Recombinant Pseudomonas syringae pv. tomato Maf-like protein PSPTO_4469 (maf-2)

What is the Maf-like protein PSPTO_4469 (maf-2) and where is it located in the P. syringae pv. tomato genome?

The Maf-like protein PSPTO_4469 (maf-2) is a 200-amino acid protein encoded in the genome of Pseudomonas syringae pv. tomato DC3000. According to genomic analysis, the maf-2 gene is located at position 5033887..5034489 on the negative strand of the bacterial chromosome . Genomic context analysis reveals that maf-2 is positioned in proximity to several genes involved in cell shape determination, including mreC (PSPTO_4471) and mreD (PSPTO_4470) . This genomic arrangement is significant as it suggests potential functional relationships between these genes and maf-2.

How does Maf-2 contribute to P. syringae pv. tomato pathogenicity?

Maf-2 contributes to P. syringae pathogenicity through its role in flagellin glycosylation, which affects bacterial motility and host interactions. In P. syringae, Maf-2 functions as part of a tripartite system that modifies flagellin post-translationally. Unlike the bipartite Maf-1 system, Maf-2 requires an additional specificity factor, GlfM, to efficiently interact with flagellin . This glycosylation process influences bacterial swimming motility, biofilm formation, and virulence. During infection, the bacterium must penetrate plant tissue to access the apoplast, a process driven by chemotaxis toward plant-derived compounds . Altered flagellin glycosylation can impact this chemotactic response, thereby affecting bacterial entry and subsequent pathogenesis in tomato plants.

How is maf-2 gene expression regulated in P. syringae?

The regulation of maf-2 involves multiple transcriptional regulators, primarily PsrA (Pseudomonas sigma regulator). PsrA functions as a DNA-binding protein that positively regulates RpoS production and negatively regulates aefR and rsmA1 . In P. syringae pv. tomato DC3000, PsrA impacts the expression of hrpL, which controls the hypersensitive response and virulence .

A regulatory network has been identified where:

• PsrA controls RpoS and AefR expression

• RpoS strongly negatively regulates AHL production, which affects quorum sensing

• AefR positively regulates AHL production

This complex regulation mechanism affects bacterial behaviors relevant to pathogenicity, including motility and biofilm formation, which are linked to Maf-2's function in flagellin glycosylation. Northern blot analysis and β-galactosidase assays have demonstrated that PsrA mutation can significantly alter the expression of these regulatory components .

What is the structural and functional relationship between Maf-2 and the flagellin glycosylation machinery?

Maf-2 functions within a tripartite glycosylation system that modifies bacterial flagellin with pseudaminic acid (Pse) derivatives. Unlike the bipartite Maf-1 system, Maf-2 requires a specificity factor called GlfM to form a functional complex with flagellin . Structurally, Maf-2 is a 441-residue protein containing a signature MAF_flag10 domain (also known as DUF115) with a predicted GT-A type glycosyltransferase-like fold .

The key structural differences between Maf-1 and Maf-2 include:

These structural differences explain the distinct flagellin modification patterns between Maf-1 and Maf-2 systems. LC-MS/MS glycopeptide analyses have confirmed that Maf-1 and Maf-2 modify different serine residues on flagellin proteins (FlaA/B) . This site-specific glycosylation has significant implications for flagellar assembly, function, and bacterial motility.

How does the function of Maf-2 in P. syringae compare with Maf proteins in other bacterial species?

In S. oneidensis MR-1, Maf-2 functions in a tripartite system requiring GlfM for flagellin glycosylation, while Maf-1 operates in a bipartite system that directly interacts with flagellin via its TPR domain . Deletion of Maf-2 in S. oneidensis slightly increases motility, suggesting a negative regulatory role, in contrast to Maf-1 deletion which severely reduces motility .

The relationship between Maf-2 and neighboring genes is conserved across multiple species. Genomic analysis shows that the coding sequences for GlfM and Maf-2 are juxtaposed in the genomes of both Gram-negative and Gram-positive bacteria , suggesting evolutionary conservation of this tripartite glycosylation system.

In P. syringae, Maf-2 specifically glycosylates flagellin with pseudaminic acid derivatives, while in other species, Maf proteins may use different nonulosonic acids as substrates, such as legionaminic acid (Leg) . These differences in substrate specificity and glycosylation targets contribute to bacterial species-specific adaptations to different ecological niches.

What is the relationship between Maf-2 activity and bacterial chemotaxis during plant infection?

Maf-2's flagellin glycosylation activity directly impacts bacterial chemotaxis during plant infection. Recent research demonstrates that P. syringae chemotaxis toward plant-derived compounds is crucial for locating plant openings and accessing the apoplast to initiate infection .

