Recombinant Azorhizobium caulinodans UPF0060 membrane protein AZC_0909 (AZC_0909)

What is AZC_0909 and which protein family does it belong to?

AZC_0909 is a UPF0060 family protein, which belongs to a group of conserved bacterial membrane proteins with unknown precise functions. It is found in Azorhizobium caulinodans, a nitrogen-fixing bacterium that forms symbiotic relationships with legume plants, particularly Sesbania rostrata. The UPF0060 designation (Uncharacterized Protein Family 0060) indicates that while the protein has been identified through genomic sequencing, its exact biological role remains to be fully elucidated.

For initial characterization, comparative genomics approaches combined with structural analysis are recommended. Sequence alignment of UPF0060 family members across bacterial species can identify conserved domains, while structural prediction using tools like AlphaFold can provide insights into potential functional motifs. The membrane localization of AZC_0909 suggests it may participate in structural or signaling processes important for bacterial physiology or host interaction.

What is known about the host organism Azorhizobium caulinodans?

Azorhizobium caulinodans is an alphaproteobacterium that fixes nitrogen and forms stem/root nodules with the legume Sesbania rostrata. This symbiotic relationship is particularly significant in lowland rice cultivation, allowing nitrogen fixation under flood conditions. The sequenced genome of A. caulinodans reveals a complex chemotactic system with 43 chemoreceptors, which plays a crucial role in bacterial colonization of the rhizosphere .

The bacterium's chemotaxis system includes CheA, CheW, CheY2, CheB, and CheR proteins organized in a che cluster, while CheY1 and CheZ are located independently from this operon . A. caulinodans has a unique chemotaxis control mechanism, with CheY1 serving as the predominant signaling protein for chemotaxis and CheA containing an unusual set of C-terminal domains that influence both chemotaxis and CheA function .

Why is AZC_0909 of interest to researchers?

Researchers are investigating AZC_0909 due to its potential involvement in the symbiotic nitrogen fixation process. While its precise function remains unknown, several key unanswered questions drive research interest:

• Mechanistic Role: Does AZC_0909 participate in nutrient transport, signal transduction, or membrane integrity maintenance?

• Protein Interactions: What are the binding partners of AZC_0909? Potential interactions with chemoreceptors like TlpA1 or flagellar components could reveal functional pathways.

• Symbiotic Relevance: Does AZC_0909 play a role in nodulation or nitrogen fixation processes? Functional knockout studies are needed to assess its importance in symbiosis.

The membrane localization of AZC_0909 suggests it may influence structural or signaling processes important for bacterial adaptation to environmental conditions during plant colonization.

What expression systems are optimal for recombinant production of AZC_0909?

The recommended expression system for AZC_0909 is Escherichia coli BL21(DE3) or similar strains designed for high-level protein expression. This bacterial expression platform offers several advantages for membrane protein production:

• High expression levels under IPTG-inducible promoters

• Reduced protease activity for improved protein stability

• Compatibility with various fusion tags (particularly His-tag) for purification

• Well-established protocols for membrane protein solubilization

The recombinant production typically involves cloning the AZC_0909 gene into a vector containing an inducible promoter and appropriate affinity tag. For optimal expression of this membrane protein, induction at lower temperatures (16-20°C) is recommended to improve proper folding.

What purification strategies ensure high purity of recombinant AZC_0909?

Purification of AZC_0909 relies on affinity chromatography utilizing the His-tag, followed by quality control validation through SDS-PAGE for purity assessment and Western blot for His-tag confirmation. The purification workflow includes:

• Cell lysis and membrane fraction isolation

• Membrane protein solubilization using appropriate detergents

• Affinity chromatography on Ni-NTA or similar matrix

• Optional size exclusion chromatography for increased purity

• Quality control analyses

For membrane proteins like AZC_0909, detergent selection is critical for maintaining protein structure and function throughout the purification process. The protein should be maintained in detergent-containing buffers throughout all chromatography steps to prevent aggregation.

What reconstitution methods ensure optimal stability and activity of purified AZC_0909?

