Recombinant Pseudomonas syringae pv. tomato Glucans biosynthesis protein G (opgG), partial

What is OpgG and what is its primary function in Pseudomonas syringae?

OpgG (also known as mdoG) is a glucans biosynthesis protein essential for the production of osmoregulated periplasmic glucans (OPGs) in Pseudomonas syringae pv. tomato. In Proteobacteria like P. syringae, OPGs are involved in osmoprotection, biofilm formation, virulence, and resistance to antibiotics. The OpgG protein specifically functions in the biosynthesis pathway of these complex glucan structures, which are produced in response to low osmolarity environments .

How does OpgG differ between Pseudomonas species and other bacteria like E. coli?

While OpgG proteins share functional similarities across bacterial species, structural and sequence variations exist. In E. coli, OpgG has been characterized as a protein composed of two beta-sandwich domains: a large N-terminal domain (residues 22-388) with a 25-stranded beta-sandwich fold found in many carbohydrate-related proteins, and a smaller C-terminal domain (residues 401-512) with a seven-stranded immunoglobulin-like beta-sandwich fold. The E. coli OpgG is suggested to function as an OPG branching enzyme. In Pseudomonas syringae, similar functions have been demonstrated, though some species-specific differences in regulation and exact enzymatic activity may exist .

What is the relationship between OpgG and bacterial pathogenicity?

OpgG plays a significant role in bacterial pathogenicity across multiple species. In various Gram-negative pathogens, including P. syringae, the knockout of opgG results in loss of pathogenicity. This is because OPGs function as essential signaling molecules in host-pathogen interactions. Studies have shown that OPGs are involved in transmitting external signals and may contribute to virulence by affecting the bacteria's ability to adapt to environmental conditions within the host. Additionally, OPGs have been implicated in biofilm formation, which further contributes to bacterial resistance and persistence during infection .

What is the domain structure of OpgG and how does it relate to its function?

OpgG protein contains two distinct domains with different structural characteristics. Based on studies of E. coli OpgG (which shares functional similarity with P. syringae OpgG):

• The N-terminal domain (residues 22-388) consists of a 25-stranded beta-sandwich fold common in carbohydrate-processing enzymes. This domain contains a large cleft with numerous aromatic and acidic residues, suggesting its role in substrate binding and catalytic activity.

• The C-terminal domain (residues 401-512) has a seven-stranded immunoglobulin-like beta-sandwich fold, commonly involved in molecular interactions.

These domains are connected by a 3₁₀ helix. The putative catalytic activity is believed to reside in the N-terminal domain, while the C-terminal domain may regulate this activity through interactions with other molecules .

What enzymatic activities have been attributed to OpgG in recent research?

Recent structural and functional analyses have revealed that OpgG and its paralog OpgD are β-1,2-glucanases with markedly different activities. Together, they establish a new glycoside hydrolase family, GH186. The reaction mechanism for OpgD involves an unusually long proton transfer pathway compared to other glycoside hydrolase families. The structural differences in the regions forming this reaction pathway between OpgG and OpgD explain the observed lower activity of OpgG. These findings have significantly enhanced our understanding of OPG biosynthesis and provide insights into the functional diversity within this novel enzyme family .

What are the recommended methods for purifying recombinant OpgG protein?

For the purification of recombinant His₆-tagged OpgG from bacterial expression systems, immobilized metal-ion affinity chromatography (IMAC) is the recommended approach. Research has shown that optimizing the purification conditions can significantly improve both yield and purity:

• Using EDTA-Mg²⁺ treatment in combination with periplasmic extraction enhances the specificity of polyhistidine tag binding to Ni-IDA (iminodiacetic acid) chromatography matrices.

• This approach eliminates the need for buffer exchange after periplasmic extraction, which is typically required to remove EDTA before IMAC purification.

• The presence of low concentrations of EDTA-Mg²⁺ in feed streams weakens non-specific adsorption while making the binding more specific toward the polyhistidine tag.

This methodology significantly improves the purity of the recombinant protein by eliminating undesired adsorption of host cell proteins. The final purified product should achieve ≥85% purity as determined by SDS-PAGE analysis .

What expression systems are most effective for producing functional recombinant OpgG?

Multiple expression systems have been successfully employed for producing recombinant OpgG protein, each with distinct advantages:

• E. coli expression systems: Most commonly used due to rapid growth, high yield, and established protocols. For OpgG specifically, E. coli has been shown to effectively produce the recombinant protein with proper folding when expressed with a His₆-tag for purification.