The perception of gamma-aminobutyric acid (GABA) and L-proline, two abundant components of the tomato apoplast, through the PsPto-PscC chemoreceptor drives the entry of P. syringae into tomato plant tissues . This chemotactic process depends on properly functioning flagella, which require appropriate glycosylation by Maf proteins.

The relationship operates as follows:

• Maf-2 glycosylates specific serine residues on flagellin proteins

• This glycosylation affects flagellar assembly and function

• Functional flagella enable chemotactic responses to plant-derived compounds

• Efficient chemotaxis facilitates bacterial entry into plant tissues

• GABA and L-proline levels significantly increase in tomato plants upon pathogen infection

Interestingly, these same plant compounds (GABA and L-proline) that serve as chemotactic signals are also involved in regulating plant defense responses, creating a complex interplay between pathogen attraction and host defense activation .

How should researchers design experiments to study Maf-2 function in P. syringae?

When designing experiments to study Maf-2 function in P. syringae, researchers should implement a comprehensive multiple-probe experimental approach with appropriate controls. The following experimental design framework is recommended:

• Gene deletion and complementation studies:

  • Generate Δmaf-2 single mutants and Δmaf-1Δmaf-2 double mutants

  • Create complementation constructs with wild-type maf-2 and specific domain mutants

  • Use appropriate vector controls in all experiments

• Phenotypic assays:

  • Motility assays on semi-solid (swarm) agar to assess flagellar function

  • Transmission electron microscopy (TEM) to visualize flagellar filament length and structure

  • Immunoblotting with antibodies against flagellin to assess glycosylation state

  • Mass spectrometry analysis to identify specific glycosylation sites and modifications

• Protein interaction studies:

  • Co-immunoprecipitation to identify Maf-2 interaction partners

  • Pull-down assays to confirm direct interactions with GlfM and flagellin

  • Bacterial two-hybrid or split-GFP assays to visualize interactions in vivo

• Virulence assessment:

  • Plant infection assays comparing wild-type and mutant strains

  • Measurement of bacterial growth in planta at multiple time points

  • Assessment of symptom development and hypersensitive response

• Data collection and analysis:

  • Implement a multiple-probe design with temporal staggering of measurements

  • Use baseline probes to establish initial conditions before experimental manipulation

  • Apply appropriate statistical analyses to determine significance

For example, when studying the impact of Maf-2 on motility, researchers should collect data in the following format:

Following the multiple-probe experimental design principles ensures that the experimental staggering maintains the fidelity of the design and allows for evolution along with skill acquisition .

What are the optimal methods for detecting and analyzing Maf-2-mediated flagellin glycosylation?

The optimal methods for detecting and analyzing Maf-2-mediated flagellin glycosylation require a combination of biochemical, mass spectrometry, and imaging techniques. The following methodological approach is recommended:

• Flagellin purification and detection:

  • Express tagged versions of flagellin (e.g., FLAG-FlaA or FLAG-FlaB) for specific detection

  • Use immunoblotting with monoclonal antibodies against the tag to detect mobility shifts indicative of glycosylation

  • Apply periodic acid-Schiff (PAS) staining to detect glycoproteins in gels

• Mass spectrometry analysis:

  • Implement LC-MS/MS (liquid chromatography followed by tandem mass spectrometry) for glycopeptide analysis

  • Use collision-induced dissociation (CID) and electron-transfer dissociation (ETD) for glycan structure determination

  • Apply multiple reaction monitoring (MRM) for quantitative analysis of specific glycopeptides

• Structural characterization:

  • Isolate glycosylated flagellin from wild-type and reconstituted systems

  • Perform enzymatic digestion with site-specific proteases

  • Analyze glycopeptides by MALDI-TOF MS and ESI-MS/MS

  • Determine glycan structures using NMR spectroscopy for detailed characterization

• In vitro glycosylation assays:

  • Express and purify recombinant Maf-2, GlfM, and flagellin proteins

  • Reconstitute the glycosylation reaction in vitro with appropriate sugar nucleotide donors

  • Analyze reaction products by mass spectrometry and gel mobility shift assays

• Data analysis and representation:

  • Generate extracted ion chromatograms (XICs) for glycopeptides of interest

  • Create mass spectra comparison tables for different experimental conditions

  • Calculate glycosylation site occupancy using label-free quantification

A representative data table for MS analysis of flagellin glycopeptides might look like this:

S with an asterisk indicates the glycosylated serine residue.

This comprehensive approach enables the precise identification of Maf-2 glycosylation sites, the characterization of glycan structures, and the quantification of glycosylation levels under different experimental conditions .

How can researchers effectively study the tripartite system involving Maf-2, GlfM, and flagellin?

