Recombinant Acidovorax citrulli UPF0391 membrane protein Aave_0978 (Aave_0978)

What is the basic structure of the Aave_0978 membrane protein?

Aave_0978 is a UPF0391 family membrane protein from Acidovorax citrulli with a full length of 61 amino acids. It is classified as a membrane protein, suggesting it contains hydrophobic domains that anchor it within the bacterial membrane structure. As a UPF0391 family protein, it belongs to a group of proteins with conserved domains but not fully characterized functions. The protein is small compared to many bacterial membrane proteins, which may indicate a specialized role rather than complex enzymatic functions .

What expression systems are suitable for producing recombinant Aave_0978?

Recombinant Aave_0978 can be successfully expressed in E. coli expression systems with a His-tag for purification purposes. The relatively small size (61 amino acids) of Aave_0978 makes it amenable to bacterial expression, though care must be taken when expressing membrane proteins as they can sometimes form inclusion bodies or exhibit toxicity to the host. For optimal expression, researchers should consider using specialized E. coli strains designed for membrane protein production, controlled induction conditions, and lower incubation temperatures to promote proper folding .

What is the taxonomic context of Acidovorax citrulli?

Acidovorax citrulli (Schaad et al. 1978) Schaad et al. 2009 has undergone several taxonomic reclassifications. It was formerly known as Pseudomonas pseudoalcaligenes subsp. citrulli, then Pseudomonas avenae subsp. citrulli, and later Acidovorax avenae subsp. citrulli before receiving its current classification. This gram-negative bacterium is a significant plant pathogen that causes bacterial fruit blotch in cucurbit plants. Understanding this taxonomic context is important when searching literature about Aave_0978, as older studies may reference the protein under different bacterial species names .

What are the optimal purification strategies for His-tagged Aave_0978?

For His-tagged Aave_0978, immobilized metal affinity chromatography (IMAC) using Ni-NTA resin is the primary purification method. The protocol should be optimized with the following considerations: 1) Use of detergents suitable for membrane proteins (e.g., n-dodecyl-β-D-maltoside or CHAPS) during cell lysis and purification; 2) Addition of glycerol (10-15%) to maintain protein stability; 3) Inclusion of low concentrations of imidazole in washing buffers to reduce non-specific binding; 4) Elution with an imidazole gradient (100-500 mM) to obtain pure protein fractions. Size exclusion chromatography can be used as a secondary purification step to achieve higher purity and to analyze the oligomeric state of the protein .

How can researchers effectively verify the structural integrity of purified Aave_0978?

To verify structural integrity of purified Aave_0978, researchers should employ multiple complementary techniques: 1) SDS-PAGE to confirm molecular weight and purity; 2) Western blot using anti-His antibodies to verify tag presence; 3) Circular dichroism (CD) spectroscopy to assess secondary structure elements typical of membrane proteins; 4) Dynamic light scattering to evaluate homogeneity and detect aggregation; 5) Limited proteolysis to examine folding quality (well-folded proteins typically show resistance to proteolytic digestion at specific sites). For membrane proteins like Aave_0978, native PAGE with appropriate detergents can also provide information about the oligomeric state under non-denaturing conditions .

What approaches can be used to study Aave_0978 protein-protein interactions in Acidovorax citrulli?

Several complementary approaches can be employed to study Aave_0978 protein-protein interactions: 1) Co-immunoprecipitation using anti-His antibodies followed by mass spectrometry to identify interacting partners; 2) Bacterial two-hybrid systems adapted for membrane proteins; 3) Cross-linking studies using membrane-permeable cross-linkers followed by identification of complexes; 4) Pull-down assays using His-tagged Aave_0978 as bait; 5) Fluorescence resonance energy transfer (FRET) with fluorescently-labeled protein partners in reconstituted membrane systems. When designing these experiments, it's crucial to maintain the native membrane environment or use appropriate detergents to preserve protein structure and interaction capabilities .

What methods are recommended for studying the membrane topology of Aave_0978?

To elucidate the membrane topology of Aave_0978, researchers should employ: 1) Computational prediction using multiple algorithms (TMHMM, MEMSAT, Phobius) to generate initial topology models; 2) PhoA/LacZ fusion analysis, creating fusion proteins at different positions to determine cytoplasmic versus periplasmic localization; 3) Cysteine scanning mutagenesis coupled with accessibility labeling; 4) Protease protection assays using proteases that cannot cross the membrane; 5) Fluorescence spectroscopy with environment-sensitive probes; 6) Cryo-electron microscopy or X-ray crystallography for high-resolution structural determination. The small size of Aave_0978 (61 amino acids) suggests it likely has 1-2 transmembrane domains, making it potentially amenable to these analytical approaches .

What experimental designs would best assess the functional complementation of Aave_0978 mutants?

