Recombinant Agrobacterium tumefaciens Putative zinc metalloprotease Atu1380 (Atu1380)

What are the structural characteristics and domain organization of Atu1380?

While the detailed three-dimensional structure of Atu1380 has not been fully characterized, analysis of its amino acid sequence and comparison with other zinc metalloproteases provides insights into its potential structural organization. The protein's domain structure likely follows patterns typical of bacterial metalloproteases with several functional regions:

• Signal Peptide/Pre-domain: The N-terminal region (approximately first 20-30 amino acids) likely functions as a signal peptide directing protein secretion from the bacterial cell. The sequence "MNTIMAATGFLTGYIVPFILVLSLLVFVHEMG" contains hydrophobic residues characteristic of signal sequences .

• Catalytic Domain: This region contains the zinc-binding motif characteristic of metalloproteases, typically with conserved histidine residues that coordinate the zinc ion essential for catalytic activity. In many metalloproteases, this includes a HEXXH consensus sequence where the two histidines coordinate zinc and the glutamic acid participates in catalysis .

• Transmembrane Regions: Analysis of the sequence suggests potential membrane-spanning domains, indicated by the presence of hydrophobic amino acid stretches. This suggests Atu1380 may be membrane-associated, which would be consistent with roles in cell surface processes or secretion .

The protein's predicted structure suggests it may participate in proteolytic activities at the bacterial cell surface or in the extracellular environment, potentially modifying either bacterial or host plant proteins during the Agrobacterium-plant interaction process.

How is Atu1380 typically expressed and purified for research applications?

Recombinant Atu1380 is typically expressed and purified using the following methodological approach:

• Expression System:

  • Host: Escherichia coli is the preferred expression system

  • Vector: Expression constructs typically incorporate an N-terminal His-tag for purification

  • Expression Region: Full-length protein (amino acids 1-377)

• Purification Protocol:

  • Immobilized Metal Affinity Chromatography (IMAC) using the His-tag

  • Buffer systems typically include Tris-based buffers at pH 8.0

  • Protein is eluted with imidazole and often lyophilized for storage

• Quality Control:

  • SDS-PAGE analysis to confirm purity (typically >90%)

  • Verification of protein identity through Western blotting or mass spectrometry

• Reconstitution:

  • The lyophilized protein should be reconstituted in deionized sterile water

  • Recommended concentration: 0.1-1.0 mg/mL

  • Addition of 5-50% glycerol for long-term storage stability

This standardized expression and purification methodology yields recombinant Atu1380 protein suitable for various research applications, including enzymatic assays, structural studies, and investigation of protein-protein interactions.

What are the optimal storage conditions for Recombinant Atu1380?

Proper storage of Recombinant Atu1380 is critical for maintaining its structural integrity and enzymatic activity. Based on empirical data, the following storage protocols are recommended:

• Short-term Storage (up to one week):

  • Store working aliquots at 4°C

  • Maintain in Tris/PBS-based buffer with 6% Trehalose, pH 8.0

• Long-term Storage:

  • Store at -20°C or preferably -80°C for extended periods

  • Add glycerol to 50% final concentration as a cryoprotectant

  • Aliquot into single-use volumes before freezing to avoid repeated freeze-thaw cycles

• Reconstitution Guidelines:

  • Briefly centrifuge vial before opening to bring contents to the bottom

  • Reconstitute in deionized sterile water to 0.1-1.0 mg/mL

  • For long-term storage, add glycerol (5-50% final concentration)

• Stability Considerations:

  • Avoid repeated freeze-thaw cycles as they significantly decrease protein activity

  • Maintain pH around 8.0 for optimal stability

  • Consider the addition of stabilizing agents such as trehalose or glycerol

Following these storage recommendations ensures maximum retention of Atu1380 structural integrity and enzymatic activity for research applications, particularly for protocols requiring active enzyme.

How does Atu1380 compare to other metalloproteases?