To effectively study the tripartite system involving Maf-2, GlfM, and flagellin, researchers should employ a systematic approach combining genetic, biochemical, and structural methods:

A systematic approach to analyzing the interactions within this tripartite system could be represented in a data table as follows:

This approach has been successfully implemented to demonstrate that Maf-2 constitutes a tripartite system that can only be functionally reconstituted in the presence of GlfM, which promotes complex formation between Maf-2 and flagellin .

How should researchers interpret contradictory findings regarding Maf-2 function in different bacterial species?

When interpreting contradictory findings regarding Maf-2 function across different bacterial species, researchers should implement a systematic comparative analysis approach:

• Phylogenetic context analysis:

  • Construct a phylogenetic tree of Maf proteins from different bacterial species

  • Map functional data onto the tree to identify evolutionary patterns

  • Consider the genomic context of maf genes in each species

• Sequence-function correlation:

  • Perform multiple sequence alignments to identify conserved and divergent regions

  • Correlate specific sequence variations with functional differences

  • Generate chimeric proteins to test domain-specific functions

• Experimental conditions assessment:

  • Carefully document all experimental conditions for each study

  • Test whether contradictory findings persist under identical conditions

  • Consider species-specific factors that might influence protein function

• Physiological context evaluation:

  • Examine the ecological niche and lifestyle of each bacterial species

  • Consider host-specific adaptations that might drive functional diversification

  • Evaluate the presence of compensatory mechanisms in different species

• Contradictory data resolution framework:

  Contradiction Type	Analysis Approach	Resolution Strategy	Example
  Different phenotypes	Side-by-side testing	Standardize conditions	Maf-2 deletion enhances motility in S. oneidensis but not in P. syringae
  Different interaction partners	Pull-down + MS analysis	Map species-specific interactomes	Maf-2 might interact with different adaptor proteins besides GlfM
  Different glycosylation targets	Glycopeptide mapping	Compare flagellin sequences	Variation in flagellin serine residues between species
  Different regulatory effects	Transcriptomics	Compare regulons	PsrA regulation of maf-2 might differ between species

As an example, studies in S. oneidensis MR-1 showed that Δmaf-2 mutants display increased motility compared to wild-type , while in some P. syringae strains, the effect might be less pronounced. These differences could be explained by:

• Variations in flagellin protein sequences between species

• Differences in the sugar nucleotide donors available in each species

• Varying regulatory networks controlling flagellar gene expression

• Ecological adaptations to different environmental conditions

By systematically addressing these factors, researchers can resolve apparent contradictions and develop a more comprehensive understanding of Maf-2 function across bacterial species .

How can researchers integrate multiple data types to build comprehensive models of Maf-2 function?

Integrating multiple data types to build comprehensive models of Maf-2 function requires a systematic multi-omics approach. Researchers should follow these methodological guidelines:

• Data integration framework:

  • Implement a hierarchical data integration approach

  • Establish connections between different data layers

  • Use computational models to predict emergent properties

• Multi-omics data collection:

  • Genomics: Sequence analysis and comparative genomics of maf-2 across species

  • Transcriptomics: RNA-seq of wild-type vs. maf-2 mutants under various conditions

  • Proteomics: Global protein abundance and post-translational modifications

  • Glycomics: Comprehensive analysis of glycosylation patterns

  • Phenomics: High-throughput phenotypic characterization

• Network analysis approaches:

  • Construct protein-protein interaction networks centered on Maf-2

  • Develop gene regulatory networks affecting maf-2 expression

  • Create metabolic networks focused on sugar nucleotide metabolism

  • Build signaling networks connecting Maf-2 to virulence phenotypes

• Machine learning applications:

  • Use supervised learning to predict functional outcomes from sequence features

  • Apply unsupervised learning to identify patterns in multi-omics data

  • Implement deep learning for complex relationship modeling

• Visualization and modeling:

  • Create interactive visualizations of integrated datasets

  • Develop mathematical models of the tripartite system dynamics

  • Build predictive models for functional outcomes

A sample data integration approach for Maf-2 research could be organized as follows:

Integration of these diverse data types enables the construction of comprehensive models that capture the complex role of Maf-2 in bacterial physiology and pathogenesis. When applied to the P. syringae pathosystem, this approach can elucidate how Maf-2-mediated flagellin glycosylation contributes to bacterial entry into plant tissues, survival in the apoplast, and interaction with host immune systems .

What are the most promising approaches for targeting Maf-2 function to control P. syringae infections?

