Recombinant Xylella fastidiosa DNA-binding protein HU (hup)

What is Xylella fastidiosa DNA-binding protein HU (hup) and what are its structural properties?

Xylella fastidiosa DNA-binding protein HU (hup) is a bacterial histone-like protein encoded by the hup gene (also identified as PD_0475 in some annotation systems). The full-length protein consists of 94 amino acids with the sequence: MNKTELIDGV AAAANLSKVE AGRAIDAVVN EITEALKEGD SVTLVGFGTF QVRQRAERPG RNPKTGEPIM IAASNNPSFK PGKALKDAVK SSAG . The protein is approximately 19.2 kDa after processing, serving functions comparable to bacterial histone-like proteins in other species.

Like other bacterial HU proteins, it likely participates in DNA-binding activities and potentially influences genome architecture. Based on studies of similar proteins in other bacterial species, HU proteins are typically small, basic proteins that bind non-specifically to DNA and can introduce bends in the DNA helix.

What expression systems have been developed for producing recombinant X. fastidiosa HU (hup) protein?

Multiple expression systems have been successfully employed to produce recombinant X. fastidiosa HU (hup) protein, each with different characteristics:

The selection of expression system depends on research requirements including post-translational modifications, protein folding considerations, and downstream applications. The protein is typically provided as a lyophilized powder and requires reconstitution in deionized sterile water to a concentration of 0.1-1.0 mg/mL with recommended addition of 5-50% glycerol for long-term storage .

How does Xylella fastidiosa HU (hup) compare functionally to HU proteins in other bacterial species?

While the search results don't provide specific functional comparisons between X. fastidiosa HU and other bacterial HU proteins, information from related systems offers insights. In Escherichia coli, the HU protein is encoded by two genes, hupA and hupB, which produce two subunits (HU-1 and HU-2) that can form homodimers or heterodimers. Studies in E. coli demonstrate that HU protein participates in site-specific DNA inversion, with either subunit being sufficient for this function .

What experimental methods are most effective for studying HU (hup) protein interactions with DNA?

Based on research approaches used in bacterial DNA-binding protein studies, the following methodological approaches are recommended for investigating HU (hup) interactions with DNA:

• Electrophoretic Mobility Shift Assays (EMSA): To determine binding affinity and specificity of purified recombinant HU protein to different DNA sequences

• Chromatin Immunoprecipitation (ChIP): To identify genomic regions bound by HU in vivo, using anti-HU antibodies

• DNase I footprinting: To identify specific DNA sequences protected by HU binding

• Atomic Force Microscopy (AFM): To visualize structural changes in DNA induced by HU binding

• Isothermal Titration Calorimetry (ITC): To measure thermodynamic parameters of HU-DNA interactions

For studying the role of HU in recombination specifically, researchers have successfully employed natural transformation assays where recombination events can be tracked at frequencies of approximately 10^-6 to 10^-7 .

What evidence suggests that HU (hup) protein plays a role in homologous recombination in X. fastidiosa?

While direct evidence specifically linking X. fastidiosa HU (hup) protein to homologous recombination is not explicitly stated in the search results, several lines of indirect evidence suggest this connection:

• The functional similarity to other bacterial HU proteins that participate in DNA architectural changes necessary for recombination

• The high rates of homologous recombination detected in X. fastidiosa populations

• The natural competence of X. fastidiosa for DNA uptake and recombination, indicating that DNA-binding proteins like HU likely participate in this process

The homologous protein in E. coli is known to participate in site-specific DNA inversion , a process that requires DNA binding and manipulation, suggesting that X. fastidiosa HU may have similar DNA-restructuring capabilities important for recombination events.

How is the recombination process in X. fastidiosa characterized at the molecular level?

Homologous recombination in X. fastidiosa has been extensively characterized through both genomic and experimental studies:

How does recombination facilitated by DNA-binding proteins affect X. fastidiosa's host range and virulence?

Recombination in X. fastidiosa, potentially facilitated by DNA-binding proteins like HU (hup), has significant implications for host range expansion and virulence:

• Host Range Expansion: Intersubspecific homologous recombination (IHR) has been associated with the invasion of new plant hosts by X. fastidiosa. For example, IHR in X. fastidiosa subsp. multiplex is linked to the colonization of blueberry and blackberry, hosts not typically infected by non-recombinant strains .

• Virulence-Related Gene Exchange: Recently recombined regions in wild-type strains include genes involved in regulation and signaling, host colonization, nutrient acquisition, and host evasion—all fundamental traits for X. fastidiosa ecology and pathogenicity .

• Emergence of Novel Variants: Highly recombinant strains pose a serious risk for the emergence of novel variants, especially when genetically distinct and formerly geographically isolated genotypes are brought into close proximity through global trade .

• Adaptive Potential: Specific recombinant genes identified in X. fastidiosa populations include those involved in vitamin B12 transport (btuD), iron acquisition (fur), biofilm formation (bigR), protein secretion (tatA-D), and proteolysis (degP) , suggesting that recombination affects multiple cellular processes critical for bacterial adaptation and virulence.

What factors influence recombination efficiency in X. fastidiosa, and how might these affect HU (hup) protein function?