Comparison of Atu1380 with other metalloproteases reveals both similarities and distinctive features that position it within the broader protease family:

• Structural Comparison:

  • Like matrix metalloproteases (MMPs), Atu1380 likely contains a catalytic domain with a zinc-binding motif

  • Unlike many eukaryotic MMPs, Atu1380 appears to lack the hemopexin domain commonly found in mammalian metalloproteases

  • As a bacterial metalloprotease, it likely has structural adaptations specific to its function in Agrobacterium

• Functional Comparison:

  • Mammalian MMPs function at neutral pH to cleave extracellular matrix components, growth factors, cytokines, and cell adhesion molecules

  • Atu1380, as a bacterial metalloprotease, may be involved in bacterial pathogenicity, potentially modifying host proteins or bacterial surface proteins during plant infection

• Comparative Features:

• Evolutionary Context:

  • Bacterial metalloproteases like Atu1380 represent a distinct evolutionary lineage compared to the more complex eukaryotic metalloproteases

  • Specialized adaptations in Atu1380 likely reflect its role in Agrobacterium's lifecycle and plant infection process

Understanding these comparative features helps researchers position studies of Atu1380 within the broader context of metalloprotease research and evolution, informing experimental design and interpretation of results.

What experimental design considerations are important when studying Atu1380 function?

When designing experiments to investigate Atu1380 function, several critical methodological considerations should be addressed to ensure robust and reproducible results:

• Randomized Complete Block Design:

  • Implement blocking to control for variables that may influence results but are not being directly manipulated

  • Ensure treatments are assigned randomly within each block to minimize bias

  • Include appropriate control groups (e.g., inactive enzyme mutants, substrate-only controls)

• Sample Size and Replication:

  • Include multiple experimental units per treatment group to ensure reliable statistical analysis

  • Provide adequate biological replicates (different bacterial cultures) and technical replicates (repeated measurements)

  • Calculate sample sizes based on anticipated effect sizes and desired statistical power

• Control of Confounding Variables:

  • Temperature, pH, and ionic strength should be strictly controlled in enzymatic assays

  • Standardize protein concentration and purity across experiments

  • Consider the influence of tags (e.g., His-tag) on enzyme activity and include tag-cleaved controls when possible

• Experimental Conditions for Enzymatic Activity:

  • Optimal conditions for metalloproteases typically include:

    • Temperature: 25-37°C

    • Buffer: 50mM HEPES, pH 7.0

    • Cofactors: 10mM CaCl₂ and zinc ions

  • Reaction times can range from 10 minutes to overnight depending on the substrate

• Substrate Selection:

  • Use both synthetic peptide substrates and potential native substrates

  • Include positive controls with well-characterized metalloproteases (e.g., MMP-1, MMP-2)

  • Consider fluorogenic or chromogenic substrates for quantitative analysis

• Data Analysis Approach:

  • Plan statistical methods in advance (t-tests, ANOVA, etc.)

  • Consider normality and variance assumptions

  • Implement appropriate controls for multiple comparisons

Adhering to these experimental design principles ensures that investigations of Atu1380 function produce reliable, interpretable, and reproducible results that can be meaningfully compared with studies of other metalloproteases.

How can transcriptomic analysis be used to investigate Atu1380 regulation and function?

Transcriptomic analysis provides powerful insights into Atu1380 regulation and function through a systematic methodological framework:

• RNA Extraction and Library Preparation:

  • Extract high-quality RNA from Agrobacterium tumefaciens under various conditions

  • Construct sequencing libraries using NEBNext Q5 Hot Start HiFi PCR Master Mix kit

  • Sequence on platforms such as Illumina Novaseq 6000 for high-throughput data generation

• Quality Control and Alignment:

  • Process raw sequencing data using FastQC (version 0.11.9)

  • Trim low-quality reads and adapters using Trimmomatic (version 0.39)

  • Align clean reads to the Agrobacterium tumefaciens strain C58 genome using Bowtie2 (version 2.4.2)

