Recombinant Xylella fastidiosa Penicillin-binding protein 1A (mrcA), partial

What is Xylella fastidiosa and what is the significance of its penicillin-binding protein 1A?

Xylella fastidiosa is a non-spore-forming, rod-shaped bacterium that lives in the xylem vessels of infected plants. The bacterium moves against the sap flow, disrupting water transport, which leads to symptoms such as dieback of branches, brown leaf edges, leaf scorch, and yellowing in host plants . Currently, four subspecies are recognized: X. fastidiosa subsp. fastidiosa, X. fastidiosa subsp. multiplex, X. fastidiosa subsp. sandyi, and X. fastidiosa subsp. pauca, collectively affecting over 300 plant species worldwide .

Penicillin-binding proteins (PBPs) are essential bacterial enzymes involved in cell wall biosynthesis. In many bacteria, PBP1A (encoded by the mrcA gene) plays crucial roles in peptidoglycan synthesis, antimicrobial resistance, and maintaining cell wall integrity. While specific information on X. fastidiosa's PBP1A is limited in current literature, understanding this protein could provide insights into bacterial survival mechanisms and potential intervention strategies.

Table 1: Xylella fastidiosa Subspecies Classification

What structural and functional characteristics define penicillin-binding protein 1A in bacteria?

Penicillin-binding proteins typically contain multiple domains with distinct functions. In most bacteria, PBP1A includes:

• A penicillin-insensitive transglycosylase domain (EC 2.4.2.-) that catalyzes glycan strand polymerization

• A penicillin-sensitive transpeptidase domain (EC 3.4.-.-) that cross-links peptidoglycan strands

The protein is typically anchored to the cytoplasmic membrane and extends into the periplasmic space where peptidoglycan synthesis occurs. While X. fastidiosa-specific structural information is limited, comparative analysis with homologous proteins from other bacteria suggests similar domain organization.

PBP1A functions primarily in peptidoglycan biosynthesis during cell growth and division. It participates in both the elongation and septation phases of bacterial cell division, making it essential for bacterial survival and a potential target for antimicrobial development.

How does X. fastidiosa's penicillin-binding protein 1A compare to those in other bacterial species?

Various bacterial species possess penicillin-binding protein 1A homologs with similar functions but differing in sequence and specific biochemical properties. For example, in Escherichia coli, PBP1A (encoded by mrcA/ponA) contains both transglycosylase and transpeptidase domains, similar to those expected in X. fastidiosa .

Other bacterial species with characterized PBP1A proteins include:

• Neisseria meningitidis (encoded by mrcA/ponA)

• Haemophilus influenzae (encoded by mrcA/ponA)

• Bacillus subtilis (encoded by ponA)

• Clostridium species (encoded by pbpA)

Each of these proteins shares the fundamental transglycosylase and transpeptidase activities but differs in sequence identity, substrate specificity, and regulation. Comparing X. fastidiosa's PBP1A with these better-characterized homologs can provide insights into its specific functions and potential unique features.

What genetic organization surrounds the mrcA gene in X. fastidiosa?

While detailed information about the genetic organization surrounding the mrcA gene in X. fastidiosa is not explicitly provided in the available literature, analysis of bacterial genome organization patterns suggests that mrcA likely exists within an operon containing other cell wall biosynthesis genes.

By comparison with other bacterial systems, potential genetic elements near mrcA might include:

• Promoter regions with regulatory elements

• Adjacent genes involved in peptidoglycan biosynthesis

• Genes encoding other penicillin-binding proteins or cell division proteins

Understanding this genetic context would be valuable for designing experiments to study mrcA expression and regulation in X. fastidiosa.

What expression systems are most effective for producing recombinant X. fastidiosa penicillin-binding protein 1A?

Based on protocols for other bacterial proteins, several expression systems could be used for X. fastidiosa PBP1A:

Table 2: Expression Systems for Recombinant Bacterial Proteins

For membrane-associated proteins like PBP1A, E. coli expression systems with specialized strains (C41/C43) designed for membrane protein expression may provide a balance between yield and proper folding. Based on commercial recombinant protein information, a purity of greater than or equal to 85% as determined by SDS-PAGE is achievable for similar proteins .

How can genetic knockout and complementation techniques be applied to study mrcA function in X. fastidiosa?

A reliable protocol for gene knockout and complementation in X. fastidiosa has been developed using overlap extension PCR and natural transformation, which can be adapted to study mrcA . This method has been successfully applied to other genes in X. fastidiosa, including pilA paralogs.

Key steps in the protocol:

• Designing the knockout construct:

  • Amplify upstream and downstream regions flanking the mrcA gene

  • Include an antibiotic resistance cassette for selection

  • Use overlap extension PCR to join these fragments

• Natural transformation:

  • Utilize X. fastidiosa's natural competence, which is mediated by type IV pili present at one of the cell poles

  • Transform cells with the knockout construct for homologous recombination

• Verification of gene deletion:

  • PCR verification of gene replacement

  • Phenotypic characterization of mutants

• Complementation:

  • Reintroduce a wild-type copy of mrcA at a neutral site in the genome using similar natural transformation approaches

  • Confirm restoration of phenotype

This approach has been shown to be rapid and reliable for generating gene knockouts and complemented mutants in X. fastidiosa strains .

What are the potential interactions between mrcA and other cell wall synthesis machinery in X. fastidiosa?

Penicillin-binding proteins typically function within complex protein networks involved in cell wall synthesis. In X. fastidiosa, PBP1A likely interacts with:

• Other PBPs: Multiple PBPs often work together in multiprotein complexes for coordinated peptidoglycan synthesis

• Cell division proteins: Coordination with divisome components during septation

• Cytoskeletal elements: Interaction with proteins that direct cell wall synthesis spatially

• Regulatory proteins: Proteins that modulate PBP activity in response to environmental conditions

Experimental approaches to study these interactions could include:

• Co-immunoprecipitation followed by mass spectrometry

• Bacterial two-hybrid assays

• Fluorescence resonance energy transfer (FRET)

• Cross-linking studies coupled with proteomic analysis

Understanding these interactions would provide insights into the broader cell wall synthesis network in X. fastidiosa.

How does σ54 regulation influence gene expression in X. fastidiosa and how might it affect mrcA?

Sigma factors are essential for directing RNA polymerase to specific promoters. In X. fastidiosa, the alternative sigma factor σ54 has been identified as a regulator for certain genes, including pilA2 . While direct evidence for σ54 regulation of mrcA is not available, the identification of σ54 binding sites in X. fastidiosa suggests this regulatory mechanism may extend to other genes involved in cell envelope biogenesis.

Putative binding sites for σ54 were identified in the promoter of PD1926 (pilA2) in both Temecula1 and WM1-1 strains, suggesting this regulatory mechanism is conserved across strains . If mrcA is similarly regulated, its expression might be coordinated with other σ54-dependent genes in response to specific environmental conditions or developmental stages.

Experimental approaches to investigate σ54 regulation of mrcA could include:

• Promoter analysis for σ54 binding motifs

• Chromatin immunoprecipitation (ChIP) with σ54 antibodies

• Expression analysis in σ54 deletion mutants

• Reporter gene assays with mrcA promoter constructs

What protocols are recommended for cloning and expressing mrcA from X. fastidiosa?

Cloning Strategy:

• Gene Amplification:

  • Design primers with appropriate restriction sites flanking the mrcA coding sequence

  • Amplify the gene from X. fastidiosa genomic DNA using high-fidelity polymerase

  • Consider codon optimization if expression will be in a heterologous host

• Vector Selection:

  • For membrane proteins like PBP1A, vectors with inducible promoters (pET, pBAD) are recommended

  • Include appropriate fusion tags (His-tag, Strep-tag) for purification

  • Consider fusion with solubility-enhancing partners (MBP, SUMO) if solubility is a concern

• Expression Optimization:

  • Test multiple E. coli strains (BL21(DE3), C41/C43, Rosetta)

  • Optimize induction conditions (temperature, inducer concentration, duration)

  • Consider detergent screening for membrane protein solubilization

• Purification Strategy:

  • Immobilized metal affinity chromatography (IMAC) for His-tagged proteins

  • Size exclusion chromatography for final polishing

  • Target purity ≥85% as determined by SDS-PAGE

How can researchers detect and measure the enzymatic activity of recombinant PBP1A?

Several methods can be employed to assess both the transpeptidase and transglycosylase activities of PBP1A:

Transpeptidase Activity:

• Bocillin-FL binding: Fluorescent penicillin derivative that binds to the active site of PBPs

• HPLC analysis: Detect products of synthetic peptide cross-linking

• Mass spectrometry: Identify cross-linked peptidoglycan fragments

Transglycosylase Activity:

• Radioactive substrate incorporation: Measure incorporation of radiolabeled lipid II into peptidoglycan

• Fluorescent lipid II analogs: Monitor polymerization using fluorescently labeled substrates

• Coupled enzyme assays: Detect released pyrophosphate during glycan strand polymerization

Integrated Assays:

• In vitro peptidoglycan synthesis assays using purified components

• Complementation of E. coli PBP1A-deficient strains with X. fastidiosa mrcA

These assays would require adaptation and optimization for the specific properties of X. fastidiosa PBP1A.

What are the best approaches for structural characterization of X. fastidiosa PBP1A?

Multiple complementary approaches can be used for structural characterization:

How can researchers design experiments to study the role of mrcA in X. fastidiosa's response to environmental stressors?

Experimental approaches could include:

• Expression Analysis Under Stress Conditions:

  • qRT-PCR to measure mrcA expression under various stressors (temperature, pH, nutrient limitation)

  • RNA-Seq for genome-wide expression patterns in wild-type vs. mrcA mutants under stress

  • Promoter-reporter fusions to monitor expression in real-time

• Phenotypic Characterization of mrcA Mutants:

  • Growth curves under various stress conditions

  • Morphological changes assessed by microscopy

  • Cell wall integrity assays (detergent sensitivity, osmotic stress tolerance)

• Biochemical Analysis:

  • Activity assays of purified PBP1A under different conditions

  • Interaction studies with potential protein partners under stress conditions

  • Post-translational modifications analysis in response to stress

• In planta Studies:

  • Colonization efficiency of mrcA mutants in host plants

  • Competitive assays between wild-type and mutant strains

  • Transcriptome analysis of bacteria recovered from plant hosts

Table 3: Potential Stress Conditions for X. fastidiosa Studies

How should researchers approach comparative genomic analysis of mrcA across X. fastidiosa strains?

Comparative genomic analysis can reveal evolutionary patterns and functional conservation of mrcA:

• Sequence Alignment and Phylogenetic Analysis:

  • Multiple sequence alignment of mrcA from different X. fastidiosa strains

  • Identification of conserved domains and variable regions

  • Construction of phylogenetic trees to understand evolutionary relationships

• Synteny Analysis:

  • Examination of gene neighborhoods around mrcA across strains

  • Identification of conserved gene clusters that may suggest functional relationships

• Selection Pressure Analysis:

  • Calculation of dN/dS ratios to identify regions under positive or purifying selection

  • Detection of recombination events that may have affected mrcA evolution

• Structure Prediction and Comparison:

  • Homology modeling based on related PBP1A structures

  • Comparison of predicted structures across strains

  • Identification of strain-specific structural features

• Tools for Analysis:

  • MEGA for phylogenetic analysis

  • PAML for selection analysis

  • Mauve or ACT for synteny visualization

  • Swiss-Model or I-TASSER for homology modeling

How can researchers interpret contradictory findings related to mrcA function?

When faced with contradictory results, researchers should consider:

• Strain-Specific Differences:

  • Different X. fastidiosa strains may have divergent mrcA functions

  • Compare experimental conditions and genetic backgrounds

• Experimental Methodology Variations:

  • Differences in knockout strategies (complete deletion vs. insertion)

  • Variations in complementation approaches

  • Differences in phenotypic assays and their sensitivity

• Redundancy and Compensation:

  • Other PBPs may compensate for mrcA loss in some conditions

  • Regulatory networks may adjust to maintain cell wall integrity

• Environmental and Physiological Context:

  • Growth conditions affect cell wall synthesis requirements

  • Developmental stage of bacteria may influence mrcA importance

• Resolution Strategies:

  • Direct comparison experiments with standardized methods

  • Epistasis studies to identify genetic interactions

  • Biochemical characterization to define molecular activities

  • In vivo studies to determine physiological relevance

What statistical approaches are most appropriate for analyzing mrcA expression data?

Appropriate statistical analyses depend on the experimental design:

• For qRT-PCR Data:

  • Relative quantification using 2^(-ΔΔCt) method

  • ANOVA for comparing expression across multiple conditions

  • Post-hoc tests (Tukey's, Bonferroni) for pairwise comparisons

  • Consider using multiple reference genes for normalization

• For RNA-Seq Data:

  • DESeq2 or edgeR for differential expression analysis

  • WGCNA for co-expression network analysis

  • GO and KEGG pathway enrichment analysis

  • Consider batch effect correction if necessary

• For Microarray Data:

  • Limma package for differential expression

  • Multiple testing correction (FDR, Bonferroni)

  • Hierarchical clustering or PCA for pattern identification

• For Promoter Activity Data:

  • Time-series analysis techniques

  • Regression models for inducer dose-response

  • Mixed-effects models for repeated measurements

• For In Planta Expression:

  • Account for plant-to-plant variability

  • Consider non-parametric tests if normality cannot be assumed

  • Use appropriate transformation methods if necessary

What phenotypic assays are most valuable for assessing the impact of mrcA mutations?

Several phenotypic assays can provide insights into PBP1A function in X. fastidiosa:

• Growth and Morphology:

  • Growth curves in liquid media

  • Colony morphology on solid media

  • Cell size and shape analysis by microscopy

  • Scanning electron microscopy for detailed morphological changes

• Cell Wall Integrity:

  • Susceptibility to β-lactam antibiotics

  • Osmotic shock tolerance

  • Detergent sensitivity (SDS, Triton X-100)

  • Lysozyme sensitivity

• Biofilm Formation:

  • Crystal violet staining assays

  • Confocal microscopy with fluorescent strains

  • Flow cell systems for real-time biofilm development

  • Biofilm matrix composition analysis

• Virulence-Related Phenotypes:

  • Twitching motility assays (similar to those used for pilA studies)

  • Attachment to plant cell surfaces

  • Xylem colonization efficiency in planta

  • Symptom development in infected host plants

• Stress Response:

  • Survival under temperature, pH, and oxidative stress

  • Nutrient limitation response

  • Stationary phase survival

Based on studies of pilA genes in X. fastidiosa, phenotypic assays should be performed in multiple strains to account for strain-specific differences .

How can understanding X. fastidiosa PBP1A contribute to control strategies for plant diseases?

Understanding PBP1A function could lead to several control strategies:

• Targeted Antimicrobials:

  • Development of compounds specifically targeting X. fastidiosa PBP1A

  • Structure-based drug design using recombinant protein crystal structures

  • Screening chemical libraries for inhibitors with specificity for X. fastidiosa PBP1A

• Engineered Resistance in Host Plants:

  • Expression of peptides that interfere with PBP1A function

  • CRISPR-based strategies targeting mrcA in planta

  • RNAi approaches to suppress mrcA expression

• Biocontrol Approaches:

  • Engineering competing microorganisms to express PBP1A inhibitors

  • Developing phage therapy targeting cell wall synthesis

• Diagnostic Tools:

  • Development of antibodies against PBP1A for detection

  • PCR-based detection of strain-specific mrcA variants

  • Point-of-care diagnostics for early disease detection

What are the emerging technologies that could advance X. fastidiosa PBP1A research?

Several cutting-edge technologies could accelerate research:

• CRISPR-Cas9 Technologies:

  • Precise genome editing for creating targeted mutations

  • CRISPRi for controlled gene repression

  • Base editors for introducing specific point mutations

• Single-Cell Technologies:

  • Single-cell RNA-Seq for heterogeneity analysis

  • Time-lapse microscopy with fluorescent reporters

  • Single-molecule tracking of labeled PBP1A

• Advanced Structural Methods:

  • Cryo-electron tomography for visualizing PBP1A in situ

  • Integrative structural biology combining multiple methods

  • Molecular dynamics simulations of membrane-embedded PBP1A

• High-Throughput Phenotyping:

  • Automated image analysis for morphological changes

  • Microfluidic devices for rapid phenotypic characterization

  • Label-free tracking of cellular changes

• Systems Biology Approaches:

  • Multi-omics integration (genomics, transcriptomics, proteomics)

  • Metabolic modeling of cell wall precursor synthesis

  • Network analysis of cell wall synthesis pathways

How can international collaboration accelerate research on X. fastidiosa proteins?

International collaboration is essential given X. fastidiosa's global impact:

• Resource Sharing:

  • Strain collections and mutant libraries

  • Standardized protocols for phenotypic assays

  • Recombinant protein production and purification expertise

• Collaborative Research Approaches:

  • Multi-laboratory validation of key findings

  • Complementary expertise in different techniques

  • Coordinated field trials across geographical regions

• Data Integration Platforms:

  • Centralized databases for genomic and phenotypic data

  • Standardized data formats and analysis pipelines

  • Machine learning approaches for integrating diverse datasets

• Regulatory Considerations:

  • Harmonized biosafety protocols for working with X. fastidiosa

  • Coordinated approach to regulatory approval for control strategies

  • Shared risk assessment methodologies

• Knowledge Transfer:

  • Training workshops and researcher exchanges

  • Open access publication of protocols and results

  • Regular international symposia focused on X. fastidiosa

What interdisciplinary approaches might enhance our understanding of X. fastidiosa PBP1A function?

Combining expertise from multiple disciplines could provide novel insights:

• Computational Biology and Biophysics:

  • Molecular dynamics simulations of membrane-embedded PBP1A

  • Machine learning prediction of protein-protein interactions

  • In silico screening for potential inhibitors

• Chemical Biology:

  • Development of activity-based probes for PBP1A

  • Click chemistry approaches for in vivo labeling

  • Photoaffinity labeling to capture transient interactions

• Plant Pathology and Immunology:

  • Understanding how PBP1A interacts with plant immune responses

  • Studying PBP1A recognition by pattern recognition receptors

  • Engineering plants with enhanced detection of PBP1A epitopes

• Synthetic Biology:

  • Creation of minimal gene sets for cell wall synthesis

  • Engineering synthetic regulation of mrcA expression

  • Development of biosensors for cell wall integrity

• Evolutionary Biology:

  • Comparative analysis across bacterial species

  • Understanding selective pressures on cell wall synthesis genes

  • Reconstructing the evolutionary history of penicillin-binding proteins

By integrating these approaches, researchers can develop a comprehensive understanding of X. fastidiosa PBP1A function and its role in bacterial pathogenicity.