Recombinant Xylella fastidiosa Cell division protein ZipA homolog (zipA), partial

What is the Cell Division Protein ZipA and what is its role in bacterial division?

ZipA (FtsZ Interacting Protein A) is an essential cell division protein that plays a critical role in bacterial cytokinesis. According to molecular characterization studies, ZipA functions to:

• Stabilize FtsZ protofilaments through cross-linking

• Serve as a cytoplasmic membrane anchor for the Z-ring structure

• Recruit downstream cell division proteins including FtsK, FtsQ, FtsL, and FtsN to the septal ring

• Protect FtsZ from degradation by ClpP by preventing recognition by ClpX

In most bacteria, ZipA contains an N-terminal transmembrane domain that anchors it to the cytoplasmic membrane, a flexible linker region, and a C-terminal globular domain that interacts with FtsZ. The protein typically doesn't affect the GTPase activity of FtsZ but rather provides structural support for the Z-ring assembly critical for successful bacterial division.

How do methods for expressing recombinant X. fastidiosa ZipA differ from other bacterial proteins?

Expression and purification of recombinant X. fastidiosa ZipA requires specific methodological considerations due to its membrane-associated nature:

Expression System Optimization:

• Selection of E. coli strains specialized for membrane protein expression (e.g., C41/C43)

• Utilization of low-copy number vectors to prevent toxicity

• IPTG concentration titration (0.1-0.5 mM) with induction at lower temperatures (16-20°C)

Purification Protocol:

• Cell lysis via sonication in buffer containing protease inhibitors

• Membrane fraction isolation through ultracentrifugation

• Detergent screening (DDM, LDAO) for optimal solubilization

• IMAC purification using His-tag affinity

• Size exclusion chromatography for final polishing

The expression challenges are similar to those faced when studying other membrane-associated proteins from X. fastidiosa, which must be adapted to the highly specialized xylem environment . Gene disruption by homologous recombination techniques can be employed to study the function of ZipA in vivo .

How does the X. fastidiosa ZipA structure compare with homologs in other bacteria?

While the exact structure of X. fastidiosa ZipA has not been fully characterized, comparative analysis with other bacterial homologs suggests:

• The protein likely contains the canonical domains: N-terminal transmembrane anchor, flexible linker, and C-terminal FtsZ-binding domain

• Sequence alignment analysis similar to that performed for other X. fastidiosa proteins shows significant homology with E. coli counterparts

• The structural prediction tools like PhyreV2 would be expected to yield high confidence models (>95%) based on the conservation patterns seen in other X. fastidiosa proteins

Structural modeling prediction using PhyreV2 software was used to analyze the MqsR/MqsA toxin-antitoxin system from X. fastidiosa, revealing high similarity with the respective proteins from E. coli with 100% confidence . A similar approach could be applied to ZipA homologs.

What functional assays are used to characterize X. fastidiosa ZipA activity?

Key experimental approaches to evaluate ZipA function include:

In vitro biochemical assays:

• FtsZ polymerization assays to measure ZipA's ability to promote FtsZ assembly

• Membrane binding assays to assess ZipA's membrane association properties

• Protein-protein interaction studies (pull-downs, SPR) to measure ZipA-FtsZ binding kinetics

Cellular and genetic approaches:

• Complementation assays in ZipA-deficient strains to confirm functional conservation

• Fluorescence microscopy to visualize ZipA localization during cell division

• Gene disruption by homologous recombination to assess ZipA essentiality

Validation techniques:

• RT-qPCR analysis following the "MIQE guidelines" to confirm expression levels

• Western blotting with specific antibodies to verify protein expression

For in vivo studies, methods similar to those used for validating other X. fastidiosa proteins can be employed, including primers validated for amplification efficiency and specificity as described for ChpA, ColS, DNAJ, and other proteins in X. fastidiosa .

How does ZipA function differ across X. fastidiosa subspecies?

X. fastidiosa comprises multiple subspecies (fastidiosa, multiplex, pauca, sandyi, and morus) that display distinct host ranges and virulence profiles. ZipA function may vary across these subspecies in the following ways:

• Sequence variations in the ZipA protein may correlate with subspecies-specific adaptation to different host plants

• Expression regulation may differ based on host environment, similar to other differentially expressed genes observed between symptomatic and asymptomatic plants

• Functional importance may vary under different stress conditions encountered in various host species

Comparative genomic analyses between divergent X. fastidiosa strains have shown that 98% of their genes are shared, with an average amino acid identity of 96% . This suggests that core cell division proteins like ZipA likely maintain their essential functions across subspecies, though subtle variations may influence host-specific interactions.

How might ZipA contribute to X. fastidiosa's survival under host-specific stress conditions?

