Recombinant Haemophilus ducreyi Single-stranded DNA-binding protein (ssb)

What is the genomic organization of the SSB gene in Haemophilus ducreyi?

The gene encoding single-stranded DNA binding protein in H. ducreyi has been designated as "plpD" in some research. This gene appears in genomic contexts alongside other genes like plpR and plpA, which are involved in various cellular functions . In H. ducreyi strain 35000HP (GenBank accession no. NC_002940), the genomic organization follows patterns similar to other Haemophilus species, with the SSB gene typically positioned near genes involved in DNA replication and repair machinery. The complete genome sequence of H. ducreyi 35000HP provides a framework for understanding the genetic context of the SSB gene and its potential regulatory elements.

What are the key structural domains of H. ducreyi SSB important for its function?

Based on structural analysis of bacterial SSB proteins, H. ducreyi SSB likely contains:

• N-terminal OB (oligonucleotide/oligosaccharide-binding) fold for ssDNA binding

• Conserved aromatic residues that stack with DNA bases

• C-terminal acidic tail for protein-protein interactions

• Potential oligomerization interfaces (typically forming homotetramers)

The interaction of these domains with DNA is essential for all processes requiring stabilization of single-stranded DNA, including replication, repair, and recombination. The OB fold particularly represents a key functional element that determines the affinity and specificity of SSB-DNA interactions.

What expression systems work best for producing recombinant H. ducreyi SSB?

Optimal expression of recombinant H. ducreyi SSB can be achieved using several approaches:

• E. coli-based expression: BL21(DE3) or similar strains with pET or pGEX vectors under the control of T7 or tac promoters

• Expression conditions: Induction at lower temperatures (16-20°C) with reduced IPTG concentrations (0.1-0.5 mM) to maximize soluble protein yield

• Fusion partners: N-terminal His6-tag or GST-tag to facilitate purification while maintaining protein solubility

• Codon optimization: May be necessary given the different codon usage preferences between H. ducreyi and E. coli

Growth in rich media like TB (Terrific Broth) rather than LB can increase yield, while supplementation with glucose (0.5-1%) can reduce basal expression before induction, improving final protein quality.

How can researchers effectively purify H. ducreyi SSB while maintaining its DNA-binding activity?

A multi-step purification strategy is recommended:

• Affinity chromatography: Using His-tag or GST-tag as the initial capture step

• Ion-exchange chromatography: Typically using a Q-Sepharose column at pH 8.0 with a salt gradient (50-500 mM NaCl)

• Size-exclusion chromatography: Final polishing step to obtain homogeneous protein preparation

Important considerations include:

• Maintaining reducing conditions throughout purification (1-5 mM DTT or β-mercaptoethanol)

• Including protease inhibitors in early purification steps

• Testing DNA-binding activity after each purification step using EMSA

• Avoiding freeze-thaw cycles that can reduce activity

• Storage in buffer containing 20-30% glycerol at -80°C for long-term stability

What methods are most effective for verifying the functional activity of purified H. ducreyi SSB?

Multiple complementary approaches should be employed:

• Electrophoretic Mobility Shift Assay (EMSA): Using labeled ssDNA oligonucleotides to detect binding and determine affinity constants

• Fluorescence-based assays: Utilizing intrinsic tryptophan fluorescence quenching upon ssDNA binding

• Surface Plasmon Resonance (SPR): For real-time binding kinetics analysis

• Single-molecule techniques: Including FRET-based approaches to study binding dynamics

• Functional complementation assays: Testing whether H. ducreyi SSB can complement E. coli SSB mutants

A quantitative comparison of binding affinity to different DNA substrates (varying in length and sequence) provides valuable information about substrate specificity and binding mode.

How does H. ducreyi SSB participate in DNA replication mechanisms?