To assess functional complementation of Aave_0978 mutants, researchers should: 1) Generate clean deletion mutants using allelic exchange techniques; 2) Characterize the mutant phenotype under various stress conditions (oxidative, osmotic, pH, temperature); 3) Create complementation constructs with wild-type Aave_0978 under native or inducible promoters; 4) Transform complementation constructs into mutant strains and verify expression; 5) Compare growth, membrane integrity, stress response, and virulence between wild-type, mutant, and complemented strains; 6) Include domain-specific point mutations in complementation constructs to identify critical functional residues. For virulence assessment, watermelon seedlings can be used as a model system to evaluate the ability of complemented strains to restore pathogenicity .

How does Aave_0978 compare to other UPF0391 family proteins in related bacterial species?

Comparative analysis of Aave_0978 with other UPF0391 family proteins should include: 1) Multiple sequence alignment to identify conserved residues using CLUSTAL, MUSCLE, or T-Coffee algorithms; 2) Phylogenetic analysis to determine evolutionary relationships; 3) Structural homology modeling using solved structures of related proteins as templates; 4) Conservation analysis of genomic context to identify potentially functionally related genes; 5) Comparison of predicted membrane topologies. Given that UPF0391 is an uncharacterized protein family, comparative analysis may reveal conserved features that suggest functional roles. Researchers should pay particular attention to differences between UPF0391 proteins from pathogenic versus non-pathogenic bacteria, which might indicate adaptation to host-microbe interactions .

What is the relationship between Aave_0978 and bacterial phage susceptibility in Acidovorax citrulli?

The relationship between Aave_0978 and bacteriophage susceptibility represents an intriguing research direction. Membrane proteins often serve as receptors or co-receptors for bacteriophages. To investigate this relationship, researchers should: 1) Compare Aave_0978 expression levels between phage-resistant and phage-susceptible A. citrulli strains; 2) Generate Aave_0978 knockout mutants and test for altered phage susceptibility; 3) Perform adsorption assays comparing phage binding to wild-type and mutant strains; 4) Conduct direct binding studies between purified Aave_0978 and bacteriophage components; 5) Analyze whether bacteriophages like ACF1, ACF8, and ACF12, which have been isolated against A. citrulli, interact with Aave_0978. This research could reveal whether Aave_0978 plays a role in the phage infection process, which has implications for both bacterial evolution and potential biocontrol strategies .

How can Aave_0978 be utilized in developing targeted antimicrobial strategies against Acidovorax citrulli?

Developing targeted antimicrobials based on Aave_0978 requires: 1) Detailed structural characterization to identify unique features that differ from host proteins; 2) High-throughput screening assays to identify small molecules that bind specifically to Aave_0978; 3) Structure-activity relationship studies to optimize lead compounds; 4) Development of peptide inhibitors that disrupt essential Aave_0978 interactions; 5) In vitro and in planta evaluation of candidate molecules for efficacy against A. citrulli without phytotoxicity. If Aave_0978 proves essential for bacterial viability or virulence, compounds that specifically target this protein could offer narrow-spectrum control options with less environmental impact than conventional bactericides. Researchers should assess whether targeting this protein affects phage susceptibility, potentially allowing for combined biocontrol approaches .

What methodologies are recommended for studying the dynamics of Aave_0978 expression during Acidovorax citrulli infection cycles?

To study Aave_0978 expression dynamics during infection, researchers should employ: 1) Quantitative RT-PCR with samples collected at different infection stages; 2) Reporter gene fusions (e.g., Aave_0978 promoter driving GFP or luciferase) to monitor expression in real-time; 3) RNA-seq analysis comparing transcription levels under various infection-relevant conditions; 4) Proteomics approaches to quantify protein levels in bacteria recovered from infected plant tissues; 5) Chromatin immunoprecipitation sequencing (ChIP-seq) to identify transcription factors regulating Aave_0978 expression; 6) Single-cell expression analysis to assess population heterogeneity during infection. Researchers should correlate expression patterns with different stages of the infection process (attachment, colonization, symptom development) to understand the temporal importance of Aave_0978 .

How can researchers effectively design experiments to resolve contradictory data regarding Aave_0978 function?

To resolve contradictory data regarding Aave_0978 function, researchers should: 1) Standardize experimental conditions across laboratories, including strain backgrounds, growth conditions, and assay parameters; 2) Employ multiple complementary methodologies to test the same hypothesis; 3) Use genetic approaches including clean deletion mutants, complementation strains, and point mutants to verify phenotypes; 4) Conduct collaborative blind studies where samples are analyzed independently by different laboratories; 5) Utilize systems biology approaches to place Aave_0978 in the context of broader cellular networks; 6) Develop in vitro reconstitution systems to test biochemical functions under defined conditions. When reporting results, researchers should explicitly describe all experimental variables and potential confounding factors to facilitate reproduction and resolution of contradictory findings .

What are the optimal conditions for studying protein-lipid interactions involving Aave_0978?