X. fastidiosa faces numerous environmental challenges within the plant xylem, and ZipA may play critical roles in stress adaptation:

Temperature stress response:

• X. fastidiosa expresses different heat shock proteins at various temperatures, including HTPG (a molecular chaperone) and HTRA (a periplasmic serine protease required at temperatures above 42°C)

• ZipA function may be coordinated with these stress responses to maintain cell division under temperature fluctuations

Oxidative stress management:

• Different X. fastidiosa strains show differential expression of oxidative stress response genes like catalase/peroxidase and alkyl-hydroperoxide reductase when grown in symptomatic versus asymptomatic plants

• Cell division proteins may be regulated in concert with these stress responses

Survival strategy coordination:
Research indicates X. fastidiosa employs "different survival strategies involving different processes... depending on plant environment" . ZipA regulation may be integrated with these adaptive strategies to ensure appropriate cell division under various host conditions.

The relationship between stress response and cell division is evidenced by findings that the cold shock protein homolog Csp1 influences both stress tolerance and biofilm formation in X. fastidiosa , suggesting integrated regulation of these processes.

What role might ZipA play in X. fastidiosa biofilm formation and virulence?

Biofilm formation is central to X. fastidiosa pathogenicity, and ZipA may influence this process through several mechanisms:

Biofilm development connection:

• X. fastidiosa host colonization involves both cell-cell aggregation and cellular adhesion to surfaces

• Cell division proteins like ZipA are essential for population expansion within biofilms

• Proper regulation of cell division timing and spatial organization is crucial for biofilm architecture

Experimental evidence from related systems:

• Deletion of the cold shock protein gene csp1 resulted in a "dispersed phenotype with visibly fewer cells attached at the air-liquid interface"

• RNA-Seq analysis revealed changes in expression of several genes important for attachment and biofilm formation in the Δcsp1 mutant compared to wild type

• Similar global regulatory effects might occur when cell division genes like zipA are disrupted

Potential mechanism in virulence:

• X. fastidiosa virulence depends on bacterial attachment to host tissue and movement within xylem vessels

• ZipA-mediated cell division regulation may influence the rate of colonization and biofilm expansion

• Cell division defects could alter the expression of attachment factors through stress response pathways

The integration of cell division with biofilm formation is suggested by observations that X. fastidiosa growth in biofilms versus planktonic states shows differential expression of various cellular components .

How can genomic and transcriptomic approaches advance understanding of ZipA function?

Modern omics approaches provide powerful tools for investigating ZipA:

Comparative genomic analysis:

• Multilocus sequence analysis of environmentally mediated genes (MLSA-E) has revealed host-based and geographic origin-based genetic relationships in X. fastidiosa

• Similar approaches focusing on cell division genes could identify correlations between zipA sequence variations and pathogenicity

• Analysis of the ratio of nonsynonymous to synonymous substitutions (dN/dS) can identify regions under selection pressure

Transcriptomic methodologies:

• Long-read nanopore transcriptome sequencing (RNA-Seq) has successfully revealed changes in gene expression patterns related to attachment and biofilm formation in X. fastidiosa mutants

• RT-qPCR following MIQE guidelines can validate expression levels of zipA and related genes under different conditions

• Transcriptomic comparison between wild-type and zipA mutant strains could identify regulatory networks connected to cell division

Integration with recombination analysis:

• X. fastidiosa undergoes homologous recombination , which can obscure phylogenetic relationships

• Tools like Gubbins can identify and remove recombined regions from genomic analyses

• This approach could help determine if zipA has been subject to recombination events that might influence its function

Can ZipA serve as a target for controlling X. fastidiosa infections?

The essential nature of ZipA makes it a potential target for controlling X. fastidiosa:

Target validation considerations:

• Essential cell division proteins present potential intervention points that may be less susceptible to resistance development

• ZipA's role in stabilizing FtsZ protofilaments provides a specific biochemical activity that could be disrupted

• The bacterial-specific nature of the protein minimizes potential off-target effects on host plants

Intervention strategies:

• Small molecule inhibitors targeting the ZipA-FtsZ interaction

• Peptide-based inhibitors mimicking the FtsZ C-terminal region

• RNA-based approaches to reduce zipA expression

Delivery challenges:

• Compounds must reach the xylem vessels where X. fastidiosa resides

• Biofilms may provide protection against inhibitors, similar to how they contribute to copper tolerance

• Systemic distribution throughout the plant vascular system is necessary for effective control

Current control strategies for X. fastidiosa mainly focus on removing infected plants and controlling vector insects , highlighting the need for more targeted molecular approaches.

What can evolutionary analysis of zipA tell us about X. fastidiosa host adaptation?

