Recombinant Photorhabdus luminescens subsp. laumondii D-alanine--D-alanine ligase (ddl)

What is D-alanine--D-alanine ligase (ddl) and what is its function in Photorhabdus luminescens?

D-alanine--D-alanine ligase (EC 6.3.2.4) is a critical enzyme involved in bacterial cell wall formation. In Photorhabdus luminescens subsp. laumondii, ddl catalyzes the ATP-dependent formation of the D-alanyl-D-alanine dipeptide, which is essential for peptidoglycan biosynthesis in bacterial cell walls. This enzyme belongs to the D-alanine--D-alanine ligase family and plays a crucial role in maintaining cellular structural integrity .

What are the optimal conditions for recombinant expression of P. luminescens ddl?

For recombinant expression of P. luminescens subsp. laumondii ddl, E. coli is the preferred heterologous host. The gene can be cloned into an appropriate expression vector containing a strong promoter (such as T7) and potentially a fusion tag to facilitate purification. Expression is typically induced in mid-log phase cultures (OD600 of 0.6-0.8) with IPTG, and cells are grown at 25-30°C post-induction to enhance protein solubility.

For optimal results, consider the following protocol:

• Transform expression plasmid into E. coli BL21(DE3) or similar expression strain

• Culture in LB medium supplemented with appropriate antibiotics at 37°C until OD600 reaches 0.6-0.8

• Induce with 0.5-1.0 mM IPTG

• Continue incubation at 25-30°C for 4-6 hours or 18°C overnight

• Harvest cells by centrifugation and proceed with protein extraction

How should recombinant P. luminescens ddl be purified for structural and functional studies?

Purification of recombinant ddl typically employs a multi-step chromatography approach:

• Cell lysis: Resuspend cell pellet in buffer (typically 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10% glycerol, 1 mM DTT) with protease inhibitors, then lyse via sonication or cell disruption

• Initial purification: If expressed with a tag (His, GST), use affinity chromatography (Ni-NTA for His-tagged proteins)

• Tag removal: Cleave tag with an appropriate protease if necessary for functional studies

• Further purification: Ion exchange chromatography followed by size exclusion chromatography

• Quality assessment: Verify purity by SDS-PAGE (>85% purity is typically required)

• Storage: Store in small aliquots with 20-50% glycerol at -80°C to prevent freeze-thaw cycles

For long-term storage, the enzyme should be maintained at -80°C, avoiding repeated freeze-thaw cycles. Working aliquots can be stored at 4°C for up to one week .

What assays can be used to measure P. luminescens ddl activity?

Several methods are available for assessing ddl activity:

• Coupled enzyme assay: Measures ADP production by coupling with pyruvate kinase and lactate dehydrogenase, monitoring NADH oxidation at 340 nm

• HPLC-based assay: Directly quantifies D-Ala-D-Ala dipeptide formation using reverse-phase HPLC

• Mass spectrometry: Detects the D-Ala-D-Ala product formation with high sensitivity

• Inorganic phosphate detection: Measures Pi released during ATP hydrolysis

Recommended protocol for coupled assay:

• Reaction buffer: 100 mM HEPES pH 7.5, 10 mM MgCl2, 10 mM KCl

• Substrates: 10 mM D-alanine, 2 mM ATP

• Coupling components: 1 mM phosphoenolpyruvate, 0.2 mM NADH, 5 units pyruvate kinase, 5 units lactate dehydrogenase

• Monitor decrease in absorbance at 340 nm

What are the kinetic parameters of P. luminescens ddl compared to other bacterial D-alanine--D-alanine ligases?

While specific kinetic data for P. luminescens ddl is not provided in the search results, typical kinetic parameters for bacterial D-alanine--D-alanine ligases include:

Determining these parameters specifically for P. luminescens ddl would require experimental kinetic analysis using the assays described above.

How conserved is ddl across Photorhabdus species and other bacterial genera?

Sequence analysis of ddl from P. luminescens subsp. laumondii compared to other bacterial species would likely reveal conserved catalytic and substrate-binding domains with variable regions that may influence substrate specificity and catalytic efficiency. The full genome of P. luminescens subsp. laumondii contains approximately 4,243 protein-coding genes, with ddl being one of the essential genes for cell wall synthesis .

Researchers should perform comparative sequence analysis using tools like BLAST, multiple sequence alignment, and phylogenetic tree construction to evaluate evolutionary relationships between ddl enzymes from different species.

