across Lgt proteins from different species can identify evolutionarily conserved residues likely involved in catalysis or substrate binding, with particular attention to the Lgt signature motif that contains invariant residues . Computational alanine scanning can predict the energetic contribution of specific residues to substrate binding. Molecular docking of prolipoprotein signal peptides and phosphatidylglycerol can identify potential binding pockets and interaction modes. Virtual screening against the modeled structure can suggest potential inhibitors for experimental validation. Machine learning approaches trained on known enzyme-substrate interactions can further refine predictions of substrate specificity. The computational predictions should guide experimental site-directed mutagenesis studies to validate the importance of predicted catalytic and binding site residues.

What are the critical residues in Lgt2 for enzyme function and how do they compare with Lgt1?

Based on studies of E. coli Lgt, several critical residues have been identified that are likely to be conserved in S. coelicolor Lgt2. The table below summarizes these residues and their functions:

What approaches should be used to analyze the metabolic changes resulting from lgt2 manipulation?

To comprehensively analyze metabolic changes resulting from lgt2 manipulation in Streptomyces coelicolor, researchers should employ a multi-omics approach. Untargeted metabolomics using liquid chromatography-mass spectrometry (LC-MS) should be performed on both intracellular and extracellular samples from wild-type and lgt2 mutant strains to detect changes in primary and secondary metabolites. Targeted analyses should focus specifically on antibiotics like actinorhodin, which was overproduced in lsp mutants , suggesting similar effects might occur with lgt2 manipulation. Stable isotope labeling experiments using 13C-glucose can track carbon flux through different metabolic pathways, revealing how lgt2 disruption affects resource allocation. Transcriptomics through RNA-seq should be employed to identify differentially expressed metabolic genes, particularly those involved in secondary metabolite biosynthesis. Chromatin immunoprecipitation sequencing (ChIP-seq) targeting key transcriptional regulators can reveal changes in metabolic gene regulation. Proteomics focusing on membrane-associated proteins should be performed to connect changes in lipoprotein processing with metabolic effects. Phenotypic microarrays testing growth on different carbon sources can identify specific metabolic capabilities affected by lgt2 manipulation. Finally, metabolic flux analysis using 13C-metabolic flux analysis (13C-MFA) can provide quantitative information about changes in metabolic pathway activities. Integration of these datasets using systems biology approaches can uncover the complex relationships between lipoprotein processing and metabolism in S. coelicolor.

What are the implications of targeting Lgt2 for antibiotic development?

Targeting Lgt2 for antibiotic development presents several promising avenues for research. The essentiality of Lgt in multiple bacterial species, including E. coli and the requirement for at least one functional Lgt in S. coelicolor , highlights this enzyme as a potential broad-spectrum antibiotic target. Since the lipoprotein biogenesis pathway is absent in humans, inhibitors of Lgt2 could potentially show selective toxicity against bacteria without affecting human cells. Natural product antibiotics like globomycin and antibiotic TA, which target the related enzyme Lsp in the same pathway, have already demonstrated antibacterial efficacy , suggesting that targeting other components of this pathway could be similarly effective. The presence of multiple Lgt enzymes in some bacteria like S. coelicolor suggests that effective Lgt inhibitors might need to target conserved catalytic sites shared between variants, or be used in combination to prevent resistance through functional redundancy. The membrane-embedded nature of Lgt with seven transmembrane segments presents challenges for inhibitor design but could also provide opportunities for developing compounds that interact with specific membrane-embedded binding sites. Future research should focus on high-throughput screening for Lgt inhibitors, structure-based drug design targeting the conserved Lgt signature motif , and in vivo testing in infection models to assess efficacy and potential resistance mechanisms.

How can understanding Lgt2 function contribute to improved heterologous protein expression in Streptomyces?

Understanding Lgt2 function can significantly contribute to improved heterologous protein expression in Streptomyces coelicolor, particularly for membrane-associated and secreted proteins. By characterizing the substrate specificity of Lgt2, researchers could engineer optimal signal peptides for efficient lipidation of recombinant proteins intended for membrane anchoring or secretion. Since approximately 20% of lipoprotein precursors in S. coelicolor are exported via the Tat pathway , which is known to export folded proteins, manipulation of the lipoprotein biogenesis pathway could enhance the secretion of correctly folded heterologous proteins with complex structures. Controlled expression of lgt2 could potentially modulate the cell envelope properties, creating more favorable conditions for heterologous protein production or secretion. For industrial enzyme production, fusion of lipoprotein signal sequences recognized by Lgt2 to target proteins could facilitate their membrane anchoring, creating whole-cell biocatalysts with improved stability and reusability. Additionally, since lsp mutants overproduce actinorhodin , targeted manipulation of the lipoprotein processing pathway might enhance the yields of heterologously expressed secondary metabolites. Researchers should develop expression vectors with optimized lipoprotein signal peptides, create host strains with engineered lipoprotein processing capabilities, and explore the effects of different membrane compositions on heterologous protein production efficiency and quality.

What novel approaches could reveal the broader role of lipoproteins in Streptomyces development and secondary metabolism?

Novel approaches to reveal the broader role of lipoproteins in Streptomyces development and secondary metabolism should combine cutting-edge techniques from various fields. Proximity-dependent biotin identification (BioID) or APEX2 labeling coupled with mass spectrometry could identify interaction partners of specific lipoproteins, revealing their functional networks. CRISPR interference (CRISPRi) libraries targeting all predicted lipoproteins would allow high-throughput screening for developmental and metabolic phenotypes. Single-cell tracking using microfluidics combined with fluorescent reporters for developmental markers and metabolite biosynthesis genes could reveal how lipoprotein function correlates with cellular differentiation and metabolite production at the single-cell level. Cryo-electron tomography of cell envelope structures in wild-type and lipoprotein mutants could visualize how lipoproteins contribute to membrane organization during development. Lipidomics analysis of membrane composition changes during development and in response to lipoprotein gene manipulation would connect lipoprotein function to membrane properties. Integration of transcriptomics, proteomics, and metabolomics data from developmental time courses of wild-type and lipoprotein pathway mutants using systems biology approaches could identify key regulatory networks. The investigation of small RNAs like scr6809, which when disrupted led to developmental phenotypes in S. coelicolor , could reveal post-transcriptional regulation of lipoprotein expression. Finally, comparative genomics across Streptomyces species with different developmental patterns and secondary metabolite profiles could identify conserved and divergent lipoprotein functions across the genus.