• Differential Expression Analysis:

  • Compare expression of Atu1380 under different conditions:

    • Various growth phases

    • Plant host interaction versus free-living

    • Response to environmental stressors

  • Identify co-regulated genes to infer functional relationships

• Functional Annotation and Enrichment:

  • Annotate genes using databases: GenBank Nonredundant, Pfam, Swiss-Prot, KOGs, GO, and KEGG

  • Conduct GO and KEGG enrichment analysis to identify pathways involving Atu1380

  • Use false discovery rate (FDR) calculations with a threshold of FDR ≤ 0.05 for significant enrichment

• Co-Expression Network Analysis:

  • Implement WGCNA (Weighted Gene Co-expression Network Analysis) using R

  • Filter genes with FPKM > 1 and coefficient of variation (CV) > 0.5

  • Use analysis parameters: similarity threshold of 0.5, minimum module size of 50, soft threshold power of 14

  • Visualize networks using Cytoscape to identify key interactions with Atu1380

• Validation by qRT-PCR:

  • Confirm RNA-seq results for Atu1380 and related genes using quantitative RT-PCR

  • Design primers using Primer Premier (v5.0) and DNAMAN (v8.0)

  • Normalize expression using reference genes such as gyrB (atu0012) and dnaC (atu1084)

This comprehensive transcriptomic approach provides insights into the regulatory mechanisms controlling Atu1380 expression, its functional relationships with other genes, and its potential roles in Agrobacterium biology and plant-microbe interactions.

What methodologies are recommended for studying Atu1380 enzymatic activity?

For rigorous characterization of Atu1380 enzymatic activity, the following methodological approaches are recommended:

• Substrate Specificity Determination:

  • Screen synthetic peptide libraries with different cleavage site sequences

  • Use fluorogenic substrates with varying amino acid sequences flanking the scissile bond

  • Test potential native protein substrates from plant hosts

  • Analyze cleavage products by HPLC, mass spectrometry, or fluorescence-based assays

• Enzyme Kinetics Analysis:

  • Determine Km, Vmax, kcat, and kcat/Km using Michaelis-Menten kinetics

  • Vary substrate concentrations (typically 0.1-10X Km)

  • Analyze data using non-linear regression to determine kinetic parameters

  • Typical assay conditions: 50mM HEPES (pH 7.0), 10mM CaCl₂, 25-37°C

• Inhibitor Studies:

  • Test general metalloprotease inhibitors (e.g., EDTA, 1,10-phenanthroline)

  • Evaluate specific MMP inhibitors for cross-reactivity

  • Determine IC₅₀ and Ki values for effective inhibitors

  • Consider time-dependent inhibition analysis for mechanism determination

• Active Site Mapping:

  • Perform site-directed mutagenesis of predicted catalytic residues

  • Focus on conserved HEXXH motif and other putative zinc-binding residues

  • Analyze effects on activity and substrate binding

  • Supplement with structural modeling when possible

• Comparison with Well-Characterized Metalloproteases:

  • Benchmark against well-characterized metalloproteases such as MMP-1, MMP-2, MMP-8, MMP-9, and MMP-13

  • Use standardized substrates and assay conditions for valid comparisons

  • Compare concentration requirements (typically 10-300nM range for metalloproteases)

• Experimental Conditions Table:

These methodological approaches provide a comprehensive framework for characterizing Atu1380 enzymatic activity, elucidating its molecular mechanism, and positioning it within the broader context of metalloprotease enzymes.

How can co-expression network analysis provide insights into Atu1380 function?

Co-expression network analysis offers a systems-level approach to understanding Atu1380 function through identification of genes with coordinated expression patterns:

• Integration with Phenotypic Data:

  • Correlate module eigengenes with phenotypic traits (e.g., virulence, plant transformation efficiency)

  • Identify conditions where Atu1380-containing modules show significant correlation with phenotypes

  • Generate testable hypotheses about Atu1380 function in specific biological contexts

This methodological framework provides a systems-level perspective on Atu1380 function, helping to elucidate its role within broader cellular processes and identify potential functional partners for further investigation.