For studying protein-lipid interactions of Aave_0978, researchers should consider: 1) Liposome binding assays using fluorescently-labeled protein and liposomes of varying composition; 2) Monolayer penetration experiments to measure protein insertion into lipid films; 3) Microscale thermophoresis to quantify binding affinities to specific lipids; 4) Hydrogen-deuterium exchange mass spectrometry to identify lipid-interacting regions; 5) Molecular dynamics simulations to predict lipid binding sites and membrane positioning. The experimental conditions should mimic the bacterial membrane composition of Acidovorax citrulli, particularly regarding phospholipid species, charge, and curvature. Temperature, pH, and ionic strength should be carefully controlled to match physiological conditions relevant to plant infection scenarios .

What adsorption rates and experimental parameters should be considered when studying phage interactions with Acidovorax citrulli expressing Aave_0978?

When studying potential phage interactions with A. citrulli expressing Aave_0978, researchers should consider the following parameters based on established phage studies: 1) Adsorption rate measurements at different temperatures (optimally 27°C based on ACF1 phage studies); 2) Measurement of adsorption over time (typical testing points at 1, 5, 10, 15, and 20 minutes); 3) Determination of optimal multiplicity of infection (MOI), with 0.002 being effective for ACF1 phage; 4) Assessment of pH stability (typically pH 5-9 for A. citrulli phages); 5) Evaluation of thermal inactivation points (around 66-67°C for known A. citrulli phages). The one-step growth curve parameters observed for A. citrulli phages include a latent period of approximately 30 minutes, a rise period of 60 minutes, and a burst size of 74 ± 5 plaque forming units per infected cell .

What are the challenges of integrating structural biology approaches in the study of Aave_0978?

Integrating structural biology approaches for Aave_0978 presents several challenges: 1) As a small membrane protein (61 amino acids), crystallization may be difficult without fusion partners or antibody fragments to increase size; 2) Detergent selection is critical, as inappropriate detergents can destabilize the native structure; 3) The protein may exist in multiple conformational states, complicating structural determination; 4) Sample heterogeneity due to post-translational modifications or flexibility may hinder crystallization; 5) NMR studies require isotopic labeling and optimization of membrane mimetics; 6) Cryo-EM typically struggles with proteins <50 kDa, though recent technological advances may overcome this limitation. Researchers should consider employing integrative structural biology, combining lower-resolution techniques (SAXS, CD spectroscopy) with computational modeling and targeted biochemical experiments like disulfide crosslinking .

How might systems biology approaches enhance our understanding of Aave_0978's role in bacterial physiology?

Systems biology approaches to understand Aave_0978's role should include: 1) Network analysis integrating transcriptomics, proteomics, and metabolomics data to position Aave_0978 within functional pathways; 2) Construction of genome-scale metabolic models with and without Aave_0978 to predict physiological impacts; 3) Protein-protein interaction network mapping using techniques like BioID or APEX proximity labeling optimized for membrane proteins; 4) Correlation analysis between Aave_0978 expression and phenotypic outcomes across multiple conditions; 5) Comparative systems analysis between A. citrulli strains with varying virulence levels. These approaches can reveal connections between Aave_0978 and broader cellular processes that might not be apparent through reductionist approaches, potentially identifying unexpected functions or regulatory roles .

What role might Aave_0978 play in bacterial responses to environmental stresses relevant to plant infection?

To investigate Aave_0978's role in stress responses, researchers should: 1) Analyze expression changes of Aave_0978 under various stresses (oxidative, osmotic, pH, temperature, plant defense compounds); 2) Compare stress sensitivity profiles between wild-type and Aave_0978 mutant strains; 3) Assess membrane integrity and permeability under stress conditions; 4) Investigate potential roles in maintaining proton motive force during stress; 5) Examine localization changes of Aave_0978 in response to stress using fluorescent protein fusions; 6) Analyze potential post-translational modifications of Aave_0978 during stress responses. As membrane proteins often function as stress sensors or in maintaining membrane homeostasis, Aave_0978 may be particularly important during transitions between environmental and host conditions experienced during the infection cycle .

How can synthetic biology approaches be applied to understand and manipulate Aave_0978 function?

Synthetic biology approaches for Aave_0978 research include: 1) Design of minimal synthetic versions to identify essential functional domains; 2) Creation of chimeric proteins with domains from related UPF0391 family members to map function to structure; 3) Engineering of controllable expression systems in A. citrulli to titrate protein levels; 4) Development of biosensors using Aave_0978 as a detection element if ligand interactions are identified; 5) Incorporation of non-natural amino acids at specific positions to probe function and create photo-crosslinkable versions; 6) Design of orthogonal membrane protein systems to test Aave_0978 function in non-native contexts. These approaches can provide unique insights into protein function that are difficult to obtain through conventional genetic methods, potentially revealing novel applications in synthetic biology and biotechnology .