What is the genomic context of the ddl gene in P. luminescens and how does it compare to other bacteria?

The genomic context of ddl in P. luminescens subsp. laumondii would likely include other genes involved in cell wall biosynthesis pathways. In many bacteria, ddl is often located in operons containing other peptidoglycan synthesis genes.

The P. luminescens subsp. laumondii genome has been sequenced (with the TT01 strain having a 5.27-Mbp genome and G+C content of 42.4%) . Researchers interested in the genomic context should:

• Identify the exact location of ddl in the genome

• Analyze neighboring genes and potential operon structures

• Compare synteny (gene order conservation) with related bacteria

• Investigate regulatory elements upstream of the ddl gene

What are the known inhibitors of bacterial D-alanine--D-alanine ligases and their effectiveness against P. luminescens ddl?

D-alanine--D-alanine ligases are attractive antibiotic targets due to their essential role in bacterial cell wall synthesis and absence in mammals. While specific inhibition data for P. luminescens ddl is not provided in the search results, known ddl inhibitors include:

• D-cycloserine: A structural analog of D-alanine that competitively inhibits ddl

• Phosphinate and phosphonate compounds: Transition state analogs that mimic the tetrahedral intermediate

• ATP-competitive inhibitors: Target the ATP-binding pocket

• Natural product inhibitors: Various compounds from microbial sources

Testing these compounds against recombinant P. luminescens ddl would require:

• Enzyme inhibition assays using methods described in section 3.1

• Determination of IC50 and Ki values

• Structure-activity relationship studies for rational design of more potent inhibitors

How can structural information about P. luminescens ddl be leveraged for structure-based drug design?

Structure-based drug design targeting P. luminescens ddl would involve:

• Structural determination: Obtain high-resolution crystal structure of P. luminescens ddl alone and in complex with substrates or inhibitors using X-ray crystallography or cryo-EM

• Active site mapping: Identify catalytic residues and substrate-binding pockets

• Virtual screening: Use computational docking to screen virtual compound libraries

• Fragment-based design: Identify small molecular fragments that bind to different regions of the active site

• Lead optimization: Iteratively modify promising compounds to improve potency and selectivity

If the crystal structure is unavailable, homology modeling based on related ddl structures could be employed, though this approach would have limitations in terms of accuracy for fine structural details.

How can recombinant P. luminescens ddl be utilized in the enzymatic synthesis of cell wall components or antimicrobial peptides?

Recombinant P. luminescens ddl has potential applications in enzymatic synthesis:

• D-Ala-D-Ala dipeptide production: For use in peptidoglycan synthesis studies or as building blocks for antimicrobial peptides

• Custom peptidoglycan fragment synthesis: Creating defined cell wall fragments for immunological studies

• Biosensors: Development of assays for antibiotic discovery

Methodology for enzymatic synthesis:

• Use purified recombinant ddl in reaction buffer (100 mM HEPES pH 7.5, 10 mM MgCl2, 10 mM KCl)

• Add ATP (5 mM) and D-alanine (20-50 mM)

• Incubate at 30°C for 1-4 hours

• Purify products by HPLC or other chromatographic methods

Similar enzymatic approaches have been used with other bacterial ligases for the synthesis of short oligopeptides, as mentioned in search result regarding the enzyme from Empedobacter brevis that efficiently produces L-alanyl-L-glutamine.

What is the potential of P. luminescens ddl in the development of novel biosynthetic pathways?

P. luminescens ddl could be integrated into synthetic biology applications:

• Cell wall engineering: Modifying peptidoglycan structure in bacteria

• Bioorthogonal chemistry: Creating non-natural peptide linkages by engineering substrate specificity

• Peptide antibiotic production: Incorporating D-amino acid containing structures into antimicrobial peptides

This approach would build upon knowledge of P. luminescens as a producer of various natural products. The bacterium contains numerous biosynthetic gene clusters (BGCs) that are regulated by bacterial enhancer binding proteins (bEBPs) . Understanding how ddl interacts with these biosynthetic pathways could inform the development of novel enzymatic production systems.

How does ddl contribute to the symbiotic relationship between P. luminescens and nematode hosts?