For optimal stability, purified AZC_0909 should be reconstituted in deionized water at concentrations of 0.1–1.0 mg/mL with 5–50% glycerol as a stabilizing agent. The glycerol percentage should be optimized based on downstream applications and storage requirements.

The protein's stability during storage is enhanced by:

• Maintaining appropriate detergent concentrations above critical micelle concentration

• Including glycerol to prevent freeze-thaw damage

• Storing in single-use aliquots to avoid repeated freeze-thaw cycles

• Considering flash-freezing in liquid nitrogen for long-term storage

For functional studies, reconstitution into liposomes composed of bacterial lipids may be necessary to recreate the native membrane environment and enable activity assessments.

What structural features define UPF0060 family proteins like AZC_0909?

The UPF0060 family, to which AZC_0909 belongs, comprises conserved bacterial membrane proteins with several characteristic structural features that can be investigated through computational and experimental approaches:

• Membrane topology analysis using prediction algorithms suggests multiple transmembrane domains

• Conserved amino acid motifs likely contribute to structural integrity or functional interactions

• Potential for oligomerization or higher-order complex formation

• Specific lipid interactions that may influence protein conformation or function

While detailed structural information specific to AZC_0909 is limited, researchers can employ techniques such as circular dichroism spectroscopy to assess secondary structure content, fluorescence spectroscopy to examine conformational changes, and potentially X-ray crystallography or cryo-electron microscopy for high-resolution structural determination.

How does AZC_0909 compare with other characterized proteins in Azorhizobium caulinodans?

Comparative analysis reveals both similarities and differences between AZC_0909 and better-characterized proteins in A. caulinodans:

While homologs like ActR regulate processes critical for host plant symbiosis, AZC_0909's precise role in symbiotic interactions requires further investigation through targeted functional studies.

What are the hypothesized functions of AZC_0909 based on current evidence?

Based on its membrane localization and comparative analysis with other bacterial membrane proteins, several potential functions have been proposed for AZC_0909:

• Nutrient transport: AZC_0909 may facilitate the movement of specific molecules across the bacterial membrane, potentially including plant-derived signals or nutrients essential during symbiosis.

• Signal transduction: The protein might participate in sensing environmental cues or host-derived signals, contributing to adaptation during colonization and nodulation processes.

• Membrane integrity: AZC_0909 could play a structural role in maintaining membrane properties necessary for bacterial survival during symbiotic interactions.

• Stress adaptation: The protein may contribute to bacterial resilience against environmental stresses encountered during plant colonization, similar to how other A. caulinodans proteins like Ohr and OhrR facilitate oxidative stress resistance and nodulation.

Experimental approaches to test these hypotheses include gene knockout studies, controlled expression analyses, transport assays with reconstituted protein, and in planta studies comparing wild-type and mutant strains.

How might AZC_0909 interact with the chemotaxis system in Azorhizobium caulinodans?

A. caulinodans possesses a sophisticated chemotactic system essential for rhizosphere colonization, raising questions about potential interactions between AZC_0909 and chemotaxis components. While no direct link has been established, several interaction mechanisms can be hypothesized:

• Physical association with chemoreceptor complexes or signaling proteins

• Influence on membrane microdomain organization affecting chemoreceptor clustering

• Transport of compounds that modulate chemotactic responses

• Participation in adaptation processes during chemotaxis

The A. caulinodans chemotaxis system includes an unusual arrangement where CheY1 functions as the predominant signaling protein, while CheA contains an atypical set of C-terminal domains (W2-Rec) that impact both chemotaxis and CheA function . Understanding whether AZC_0909 influences this system requires specialized interaction studies using techniques like bacterial two-hybrid screening, co-immunoprecipitation, or FRET analysis with fluorescently tagged proteins.

What experimental designs can assess AZC_0909's role in symbiotic nitrogen fixation?