• Yeast expression systems: Beneficial when post-translational modifications are required, though not typically necessary for bacterial proteins like OpgG.

• Baculovirus expression systems: Useful for producing larger quantities of properly folded proteins when E. coli systems prove challenging.

• Mammalian cell expression systems: Generally reserved for proteins requiring complex folding or extensive post-translational modifications.

For most research applications involving recombinant OpgG, the E. coli expression system provides the best balance of yield, cost-effectiveness, and functional protein production .

How can I design an experimental approach to study OpgG gene function in Pseudomonas syringae?

To study OpgG function in P. syringae, a comprehensive experimental design should include:

• Gene deletion strategies:

  • Create opgG knockout mutants using homologous recombination techniques

  • Utilize suicide plasmids (like pDS132-ΔopgGH) for gene deletion

  • Verify deletions by PCR and sequencing

• Complementation studies:

  • Reintroduce the opgG gene under an inducible promoter (like araBAD)

  • Create plasmid constructs (e.g., pBAD24-opgG) for controlled expression

  • Transform into mutant strains to confirm phenotype restoration

• Phenotypic characterization:

  • Assess bacterial growth under various osmotic conditions

  • Evaluate biofilm formation capabilities

  • Measure virulence in appropriate plant host models

  • Analyze OPG production using biochemical assays

• Signal transduction analysis:

  • Investigate interactions with regulatory systems (e.g., Rcs pathway)

  • Create double mutants (opgG plus regulatory genes) to study pathway interactions

This experimental approach allows for comprehensive functional characterization of OpgG while adhering to established one-group experimental designs for proper scientific rigor .

How can recombinant OpgG be used to study bacterial adaptation to osmotic stress?

Recombinant OpgG provides a valuable tool for investigating bacterial osmoadaptation mechanisms:

• In vitro enzymatic assays: Purified recombinant OpgG can be used to study the kinetics and substrate specificity of glucan synthesis under varying osmotic conditions, providing insights into how the protein's activity is regulated by environmental osmolarity.

• Structure-function analysis: Site-directed mutagenesis of recombinant OpgG can help identify key residues involved in sensing osmotic changes or catalyzing glucan synthesis, particularly focusing on the N-terminal domain that contains the putative catalytic site.

• Reconstitution experiments: Adding purified recombinant OpgG to membrane preparations or liposome systems can help determine how the protein interacts with membrane components during osmotic adaptation.

• Protein-protein interaction studies: Using recombinant OpgG as bait in pull-down assays or yeast two-hybrid screens can identify interaction partners that may regulate its activity in response to osmotic changes.

• Comparative studies: Analyzing recombinant OpgG from different bacterial species that inhabit diverse osmotic environments (from 10 mOsM in freshwater to 1,000 mOsM in seawater) can reveal evolutionary adaptations in protein function .

What is the current understanding of OpgG's role in horizontal gene transfer and plasmid integration?

Research has revealed complex relationships between OpgG and genomic plasticity in Pseudomonas species. Studies have shown that:

• In some virulent strains of P. syringae pv. phaseolicola, cryptic plasmids (like the 98-megadalton pMC7105) can integrate into the chromosome after exposure to plasmid-curing agents such as mitomycin C.

• This integration process is followed by imprecise excision events that can result in the formation of smaller derivative plasmids, while parts of the original plasmid remain integrated into the chromosome.

• While direct evidence linking OpgG to these processes is still emerging, the periplasmic location of OPGs and their role in membrane organization suggests they may influence the cell's ability to acquire and maintain foreign DNA.

• The relationship between OpgG-dependent OPG synthesis and plasmid transfer/maintenance systems represents an important area for future research, particularly in understanding the evolution of virulence factors and antibiotic resistance genes in plant pathogens .

How does OpgG interact with plant host defense systems during infection?

OpgG-dependent OPG production influences plant-pathogen interactions through several mechanisms:

• PAMP recognition evasion: OPGs may mask pathogen-associated molecular patterns (PAMPs) that would otherwise trigger plant immune responses. By altering the bacterial cell surface properties, OpgG-produced glucans can help bacteria avoid recognition by plant pattern recognition receptors.

• Signal transduction interference: OPGs can intercept or modify plant defense signaling molecules, affecting the host's ability to mount effective immune responses. This interference may occur through molecular mimicry or by physically blocking signal transduction pathways.

• Biofilm matrix components: OpgG-produced glucans contribute to biofilm formation, creating a protective environment that shields bacteria from plant antimicrobial compounds and environmental stresses during colonization.