What challenges exist in determining Atu1380 substrate specificity?

Determining the substrate specificity of Atu1380 presents several methodological challenges that researchers should consider when designing studies:

• Limited Prior Knowledge:

  • Unlike well-characterized metalloproteases, Atu1380 has minimal published data on natural substrates

  • Prediction algorithms have limited accuracy for bacterial metalloproteases

  • Homology-based predictions require validation due to potential functional divergence

• Technical Challenges in Substrate Identification:

  • Natural substrates may be plant proteins not readily available in standard assays

  • Low abundance substrates might be missed in untargeted approaches

  • Multiple approaches are needed for comprehensive identification

  • Dynamic interactions may depend on specific physiological conditions

• Methodological Approaches and Limitations:

• Experimental Design Considerations:

  • Compare with characterized MMPs (MMP-1, MMP-2, MMP-8, MMP-9, MMP-13) as reference points

  • Use enzyme concentrations between 10-300nM for comparative studies

  • Include both bacterial and plant proteins as potential substrates

  • Design experiments that mimic the Agrobacterium-plant interface environment

• Validation Requirements:

  • Confirm cleavage sites by mass spectrometry

  • Demonstrate concentration and time-dependent activity

  • Show inhibition by metalloprotease inhibitors

  • Validate with site-directed mutagenesis of catalytic residues

  • Correlate in vitro findings with in vivo phenotypes

Addressing these challenges requires a multi-faceted approach combining biochemical, proteomic, and computational methods to comprehensively characterize Atu1380 substrate specificity and its biological implications in Agrobacterium-plant interactions.

How can qRT-PCR be used to validate Atu1380 expression studies?

Quantitative reverse transcription PCR (qRT-PCR) provides a robust method for validating Atu1380 expression findings through the following methodological workflow:

• Experimental Design for Validation:

  • Select conditions that showed significant differential expression in transcriptomic data

  • Include biological replicates (minimum n=3) for statistical validation

  • Design time-course experiments if temporal expression patterns are of interest

  • Include appropriate controls (e.g., different growth conditions, host interactions)

• Primer Design and Validation:

  • Design primers specific to Atu1380 using primer design software (Primer Premier v5.0, DNAMAN v8.0)

  • Optimal primer characteristics:

    • Length: 18-25 nucleotides

    • GC content: 40-60%

    • Tm: ~60°C with <5°C difference between pairs

    • Amplicon size: 80-200 bp for efficient amplification

  • Validate primer specificity through melt curve analysis and gel electrophoresis

• Reference Gene Selection:

  • Use established reference genes for Agrobacterium tumefaciens:

    • gyrB (atu0012): DNA gyrase B subunit

    • dnaC (atu1084): DNA replication protein

  • Validate reference gene stability across experimental conditions

  • Consider using multiple reference genes for robust normalization

• RNA Extraction and cDNA Synthesis:

  • Extract high-quality RNA (RIN > 8)

  • Treat with DNase to remove genomic DNA contamination

  • Synthesize cDNA using the PrimeScriptTM RT Reagent Kit (Takara)

  • Include no-RT controls to detect genomic DNA contamination

• qRT-PCR Experimental Protocol:

  • Use TB Green TM Premix Ex Taq TM II kit (Takara) or similar SYBR Green-based systems

  • Perform reactions on a quantistudio 3 Real-Time PCR system or equivalent

  • Include technical replicates (minimum n=3) for each biological sample

  • Implement inter-run calibrators if multiple plates are required

• Data Analysis Workflow:

This comprehensive qRT-PCR methodology ensures robust validation of Atu1380 expression patterns identified in transcriptomic studies, providing a foundation for further functional characterization of this putative zinc metalloprotease in Agrobacterium tumefaciens .