Investigating AZC_0909's potential contribution to symbiotic nitrogen fixation requires a multi-faceted experimental approach:

• Genetic manipulation:

  • Creation of clean deletion mutants lacking AZC_0909

  • Development of complemented strains expressing wild-type or modified versions

  • Generation of reporter fusions to monitor expression patterns during symbiosis

• Symbiotic performance analysis:

  • Quantification of nodulation efficiency on Sesbania rostrata

  • Measurement of nitrogen fixation rates using acetylene reduction assays

  • Assessment of competitive ability against wild-type for nodule occupancy

  • Microscopic examination of bacteroid differentiation within nodules

• Molecular interactions:

  • Identification of proteins that co-localize or directly interact with AZC_0909

  • Transcriptomic analysis comparing wild-type and mutant strains during symbiosis

  • Examination of protein expression and modification under symbiotic conditions

The symbiotic relationship between A. caulinodans and S. rostrata is particularly significant in rice cultivation under flood conditions, making functional characterization of proteins like AZC_0909 valuable for agricultural applications .

What methodological approaches can identify AZC_0909 binding partners?

Identifying protein-protein interactions is crucial for understanding AZC_0909's function. Several complementary approaches can be employed:

• Affinity-based methods:

  • Pull-down assays using His-tagged AZC_0909 as bait

  • Co-immunoprecipitation with specific antibodies against AZC_0909

  • Chemical crosslinking to capture transient interactions

• Proximity-based labeling:

  • APEX2 or BioID fusions for proximity-dependent biotinylation

  • Photo-crosslinking with unnatural amino acids incorporated at specific positions

  • Fluorescence-based interaction detection in living cells

• Functional screening:

  • Bacterial two-hybrid or split-protein complementation assays

  • Suppressor mutation analysis to identify genetic interactions

  • Synthetic lethality screening with other membrane protein mutants

The membrane localization of AZC_0909 presents technical challenges for interaction studies, as membrane proteins often form complexes that are difficult to maintain during solubilization. Specialized approaches using membrane-compatible detergents or lipid nanodiscs may be necessary to preserve physiologically relevant interactions.

What controls are essential when studying AZC_0909 through mutagenesis?

When designing mutagenesis experiments for AZC_0909 functional studies, implementing appropriate controls is critical:

• Genetic controls:

  • Clean deletion mutant with precise removal of AZC_0909

  • Complementation with wild-type AZC_0909 expressed from native or controlled promoter

  • Site-directed mutagenesis targeting conserved residues to distinguish functional domains

  • Empty vector controls for plasmid-based complementation

• Phenotypic verification:

  • Confirmation of AZC_0909 absence at both transcript and protein levels

  • Demonstration of phenotype reproducibility across multiple independent mutants

  • Testing under various growth conditions to identify condition-specific effects

  • Comparison with mutants of unrelated membrane proteins to distinguish specific from general membrane disruption effects

• Biochemical validation:

  • Verification of protein expression levels in complemented strains

  • Assessment of membrane integrity and composition in mutants

  • Examination of potential polar effects on adjacent genes

These controls help distinguish direct effects of AZC_0909 deletion from indirect consequences of membrane disruption or secondary mutations.

How should experiments be designed to investigate AZC_0909's potential role in membrane integrity?

If investigating AZC_0909's contribution to membrane structure or function, consider the following experimental design elements:

• Membrane permeability assessments:

  • Fluorescent dye uptake assays (e.g., propidium iodide)

  • Sensitivity to membrane-targeting antibiotics

  • Resistance to environmental stresses that challenge membrane integrity

  • Osmotic shock tolerance measurements

• Membrane composition analysis:

  • Lipidomic profiling comparing wild-type and mutant membranes

  • Membrane fluidity measurements using fluorescence anisotropy

  • Phase transition temperature determination using differential scanning calorimetry

  • Electron microscopy to visualize membrane ultrastructure

• Biophysical characterization:

  • Membrane potential measurements in whole cells

  • Protein mobility within membranes using FRAP (Fluorescence Recovery After Photobleaching)

  • Microdomain organization using specialized fluorescent lipid probes

When interpreting results, remember that membrane properties may differ between free-living bacteria and bacteroids within nodules, necessitating examination under both conditions to fully understand AZC_0909's role in symbiosis.

What approaches can address the technical challenges of studying membrane protein function?