• Effector protein delivery: The proper functioning of type III secretion systems, which deliver bacterial effector proteins into plant cells, depends on membrane integrity and periplasmic conditions that are influenced by OPGs.

• Symbiotic relationship modulation: In some bacterial species, OPGs serve as symbiotic factors, suggesting that OpgG may play dual roles in pathogenicity and mutualistic interactions depending on the bacterial species and host context .

What are common challenges in producing soluble recombinant OpgG and how can they be addressed?

Researchers frequently encounter several challenges when attempting to produce soluble recombinant OpgG:

• Inclusion body formation: OpgG often forms insoluble aggregates when overexpressed in E. coli.

  • Solution: Optimize growth conditions by reducing expression temperature (16-20°C), using weaker promoters, or co-expressing molecular chaperones like GroEL/GroES.

• Improper folding: The complex beta-sandwich domains of OpgG can misfold.

  • Solution: Express the protein as fusion constructs with solubility enhancers like MBP (maltose-binding protein) or SUMO, which can be later cleaved with specific proteases.

• Low expression levels: Sometimes OpgG expression is poor despite optimized vectors.

  • Solution: Codon optimization for the expression host, use of specialized expression strains like BL21(DE3) pLysS, or testing different E. coli strains like Rosetta for rare codon supplementation.

• Protein degradation: OpgG may be subject to proteolytic degradation.

  • Solution: Include protease inhibitors during purification, use protease-deficient host strains, or optimize extraction conditions with different buffer compositions.

• Periplasmic targeting issues: As a periplasmic protein, proper targeting can be problematic.

  • Solution: Use periplasmic expression vectors with appropriate signal sequences (like pelB) and optimize periplasmic extraction procedures using EDTA-Mg²⁺ treatments as described in literature .

How can proteomics data be integrated with functional studies of OpgG to better understand its role in bacterial physiology?

Integrating proteomics with functional studies of OpgG provides a comprehensive understanding of its physiological role:

• Quantitative proteomics approaches:

  • Utilize techniques like LC-MS/MS to compare protein expression levels between wild-type and opgG mutant strains

  • Raw data analysis can identify proteins with significantly altered expression, as shown in the provided proteomics dataset where OpgG (Q87UY0) in P. syringae pv. tomato DC3000 showed distinct expression patterns compared to other proteins

• Protein-protein interaction networks:

  • Employ affinity purification-mass spectrometry (AP-MS) with tagged OpgG to identify interaction partners

  • Create interaction maps to visualize OpgG's position within bacterial cellular networks

• Post-translational modification analysis:

  • Identify potential regulatory modifications of OpgG using phosphoproteomics or glycoproteomics

  • Correlate modifications with changes in environmental conditions or osmotic stress

• Multi-omics data integration:

  • Combine proteomics data with transcriptomics and metabolomics to build comprehensive models of glucan metabolism

  • Use statistical approaches to correlate OpgG expression with specific metabolic pathways

• Evolutionary proteomics:

  • Compare OpgG protein sequences and expression patterns across bacterial species to understand evolutionary adaptations

  • Identify conserved regions that may be essential for function versus variable regions that might confer species-specific advantages .

What strategies can be employed to resolve contradictory findings about OpgG function across different Pseudomonas strains?

When faced with contradictory findings about OpgG function across different Pseudomonas strains, researchers should implement these systematic approaches:

• Standardized experimental protocols:

  • Develop and adopt consistent methodologies for OpgG purification, activity assays, and phenotypic characterization

  • Ensure that growth conditions, media compositions, and assay parameters are comparable across studies

• Comprehensive genetic context analysis:

  • Sequence the complete opgG locus and surrounding regions in each strain

  • Identify strain-specific genetic elements (promoters, regulatory regions, operon structure) that may affect OpgG expression or function

  • Consider the presence of paralogous genes like opgD that may compensate for OpgG function in some strains

• Cross-complementation studies:

  • Perform reciprocal gene replacement experiments where the opgG gene from one strain is expressed in the opgG mutant of another strain

  • Assess whether the phenotypic differences are due to the opgG gene itself or the genetic background

• Structural biology approaches:

  • Determine and compare the three-dimensional structures of OpgG proteins from different strains

  • Identify structural variations that might explain functional differences

• Environmental context considerations:

  • Evaluate whether contradictory findings might result from strain-specific adaptations to different ecological niches

  • Test OpgG function under a range of environmental conditions relevant to each strain's natural habitat

This methodical approach can help reconcile seemingly contradictory findings by distinguishing between true biological variation and experimental artifacts .