Evolutionary analysis of zipA can provide insights into X. fastidiosa's adaptive processes:

Subspecies diversification:

• The five recognized X. fastidiosa subspecies show differences in host range and geographic distribution

• Analysis of selection signatures in zipA could reveal whether this gene has contributed to host adaptation

• Comparative genomics across strains with different host preferences could identify correlations with zipA sequence variations

Horizontal gene transfer considerations:

• X. fastidiosa has acquired a significant portion of its genome through horizontal genetic transfer from distantly related bacteria

• Analysis can determine whether zipA has been vertically inherited or horizontally acquired

• Cell division genes might show different evolutionary patterns compared to virulence factors

Integration with recombination analysis:

• Intersubspecific homologous recombination has been associated with X. fastidiosa adaptation to novel hosts

• Tools like Gubbins can identify potential recombination events affecting zipA

• Understanding whether zipA has been subject to recombination could clarify its role in host adaptation

What expression systems are optimal for recombinant X. fastidiosa ZipA production?

Optimal expression of recombinant X. fastidiosa ZipA requires careful system selection:

Bacterial expression systems:

• E. coli BL21(DE3) derivatives optimized for membrane proteins

• Cold-adapted strains (Arctic Express) for expression at lower temperatures

• C41/C43 strains specifically developed for toxic or membrane proteins

Vector considerations:

• pET vectors with T7 promoter for high-level controlled expression

• Fusion tags: N-terminal His6 tag for purification, with TEV cleavage site

• Codon optimization for E. coli expression if rare codons are identified

Induction parameters:

• Temperature: 16-20°C for optimal folding

• IPTG concentration: 0.1-0.5 mM, with extended expression periods (16-24 hours)

• Media supplementation: Additional zinc may benefit proper folding based on the zinc-finger domains found in some X. fastidiosa proteins

When studying X. fastidiosa proteins, validation of expression using techniques such as RT-qPCR and protein-specific antibodies is essential to confirm successful production .

What specific challenges arise when studying ZipA in the context of X. fastidiosa's unique lifestyle?

X. fastidiosa's xylem-limited lifestyle presents unique research challenges:

Cultivation challenges:

• X. fastidiosa grows slowly in culture, taking 7-10 days to form visible colonies

• The bacterium shows decreased viability in extended stationary phase, with significant decreases observed by day 13 in culture

• Specialized media (PD3) is required for optimal growth

Host environment replication:

• Different gene expression patterns have been observed in bacteria from symptomatic versus asymptomatic plants

• Laboratory conditions may not accurately replicate the xylem environment

• Cell-to-cell signaling within bacterial communities affects X. fastidiosa virulence regulation

Technical approaches:

• In vivo survival can be assessed by comparing cell viability at different time points post-inoculation

• Biofilm formation can be quantified by measuring surface attachment and aggregation

• Vector transmission studies require specialized insect rearing facilities

Understanding ZipA function within this context requires integrating knowledge about X. fastidiosa's unique adaptation to the xylem environment with general principles of bacterial cell division.

How might CRISPR-Cas systems advance functional studies of ZipA in X. fastidiosa?

CRISPR-Cas technology offers promising approaches for X. fastidiosa research:

Genetic manipulation advantages:

• Precise genome editing to create clean deletions, point mutations, or tagged versions of zipA

• Conditional knockdown systems to study essential genes like zipA

• Integration of reporter genes for real-time monitoring of expression

Methodological considerations:

• Delivery methods must be optimized for X. fastidiosa transformation

• PAM site availability in the zipA gene region

• Selection markers compatible with X. fastidiosa physiology

Potential applications:

• Creating zipA mutant libraries to identify critical functional residues

• Tagging endogenous ZipA with fluorescent proteins for localization studies

• Engineering strains with regulatable zipA expression to study concentration-dependent effects

Gene disruption by homologous recombination has been successfully applied to X. fastidiosa , suggesting that CRISPR-based approaches could further enhance genetic manipulation capabilities.

What interdisciplinary approaches could yield new insights into ZipA's role in X. fastidiosa pathogenicity?

Integrating multiple research disciplines offers comprehensive understanding:

Systems biology integration:

• Combining transcriptomics, proteomics, and metabolomics data to build network models of cell division regulation

• Mathematical modeling of ZipA's role in biofilm formation and bacterial population dynamics

• Correlation of zipA expression patterns with virulence in different host plants

Structural biology and biophysics:

• Cryo-electron microscopy to visualize ZipA-FtsZ interactions in native-like environments

• Advanced fluorescence techniques (FRET, FLIM) to study protein dynamics in living cells

• Single-molecule approaches to measure ZipA-membrane interactions

Host-pathogen interaction studies:

• Microscopy of X. fastidiosa division within the plant xylem environment

• Effects of plant-derived compounds on ZipA function and bacterial division

• Co-evolution analysis between zipA sequences and host plant resistance factors

Integrating these approaches could help explain observations that X. fastidiosa adopts "different survival strategies involving different processes... depending on plant environment" , potentially involving cell division regulation.