P. luminescens subsp. laumondii forms a symbiotic association with Heterorhabditis nematodes, particularly Heterorhabditis bacteriophora . While ddl itself has not been directly implicated in this symbiotic relationship based on the provided search results, it likely plays an indirect role:

• Cell wall integrity: By ensuring proper cell wall structure, ddl helps P. luminescens maintain its cellular integrity within the nematode host

• Stress response: Proper cell wall synthesis is essential for bacterial survival under the stress conditions encountered within the host

• Colonization ability: Cell wall properties affect the bacterium's ability to colonize the nematode intestine

The symbiotic relationship is complex, with P. luminescens providing benefits to the nematode host while also contributing to its pathogenicity against insects. The bacterium has been isolated from H. bacteriophora found in various locations, including New Jersey, USA .

What is the impact of ddl expression levels on bacterial growth, stress response, and antibiotic resistance in P. luminescens?

While specific data on ddl expression in P. luminescens is not available in the search results, the impact of ddl expression can be inferred from studies in other bacteria:

• Growth rate: Optimal ddl expression is critical for normal growth rates; under-expression leads to cell wall defects while over-expression may divert resources from other cellular processes

• Stress response: Altered ddl expression affects cell wall integrity under various stresses (osmotic, pH, temperature)

• Antibiotic susceptibility: Changes in ddl expression can modify susceptibility to cell wall-targeting antibiotics

To study these effects experimentally in P. luminescens:

• Create conditional expression strains or gene knockdowns

• Monitor growth curves under various conditions

• Perform antibiotic susceptibility testing

• Analyze cell morphology using microscopy

• Measure peptidoglycan composition using HPLC or mass spectrometry

What are the cutting-edge techniques for studying the in vivo dynamics of ddl in P. luminescens?

Advanced methodologies for investigating ddl dynamics in P. luminescens include:

• CRISPR-Cas9 gene editing: Creating precise mutations or tagged versions of ddl

• Fluorescent protein tagging: Monitoring ddl localization within bacterial cells

• Quantitative proteomics: Measuring changes in ddl expression under different conditions

• Single-cell analysis: Examining cell-to-cell variability in ddl expression

• Metabolic labeling: Using D-alanine analogs to track peptidoglycan synthesis

P. luminescens can be cultured under laboratory conditions at 28°C using specific media such as Medium 535b or Medium 1a , facilitating these experimental approaches.

How can systems biology approaches integrate ddl function into the broader metabolic network of P. luminescens?

Systems biology approaches to study ddl in the context of P. luminescens metabolism include:

• Genome-scale metabolic modeling: Integrating ddl into metabolic flux models of P. luminescens

• Transcriptomics: RNA-seq analysis to identify genes co-regulated with ddl

• Proteomics: Analyzing protein-protein interactions involving ddl

• Metabolomics: Measuring changes in metabolite pools related to cell wall synthesis

• Multi-omics integration: Combining datasets to create comprehensive models

These approaches would help place ddl within the context of P. luminescens' complex metabolism, including its production of various natural products from biosynthetic gene clusters that are regulated by bacterial enhancer binding proteins (bEBPs) .

What factors regulate ddl expression in P. luminescens under different environmental conditions?

While specific regulatory mechanisms for ddl in P. luminescens are not detailed in the search results, regulation likely involves:

• Nutrient availability: Adjustments in cell wall synthesis based on available resources

• Growth phase: Differential expression during exponential versus stationary phase

• Stress responses: Changes in expression under cell envelope stress

• Host-associated signals: Potential regulation during symbiotic or pathogenic interactions

P. luminescens has a complex regulatory network including bacterial enhancer binding proteins (bEBPs) that control gene expression. Six bEBPs have been identified in P. laumondii TT01, including GlrR, GlnG, TyrR, PspF, PrpR, and PLU_RS06090 . These regulatory proteins may directly or indirectly influence ddl expression under various conditions.

How does ddl expression coordinate with other cell wall synthesis enzymes in P. luminescens?

Coordination of ddl with other cell wall synthesis enzymes likely involves:

• Transcriptional co-regulation: Common regulatory elements controlling expression of multiple cell wall synthesis genes

• Operon structure: Potential organization of ddl with other cell wall synthesis genes in operons

• Protein-protein interactions: Physical interactions between ddl and other peptidoglycan biosynthesis enzymes

• Metabolic feedback: Regulation based on intermediate concentrations in the peptidoglycan synthesis pathway

Experimental approaches to study this coordination include:

• RNA-seq analysis under various conditions

• Chromatin immunoprecipitation (ChIP-seq) to identify transcription factor binding sites

• Co-immunoprecipitation to detect protein-protein interactions

• Metabolic profiling of cell wall precursors