H. ducreyi SSB likely plays multiple critical roles in DNA replication similar to those observed in related bacteria:

• Replication fork stabilization: Binding to ssDNA generated at replication forks to prevent secondary structure formation

• Helicase activity enhancement: Stimulating DNA helicase activity by reducing ssDNA reannealing

• Polymerase processivity: Potentially enhancing DNA polymerase activity through direct or indirect interactions

• Replication restart: Facilitating replisome reassembly following replication fork collapse

The protein likely interacts with multiple components of the replication machinery, including DNA polymerases and helicases, similar to homologous proteins in related bacterial species. These interactions would be mediated primarily through the C-terminal domain of the SSB protein.

What role does SSB play in H. ducreyi DNA repair pathways?

Based on comparative analysis of bacterial DNA repair systems, H. ducreyi SSB likely functions in multiple repair pathways:

• Base Excision Repair (BER): Stabilizing ssDNA gaps generated during repair

• Nucleotide Excision Repair (NER): Facilitating damage recognition and excision

• Recombinational Repair: Essential for RecA-mediated strand exchange

• SOS response: Potentially upregulated during stress conditions

H. ducreyi's genome contains homologs of various repair proteins that typically interact with SSB. The study of cellular responses to DNA damage in H. ducreyi, including those induced by cytolethal distending toxin (which causes DNA double-strand breaks), suggests the presence of sophisticated DNA repair mechanisms in which SSB would play a central role .

How might SSB function relate to H. ducreyi genome stability and mutation rates?

SSB deficiency or dysfunction could potentially:

• Increase spontaneous mutation rates due to impaired DNA repair

• Lead to genomic instability through insufficient protection of ssDNA regions

• Affect recombination frequencies and horizontal gene transfer

• Influence the integrity of integrated genetic elements

The presence of integrated genetic elements in H. ducreyi, such as the tandem copies of pNAD1 plasmid in the H. ducreyi 35000HP genome , suggests that SSB may play a role in maintaining the stability of these elements and preventing adverse recombination events.

How can researchers study SSB-protein interactions in H. ducreyi systems?

Several complementary approaches can be employed to identify and characterize SSB-protein interactions:

• Bacterial two-hybrid assays: For initial screening of potential interaction partners

• Pull-down assays: Using tagged SSB to isolate protein complexes followed by mass spectrometry

• Co-immunoprecipitation: With specific antibodies against H. ducreyi SSB

• Surface Plasmon Resonance (SPR): For quantitative interaction analysis

• Crosslinking mass spectrometry: To identify interaction interfaces at amino acid resolution

The methodologies used in H. ducreyi proteomics studies, such as the comparative proteomic analysis mentioned in the literature , provide foundations for studying protein-protein interactions involving SSB. Researchers should consider both direct physical interactions and functional interactions that may not involve stable complex formation.

What experimental strategies can assess the role of SSB in H. ducreyi pathogenesis?

An integrated approach combining multiple methodologies is recommended:

• Creation of conditional SSB mutants: Using techniques similar to those employed for creating other H. ducreyi mutants (like the porin-deficient mutant)

• Transcriptomic analysis: Comparing wild-type and SSB-deficient strains under various stress conditions

• Animal and human infection models: Testing mutant strains in established experimental systems, such as the human volunteer model mentioned in the literature

• Ex vivo infection assays: Using human skin explants or cell culture systems

• DNA damage sensitivity assays: Comparing wild-type and mutant strains' response to DNA-damaging agents

These approaches would help determine whether SSB plays a specialized role in pathogenesis beyond its essential functions in DNA metabolism.

How does SSB function relate to H. ducreyi's ability to adapt to host environments?

SSB likely contributes to H. ducreyi's adaptive capabilities through:

• Support of DNA repair: Enabling survival under host-induced DNA damage (oxidative stress, neutrophil extracellular traps)

• Genome plasticity: Facilitating genetic adaptation through recombination

• Stress response: Potentially participating in global stress responses that occur during infection

• Horizontal gene transfer: Possibly facilitating acquisition of adaptive genes

The suppurative granuloma-like niche that H. ducreyi exploits during infection contains immune cells that can produce DNA-damaging reactive oxygen and nitrogen species . SSB would be crucial for DNA protection and repair in this challenging environment.