Membrane proteins like AZC_0909 present unique technical challenges that require specialized approaches:

• Protein solubilization strategies:

  • Optimization of detergent type and concentration

  • Use of amphipols or nanodiscs for detergent-free handling

  • Reconstitution into liposomes of defined composition

  • Extraction using styrene-maleic acid copolymers to maintain native lipid environment

• Functional assay development:

  • Proteoliposome-based transport assays if AZC_0909 functions as a transporter

  • Binding studies with potential ligands using surface plasmon resonance

  • Electrophysiological measurements if channel activity is suspected

  • Monitoring conformational changes using engineered reporter groups

• Structural analysis approaches:

  • Lipidic cubic phase crystallization for X-ray diffraction studies

  • Single-particle cryo-electron microscopy of protein-nanodisc complexes

  • Solid-state NMR for studying membrane-embedded proteins

  • Hydrogen-deuterium exchange mass spectrometry for identifying flexible regions

For recombinant production, expressing AZC_0909 in E. coli BL21(DE3) with appropriate solubilization and purification protocols has been established as an effective approach. Quality control using SDS-PAGE and Western blot confirmation of the His-tag ensures proper protein production before functional studies.

How can researchers distinguish direct from indirect effects in AZC_0909 mutant phenotypes?

A significant challenge when studying membrane proteins like AZC_0909 is determining whether observed phenotypes result directly from protein function or indirectly from membrane disruption. Several strategies can help address this challenge:

• Complementation analysis:

  • Test whether reintroduction of wild-type AZC_0909 restores normal phenotypes

  • Create point mutations in specific domains to identify critical functional regions

  • Use controlled expression systems to establish dose-dependency of phenotypes

• Comparative mutant analysis:

  • Compare phenotypic signatures of AZC_0909 mutants with those of other membrane protein mutants

  • Identify unique phenotypes specific to AZC_0909 disruption

  • Construct combination mutants to test for genetic interactions

• Temporal control strategies:

  • Use inducible or repressible systems to control AZC_0909 expression timing

  • Monitor phenotype development after expression changes

  • Determine reversibility of phenotypes upon restored expression

These approaches can help build a stronger case for direct functional roles versus indirect effects resulting from general membrane disruption.

What statistical approaches are recommended for analyzing complex phenotypic data?

When analyzing complex phenotypic data from AZC_0909 studies, robust statistical approaches are essential:

• For multi-parameter phenotypic analysis:

  • Principal Component Analysis (PCA) to identify major sources of variation

  • Hierarchical clustering to group related phenotypes

  • MANOVA for simultaneously comparing multiple dependent variables

  • Post-hoc tests with appropriate corrections for multiple comparisons

• For time-course experiments:

  • Repeated measures ANOVA for longitudinal data

  • Growth curve parametrization and parameter comparison

  • Area under the curve (AUC) calculations for cumulative effect measurement

  • Mixed effects models for data with multiple random factors

• For symbiotic performance assessment:

  • Non-parametric tests for count data (e.g., nodule numbers)

  • Competitive index calculations for mixed inoculation experiments

  • Appropriate transformations for data that violate normality assumptions

Statistical power analysis should guide experimental design, ensuring sufficient replication to detect biologically meaningful effects while controlling both Type I and Type II error rates.

How can researchers integrate multiple data types to develop a comprehensive model of AZC_0909 function?

Building a complete understanding of AZC_0909 function requires integration of diverse data types:

• Data integration strategies:

  • Pathway enrichment analysis across multiple datasets

  • Network analysis to identify functional modules and interaction partners

  • Correlation analysis between transcriptomic, proteomic, and phenotypic changes

  • Predictive modeling to generate testable hypotheses

• Visualization approaches:

  • Integrated pathway maps highlighting changes at multiple levels

  • Heat maps with hierarchical clustering of multi-omics data

  • Network diagrams showing protein-protein interactions with overlaid expression data

  • Custom visualizations specific to membrane protein localization and dynamics

• Model validation strategies:

  • Design targeted experiments to test specific model predictions

  • Compare findings with related proteins in other bacterial species

  • Iteratively refine models as new data becomes available

By combining structural data, functional characterization, interaction studies, and phenotypic analysis, researchers can develop increasingly refined models of how AZC_0909 contributes to A. caulinodans biology and symbiotic nitrogen fixation .