Several factors have been identified that influence recombination efficiency in X. fastidiosa, which might also impact HU (hup) protein function:

• Nutrient Availability: Transformation and recombination efficiencies are affected by nutrient conditions, suggesting that metabolic state influences DNA uptake and processing mechanisms .

• Growth Stage: The bacterial growth phase impacts recombination efficiency, likely through changes in gene expression patterns, including those of DNA-binding proteins .

• DNA Methylation: Methylation of transforming DNA affects recombination efficiency , indicating that epigenetic factors interact with DNA-binding proteins during recombination.

• Type I Restriction-Modification Systems: These systems, encoded in the X. fastidiosa genome, influence horizontal gene transfer and recombination by determining which DNA sequences can be maintained after uptake. These systems themselves undergo recombination, exchanging target recognition domains to generate novel alleles with new target specificities .

• Genome Methylation Patterns: DNA methylation patterns are associated with type I restriction-modification system allele profiles, creating distinct epigenetic landscapes across X. fastidiosa lineages that likely interact with DNA-binding proteins like HU.

How can protein engineering of HU (hup) be used to study recombination mechanisms in X. fastidiosa?

Protein engineering approaches for studying HU (hup) role in recombination could include:

• Site-Directed Mutagenesis: Introducing specific mutations in DNA-binding domains to alter affinity or specificity, potentially modifying recombination rates or patterns.

• Domain Swapping: Exchanging domains between HU proteins from different bacterial species to identify regions critical for X. fastidiosa-specific recombination functions.

• Fluorescent Protein Fusions: Creating HU-GFP fusion proteins to visualize DNA-binding dynamics in living cells during recombination events.

• Conditional Expression Systems: Developing inducible or repressible HU expression systems to control protein levels and study dosage effects on recombination.

• Affinity-Tagged Variants: Generating biotinylated or otherwise tagged HU proteins (as in CSB-EP803233XAT-B) to facilitate pull-down experiments that identify interaction partners during recombination.

These approaches could help elucidate the specific mechanisms by which HU influences DNA architecture during recombination events in X. fastidiosa.

What are the implications of understanding HU (hup) function for developing novel control strategies against X. fastidiosa infections?

Understanding HU (hup) function could inform several innovative control strategies:

• Recombination Inhibitors: If HU plays a crucial role in recombination, small molecules that specifically inhibit its DNA-binding activity could potentially reduce bacterial adaptation rate to host defenses or environmental stresses.

• Host Range Prediction: Knowledge of how DNA-binding proteins facilitate recombination could help predict which X. fastidiosa strains might gain the ability to infect new plant hosts through recombination events.

• Genetic Barriers: Engineering plants to express inhibitors of bacterial recombination machinery could potentially reduce adaptation rates of the pathogen.

• Diagnostic Tools: Understanding the diverse allele profiles resulting from recombination could lead to more precise molecular diagnostic tools that better identify and characterize X. fastidiosa strains.

• Risk Assessment Models: Information about recombination rates and patterns could inform models predicting the likelihood of emergence of novel pathogenic variants, especially in regions where multiple X. fastidiosa subspecies coexist.

Research has identified that intersubspecific recombination in X. fastidiosa is associated with the invasion of at least two new native plant hosts , highlighting the importance of understanding recombination mechanisms for predicting and managing disease emergence.

How does the recombination system involving HU in X. fastidiosa compare to similar systems in other bacterial pathogens?

Comparative analysis reveals both similarities and differences between X. fastidiosa's recombination system and those of other bacterial pathogens:

This comparison highlights X. fastidiosa's particularly active recombination system, with larger recombination fragments than typically seen in many bacteria, suggesting potentially unique mechanisms involving DNA-binding proteins like HU.

What methodological approaches have been most successful in characterizing the role of DNA-binding proteins in bacterial recombination?

The most successful methodological approaches for characterizing DNA-binding proteins in bacterial recombination include:

• Whole-Genome Sequencing (WGS): Provides superior detection of homologous recombination compared to targeted gene approaches. In X. fastidiosa studies, WGS revealed extensive inter- and intrasubspecific recombination of both ancient and recent origins .

• Natural Transformation Assays: Demonstrated X. fastidiosa's ability to naturally transform and homologously recombine exogenous DNA, with recombination observed in approximately one out of every 10^6 cells when exogenous plasmid DNA was supplied .

• Experimental In Vitro Recombination: In vitro experiments generating recombinants through natural transformation have been valuable for identifying recombination patterns and hotspots. These experimentally generated recombinants displayed similar recombination patterns to wild-type strains .

• Bioinformatic Analysis Tools: Programs like BratNextGen and fastGEAR have been successfully used to identify recombination events in bacterial genomes, detecting random recombination events away from selection markers .

• Multilocus Sequence Typing (MLST): While less comprehensive than WGS, MLST has successfully identified recombinant strains of X. fastidiosa, defined as those with evidence of intersubspecific recombination at multiple loci .

• Introgression Test: A more sensitive method for detecting intersubspecific recombination, especially when other recombination detection methods (such as those in RDP4 and PHI programs) fail to detect recombination events .