How does H. ducreyi SSB compare to SSB proteins from other pathogenic bacteria?

Key points of comparison include:

• Sequence conservation: Typically 30-60% identity with SSBs from other gram-negative pathogens

• Binding mode differences: Variations in binding site size and cooperativity

• Interaction partner specificity: Differences in C-terminal interaction motifs

• Expression regulation: Potential pathogen-specific regulatory mechanisms

While SSB proteins are functionally conserved across bacteria, species-specific adaptations may exist that reflect the particular DNA metabolism requirements of H. ducreyi. Comparative analysis with SSB proteins from related pathogens like H. influenzae could reveal adaptations specific to the H. ducreyi lifestyle.

What techniques are available for studying SSB-DNA interactions at the single-molecule level?

Advanced biophysical approaches include:

• Single-molecule FRET: For studying conformational dynamics during DNA binding

• Optical tweezers: To measure forces involved in SSB-DNA interactions

• DNA curtains: For visualizing multiple SSB proteins on long DNA molecules

• Atomic Force Microscopy (AFM): For structural analysis of SSB-DNA complexes

• Single-molecule pull-down (SiMPull): For analyzing composition of SSB-containing complexes

These techniques can reveal mechanistic details not accessible through bulk biochemical assays, such as binding cooperativity, sliding dynamics, and interaction with other proteins at the replication fork.

How might SSB contribute to H. ducreyi's resistance to antimicrobial treatments?

SSB could influence antimicrobial resistance through several mechanisms:

• DNA damage repair: Enhancing survival following treatment with DNA-damaging antibiotics

• Stress response coordination: Facilitating adaptation to antibiotic-induced stress

• Genomic plasticity: Supporting acquisition and recombination of resistance genes

• Biofilm formation: Potentially participating in DNA metabolism during biofilm development

The role of SSB in DNA repair pathways suggests it could influence H. ducreyi's response to antibiotics that target DNA replication or induce DNA damage. This consideration is particularly relevant given the rising antimicrobial resistance observed in H. ducreyi clinical isolates.

What are the key unanswered questions regarding H. ducreyi SSB function?

Critical areas requiring further investigation include:

• Strain variations: Whether SSB sequence and function vary among clinical isolates

• Regulation mechanisms: How SSB expression is regulated during infection and stress

• Specialized functions: Whether H. ducreyi SSB has pathogen-specific functions beyond DNA metabolism

• Interaction network: The complete set of proteins that interact with SSB during infection

• Potential as drug target: Whether SSB functions can be specifically inhibited for therapeutic purposes

Understanding these aspects would provide a more complete picture of SSB's role in H. ducreyi biology and potentially reveal new approaches for intervention.

How can high-throughput screening approaches identify inhibitors of H. ducreyi SSB?

Effective screening strategies include:

• Fluorescence-based binding assays: Monitoring displacement of fluorescently labeled DNA

• AlphaScreen technology: For detecting disruption of SSB-DNA or SSB-protein interactions

• Surface plasmon resonance: For direct measurement of binding inhibition

• In silico screening: Using structural models to identify potential binding pockets

• Bacterial growth assays: Secondary screening of compounds that inhibit SSB function

Careful counter-screening against human ssDNA-binding proteins would be essential to identify compounds with sufficient selectivity for the bacterial protein.

What novel techniques could advance our understanding of H. ducreyi SSB dynamics in vivo?

Emerging methodologies with potential application include:

• CRISPRi approaches: For controlled depletion of SSB in vivo

• Fluorescent protein fusions: For tracking SSB localization during infection

• Super-resolution microscopy: For visualizing SSB distribution at DNA replication and repair sites

• In vivo crosslinking: For capturing transient interactions during infection

• RNA-seq and ChIP-seq: For genome-wide analysis of SSB effects on transcription and DNA metabolism