The most promising approaches for targeting Maf-2 function to control P. syringae infections leverage our understanding of the tripartite glycosylation system. Strategic research directions include:

• Small molecule inhibitor development:

  • Design inhibitors targeting the Maf-2 active site based on structural information

  • Develop compounds disrupting the Maf-2/GlfM interaction

  • Screen for molecules that compete with sugar nucleotide donors

• Peptide-based intervention strategies:

  • Design peptides mimicking the GlfM interaction interface to disrupt complex formation

  • Develop flagellin-derived peptides that compete for Maf-2 binding

  • Create cell-penetrating peptides to deliver inhibitory molecules into bacteria

• Genetic control strategies:

  • Engineer plant varieties with enhanced recognition of non-glycosylated flagellin

  • Develop CRISPR-Cas systems targeting maf-2 genes for bacterial control

  • Create phage-based delivery systems for anti-maf-2 interventions

• Methodological approaches for validation:

  • Implement high-throughput screening assays for inhibitor discovery

  • Develop in vitro glycosylation assays for mechanism validation

  • Create plant infection models to assess efficacy in planta

• Combination strategies with existing control methods:

  • Test synergy between Maf-2 inhibitors and conventional antibiotics

  • Explore integration with biological control agents

  • Evaluate compatibility with plant defense activators

A methodological framework for evaluating potential Maf-2 inhibitors could include:

This integrative approach to targeting Maf-2 function holds promise for developing new strategies to control P. syringae infections in agriculturally important crops like tomato, potentially reducing reliance on conventional pesticides and improving food security .

How might research on bacterial Maf proteins inform our understanding of protein glycosylation systems more broadly?

Research on bacterial Maf proteins offers unique insights into protein glycosylation systems with broad implications for understanding these processes across domains of life:

• Evolutionary perspectives:

  • Trace the evolution of glycosylation systems from prokaryotes to eukaryotes

  • Identify fundamental mechanisms conserved across diverse species

  • Understand how bipartite systems evolved into tripartite systems through acquisition of adaptor proteins

• Mechanistic insights:

  • Elucidate the structural basis for glycosyltransferase specificity

  • Determine how adaptor proteins like GlfM direct glycosylation to specific targets

  • Understand the catalytic mechanisms of Maf-family glycosyltransferases

• Comparative analysis approaches:

  • Compare bacterial O-glycosylation with eukaryotic systems

  • Analyze substrate recognition across different glycosylation pathways

  • Identify common principles in quality control mechanisms

• Biotechnological applications:

  • Engineer Maf proteins for custom glycosylation patterns

  • Develop bacterial glycosylation systems for recombinant protein production

  • Create semi-synthetic glycoproteins using bacterial enzymes

• Medical and agricultural relevance:

  • Understand how glycosylation affects pathogen recognition by host immune systems

  • Develop glycosylation-based strategies for vaccine development

  • Create diagnostic tools based on glycan detection

The study of the Maf-2/GlfM/flagellin tripartite system provides a unique model system because:

• It represents a simpler glycosylation system compared to eukaryotic pathways

• The tripartite nature reveals fundamental principles about adaptor protein function

• The site-specificity demonstrates mechanisms of glycosylation target selection

• The bacterial system is amenable to genetic manipulation and in vitro reconstitution

By systematically investigating Maf protein function across different bacterial species, researchers can build a comprehensive understanding of protein glycosylation that bridges prokaryotic and eukaryotic systems, potentially leading to novel biotechnological applications and therapeutic strategies .

What are the most effective heterologous expression systems for producing active recombinant Maf-2 protein?

The selection of appropriate heterologous expression systems is critical for producing functional recombinant Maf-2 protein for structural and biochemical studies. Based on current research, the following methodological approaches are recommended:

• E. coli expression systems:

  • BL21(DE3) strain for standard expression with optimization of induction conditions

  • Arctic Express or C41/C43 strains for improved folding at lower temperatures

  • SHuffle strain for enhanced disulfide bond formation if required

  • Codon-optimization of the maf-2 gene for E. coli expression

• Expression vector considerations:

  • Use vectors with tightly controlled inducible promoters (T7, araBAD)

  • Include solubility-enhancing fusion tags (MBP, SUMO, TrxA)

  • Incorporate precision protease cleavage sites for tag removal

  • Consider expression as part of operons including GlfM for co-expression

• Purification strategies:

  • Implement multi-step purification incorporating affinity, ion exchange, and size exclusion chromatography

  • Use on-column refolding if necessary for inclusion body purification

  • Apply tag-specific purification methods followed by tag removal

  • Consider native purification from closely related non-pathogenic Pseudomonas species

• Functional validation:

  • Develop activity assays to confirm glycosyltransferase function

  • Test protein-protein interactions with purified flagellin and GlfM

  • Perform circular dichroism to confirm proper folding

  • Use mass spectrometry to verify post-translational modifications

• Scale-up considerations for structural studies:

  • Optimize for high-yield production (>10 mg/L culture)

  • Ensure homogeneity for crystallization attempts

  • Label with isotopes for NMR studies if required

  • Test different buffer conditions for stability optimization

A representative expression optimization table might include: