Recombinant Haemophilus influenzae Uncharacterized protein HI_0939 (HI_0939)

What is the genetic organization of HI_0939 and its neighboring genes?

HI_0939 (comO) is part of a competence-regulated operon in Haemophilus influenzae. The gene is located within a 2,614-bp region of the H. influenzae Rd KW20 genome, consisting of genes designated HI0937 to HI0940 . This operon includes comN (HI0938), comO (HI0939), and comQ (HI0941), which are all involved in the DNA uptake machinery . These genes are co-regulated as part of the competence stimulon and are induced during the development of natural competence in H. influenzae.

This region can be PCR-amplified using primers such as 0938CloneF and 0938CloneR, which have been successfully used to clone this genomic region into vectors like pCR2.1-TOPO, resulting in plasmids such as pTV05 . Understanding this genetic organization is crucial for designing experiments that target comO and related genes.

What are the structural and biochemical properties of the ComO protein?

Unlike many other competence proteins in H. influenzae, ComO lacks clear homologs in other well-studied naturally competent bacteria such as Neisseria or Pseudomonas species . This uniqueness suggests that ComO may represent a specialized adaptation in the H. influenzae competence system, distinct from the more conserved components of the DNA uptake machinery.

What experimental methods are used to create and verify HI_0939 mutations?

Creating mutations in HI_0939 requires precise genetic manipulation techniques. The standard approach involves:

• PCR amplification of the target region (HI0937-HI0940)

• Cloning into a suitable vector (e.g., pCR2.1-TOPO)

• Insertion of an antibiotic resistance marker (e.g., spectinomycin cassette) into a unique restriction site within HI_0939

• Transformation of the construct into H. influenzae

• Selection of transformants using the appropriate antibiotic

• Verification of the mutation by PCR analysis

For example, researchers have created insertions into HI0939 by cloning a spectinomycin cassette from pSPECR into the unique SwaI site in pTV05 (located in HI0939) to create pTV23 . Following transformation into Rd KW20, the presence of the correct insertion can be verified by PCR analysis with primers specific to HI0939, such as QPCR-0939-F and 0938Clone-R .

Additionally, unmarked nonpolar mutations can be created using FLP recombinase to remove the antibiotic resistance marker after verification of the initial insertion .

How does the transformation deficiency phenotype of HI_0939 mutants compare to other competence gene mutants?

The transformation phenotypes of various competence gene mutants in H. influenzae show distinctive patterns, as summarized in the following table:

*Later study confirmed transformation defects in comN mutants

What molecular mechanisms underlie the role of ComO in DNA transformation?

The precise molecular mechanism of ComO in DNA transformation remains incompletely understood, but several lines of evidence suggest important functions. Studies have shown that an insertion in HI0939 abolished both DNA binding and uptake, indicating that ComO plays a critical role in the early stages of the transformation process . The protein's predicted signal peptidase I signal sequence suggests it may be localized to either the periplasmic space or the outer membrane, where it could potentially interact with incoming DNA .

Given its basic pI (9.6), ComO may directly bind DNA through electrostatic interactions. Alternatively, it might function as part of a larger protein complex involved in DNA processing during transformation. ComO may work in conjunction with other competence proteins like ComN (HI0938) and ComQ (HI0941), which are encoded in the same operon and likely function in the same pathway .

Research approaches to elucidate these mechanisms should include protein-protein interaction studies, DNA-binding assays, and subcellular localization experiments. Crosslinking studies followed by immunoprecipitation or bacterial two-hybrid analyses could reveal binding partners of ComO within the transformation machinery.

How can recombinant ComO be expressed and purified for functional studies?

Expressing and purifying recombinant ComO presents several challenges due to its membrane association and potential toxicity. Based on current methodologies for similar proteins, the following protocol is recommended:

• Construct Design:

  • Clone the mature coding sequence (without signal sequence) into an expression vector with an N-terminal His-tag

  • Consider using vectors with tightly regulated promoters (e.g., pET with T7lac promoter)

  • Include a TEV protease cleavage site for tag removal

• Expression Systems:

  • E. coli BL21(DE3) or derivatives for initial trials

  • For improved expression of membrane-associated proteins, consider C41(DE3) or C43(DE3) strains

  • Test expression at lower temperatures (16-25°C) to improve protein folding

• Purification Strategy:

  • Immobilized metal affinity chromatography (IMAC) for initial capture

  • Ion exchange chromatography as a second step (given the high pI, use cation exchange)

  • Size exclusion chromatography for final polishing

• Functional Verification:

  • DNA binding assays using electrophoretic mobility shift assays (EMSAs)

  • Assessment of oligomeric state by analytical ultracentrifugation

  • Structural characterization using circular dichroism or thermal shift assays

Researchers should be aware that the presence of the signal peptide may cause inclusion body formation in E. coli, and strategies to refold the protein may be necessary if expression in the soluble fraction is unsuccessful.

What are the comparative genomics insights regarding ComO homologs across bacterial species?

Comparative genomics analyses reveal that ComO (HI0939) appears to be relatively unique to Haemophilus influenzae, with no clear homologs found in other well-studied naturally competent bacteria such as Neisseria or Pseudomonas species . This evolutionary distinctiveness suggests ComO may represent a specialized adaptation in the H. influenzae competence system.

When analyzing USS (Uptake Signal Sequence) recognition systems across related bacteria, significant differences are observed. H. influenzae recognizes the Hin-type USS (5'-AAGTGCGGT-3'), while related Pasteurellaceae such as Actinobacillus pleuropneumoniae recognize the Apl-type USS (5'-ACAAGCGGT-3') . The specialized nature of ComO might be connected to these species-specific USS recognition patterns.

To identify distant homologs, researchers should employ sensitive sequence analysis tools like PSI-BLAST, HHpred, or AlphaFold structure prediction, which might reveal structural similarities despite low sequence conservation. Additionally, functional complementation studies, where comO genes from related species are expressed in H. influenzae comO mutants, could provide insights into functional conservation despite sequence divergence.

What experimental approaches can resolve contradictory data regarding ComO function?

When facing contradictory results regarding ComO function, researchers should implement a systematic troubleshooting approach:

• Strain Verification:

  • Confirm the genotype of mutant strains by PCR and sequencing

  • Verify that no secondary mutations have occurred using whole-genome sequencing

  • Create new mutations using different strategies (e.g., clean deletions vs. insertions)

• Complementation Analysis:

  • Express wild-type ComO from a plasmid in the mutant background

  • Use inducible promoters to test dose-dependent effects

  • Create point mutations to identify critical functional residues

• Standardized Transformation Assays:

  • Implement rigorously controlled transformation protocols

  • Use MIV-induced competence or electroporation as appropriate

  • Include positive controls (wild-type) and negative controls (known transformation-deficient mutants)

  • Quantify transformation efficiency using standardized metrics

• Multi-technique Validation:

  • Combine genetic, biochemical, and microscopy approaches

  • Implement CRP-S-dependent expression analysis to confirm regulation

  • Use fluorescently labeled DNA to track uptake in real-time

• Collaborative Cross-Validation:

  • Have independent laboratories reproduce key findings

  • Exchange strains and protocols to ensure consistency

By implementing these approaches, researchers can identify sources of variability and establish a consensus regarding ComO function in natural transformation.

What are the optimal conditions for studying natural competence in H. influenzae HI_0939 mutants?

When studying natural competence in H. influenzae HI_0939 (comO) mutants, several critical experimental parameters must be controlled:

• Growth and Competence Induction:

  • Use fresh colonies for inoculation (18-24 hours old)

  • Grow cultures to an optical density at 600 nm of 0.2-0.3 before competence induction

  • For MIV-induced competence, wash cells and resuspend in MIV medium for 100 minutes at 37°C

  • Maintain consistent cell densities between experiments (approximately 2×10^9 cells/ml)

• DNA Transformation Parameters:

  • Use standardized DNA concentrations (typically 1 μg/ml)

  • Ensure DNA is high quality and free of contaminants

  • Allow sufficient time for DNA uptake (20-30 minutes)

  • Include a 2-hour expression period at 37°C before plating on selective media

• Controls and Comparisons:

  • Include wild-type H. influenzae Rd KW20 as a positive control

  • Use known transformation-deficient mutants (e.g., comE mutants) as negative controls

  • Test multiple DNA substrates (chromosomal, plasmid, PCR products)

  • Quantify both uptake and transformation when possible

• Data Analysis:

  • Calculate transformation frequencies as the number of transformants per total viable cells

  • Use appropriate statistical tests (typically t-tests or ANOVA)

  • Present data on logarithmic scales when comparing efficiencies across strains

For electroporation experiments, cells should be grown to an optical density at 600 nm of 0.4, and electroporation performed at 14 kV/cm, with a 2-hour expression period before selection .

How should researchers design experiments to study the interaction between ComO and other competence proteins?

To investigate potential interactions between ComO and other competence proteins, researchers should employ a multi-faceted approach:

• Genetic Interaction Studies:

  • Create double mutants combining comO mutations with mutations in other competence genes

  • Analyze epistatic relationships through transformation efficiency measurements

  • Implement suppressor screens to identify mutations that restore function in comO mutants

• Protein-Protein Interaction Methods:

  • Bacterial two-hybrid analysis using ComO as bait against a library of other competence proteins

  • Co-immunoprecipitation with epitope-tagged ComO expressed at physiological levels

  • Split-GFP complementation assays for in vivo interaction visualization

  • Crosslinking followed by mass spectrometry for interaction partner identification

• Localization Studies:

  • Fluorescent protein fusions to determine ComO subcellular localization

  • Co-localization analysis with other competence proteins

  • Super-resolution microscopy to visualize potential competence complexes

  • Fractionation studies to determine membrane association

• Temporal Dynamics:

  • Time-course expression analysis of ComO relative to other competence proteins

  • Inducible expression systems to control timing of ComO production

  • Single-cell imaging to track competence protein dynamics during transformation

Focus particularly on potential interactions with ComN (HI0938) and ComQ (HI0941), as they are encoded in the same operon and likely function in the same pathway . Additionally, interactions with the secretin ComE (HI0435) and the DNA channel protein Rec2 (HI0061) should be investigated given their roles in DNA uptake .

What strategies can be used to generate unmarked, non-polar mutations in HI_0939?

Creating unmarked, non-polar mutations in HI_0939 requires sophisticated genetic tools to avoid polar effects on downstream genes. Based on established protocols, the following methodology is recommended:

• Initial Marked Mutation:

  • Amplify a 2-5 kb region containing HI_0939 with 400-1,500 bp flanking homology on either side

  • Clone the PCR product into a suitable vector (e.g., pGEMT-Easy or pCR2.1-TOPO)

  • Insert a spectinomycin resistance cassette (Spec^r) with FRT sites at a unique restriction site within HI_0939

  • Transform the construct into H. influenzae and select for Spec^r transformants

• FLP-Mediated Marker Removal:

  • Prepare electrocompetent cells of the marked HI_0939 mutant

  • Transform with FLP-encoding plasmid pRSM2947 and select for kanamycin resistance

  • Induce FLP expression with 200 ng/ml anhydrotetracycline for 2 hours at 30°C

  • Plate on non-selective media and incubate at 37°C

  • Screen colonies for loss of spectinomycin resistance (indicating successful marker removal)

  • Confirm the loss of pRSM2947 by testing for kanamycin sensitivity

• Mutation Verification:

  • Confirm the genotype by PCR analysis using primers flanking HI_0939

  • Verify the correct sequence by Sanger sequencing

  • Check for expression of downstream genes to confirm non-polarity

  • Phenotypically characterize the mutant for transformation deficiency

This approach leaves behind only a small FRT "scar" sequence (typically <100 bp) in place of HI_0939, minimizing effects on the expression of neighboring genes while completely inactivating the target gene .

How can researchers troubleshoot unsuccessful transformation experiments with HI_0939 mutants?

When troubleshooting transformation experiments with HI_0939 mutants, consider the following systematic approach:

• Strain Verification Issues:

  • Confirm the HI_0939 mutation by PCR and sequencing

  • Check for contamination by plating on selective and non-selective media

  • Verify strain identity using species-specific PCR

  • Ensure no secondary mutations have occurred during manipulation

• Media and Growth Conditions:

  • Verify that sBHI medium contains all required supplements (hemin and NAD)

  • Check pH of all media (optimal pH range: 7.2-7.4)

  • Monitor growth curves to ensure cultures reach appropriate density

  • Verify MIV medium components for competence induction

• Competence Development Issues:

  • Confirm that wild-type control strains transform efficiently

  • Test competence development using CRP-S-regulated gene expression

  • Verify proper timing of competence development (peak at 100-120 minutes in MIV)

  • Check cell viability before and after competence induction

• DNA Quality and Handling:

  • Ensure transformation DNA is high quality and free of inhibitors

  • Verify DNA concentration using accurate methods (e.g., fluorometric quantification)

  • Test multiple types of DNA substrates

  • Ensure proper timing for DNA addition and uptake periods

• Selection and Expression:

  • Verify antibiotic concentration and activity

  • Allow sufficient expression time (2 hours minimum) before selection

  • Plate appropriate dilutions to obtain countable colonies

  • Include controls for spontaneous resistance

• Advanced Troubleshooting:

  • Test alternative methods (e.g., electroporation vs. MIV-induced competence)

  • Implement qPCR to quantify DNA uptake independent of transformation

  • Consider complementation with wild-type HI_0939 to verify phenotype reversibility

  • Examine potential suppressor mutations that might restore function

What analytical methods are most appropriate for studying the DNA binding properties of recombinant ComO?

To characterize the DNA binding properties of recombinant ComO, researchers should employ multiple complementary techniques:

• Electrophoretic Mobility Shift Assays (EMSA):

  • Use fluorescently labeled DNA fragments (30-50 bp)

  • Test binding to H. influenzae USS-containing vs. non-USS DNA

  • Perform competition assays with unlabeled DNA

  • Analyze binding kinetics by varying protein concentration

  Protocol considerations:

  • Use HEPES or Tris buffer (pH 7.5-8.0) with 50-150 mM NaCl

  • Include 1-5 mM MgCl₂ and 0.1-1 mM DTT

  • Run gels at 4°C to stabilize complexes

• Surface Plasmon Resonance (SPR):

  • Immobilize biotinylated DNA on streptavidin chips

  • Flow ComO protein at various concentrations

  • Determine association and dissociation rates

  • Calculate binding affinities (K_D)

• Fluorescence Anisotropy:

  • Use fluorescently labeled DNA oligonucleotides

  • Titrate with increasing concentrations of ComO

  • Analyze changes in anisotropy to determine binding constants

  • Perform in solution without separation steps

• DNA Footprinting:

  • Use DNase I or hydroxyl radical footprinting

  • Identify specific DNA sequences protected by ComO binding

  • Map interaction sites at single-nucleotide resolution

  • Compare USS and non-USS containing sequences

• Microscale Thermophoresis (MST):

  • Label either DNA or protein with fluorescent dye

  • Measure changes in thermophoretic mobility upon binding

  • Determine binding constants in near-native conditions

  • Require minimal sample amounts

When interpreting binding data, consider:

• The effect of salt concentration on binding (electrostatic vs. specific interactions)

• Potential cooperativity in binding (Hill coefficient)

• Competition with other DNA-binding proteins from the competence system

• The influence of DNA structure and sequence context around binding sites

How can researchers distinguish between direct and indirect effects of HI_0939 mutations on transformation?

Distinguishing between direct and indirect effects of HI_0939 mutations on transformation requires careful experimental design:

• Complementation Analysis:

  • Express wild-type ComO from an inducible promoter in the mutant background

  • Determine whether transformation is restored to wild-type levels

  • Test timing requirements by inducing expression at different stages

  • Use point mutations to identify critical functional residues

• Pathway Analysis:

  • Examine expression of other competence genes in the HI_0939 mutant

  • Test functionality of other competence components individually

  • Analyze epistatic relationships through double mutant analysis

  • Determine whether defects are confined to specific steps in transformation

• Biochemical Approaches:

  • Perform in vitro reconstitution experiments with purified components

  • Test DNA binding, uptake, and processing separately

  • Use radiolabeled or fluorescently labeled DNA to track movement

  • Examine protein-protein interactions with other competence components

• Conditional Depletion:

  • Create conditional HI_0939 alleles (e.g., degradation tags)

  • Deplete ComO at specific stages of competence development

  • Determine the immediate effects of ComO loss

  • Identify the earliest detectable defect in the transformation pathway

• High-Resolution Phenotyping:

  • Quantify DNA binding to the cell surface

  • Measure DNA uptake into the periplasm

  • Assess DNA transport across the inner membrane

  • Monitor DNA integration into the chromosome

By implementing these approaches, researchers can determine whether ComO acts directly in DNA binding and uptake (as suggested by current data) or whether its effects are mediated through interactions with other competence components.

What structural biology approaches would be most informative for understanding ComO function?

Understanding the three-dimensional structure of ComO would significantly advance knowledge of its function. Several complementary structural biology approaches are recommended:

These structural studies should target both the mature ComO protein (without signal sequence) and potential complexes with other competence proteins, particularly those encoded in the same operon (ComN and ComQ).

How could systems biology approaches advance understanding of ComO in the competence regulon?

Systems biology approaches offer powerful tools for understanding ComO in the broader context of the competence regulon:

• Transcriptomics:

  • Compare RNA-seq profiles of wild-type and comO mutant strains during competence development

  • Identify genes with altered expression patterns in the absence of ComO

  • Analyze temporal dynamics of competence gene expression

  • Implement ribosome profiling to assess translational effects

• Proteomics:

  • Quantitative proteomics to measure protein abundance changes in comO mutants

  • Phosphoproteomics to identify potential regulatory mechanisms

  • Protein-protein interaction mapping using proximity labeling (BioID, APEX)

  • Membrane proteomics to characterize competence complex formation

• Metabolomics:

  • Measure changes in cellular metabolism during competence development

  • Identify metabolic requirements for transformation

  • Investigate energy utilization in DNA uptake and processing

  • Connect metabolic state to competence regulation

• Network Analysis:

  • Construct gene regulatory networks of the competence regulon

  • Predict new interactions based on co-expression patterns

  • Identify feedback loops and regulatory motifs

  • Model the dynamics of competence development

• Single-Cell Analysis:

  • Characterize cell-to-cell variability in competence development

  • Measure correlation between ComO levels and transformation efficiency

  • Track DNA uptake in real-time at the single-cell level

  • Identify stochastic elements in competence regulation

• Comparative Genomics:

  • Analyze the evolution of comO across Haemophilus species

  • Identify co-evolving genes that may function with ComO

  • Compare natural competence systems across diverse bacteria

  • Study horizontal gene transfer patterns mediated by ComO-dependent transformation

These approaches should be integrated to develop a comprehensive model of ComO's role in the competence regulon, potentially revealing unexpected connections to other cellular processes.

What potential applications could emerge from detailed understanding of ComO function?

Detailed characterization of ComO function could lead to several innovative applications:

• Antimicrobial Development:

  • Design inhibitors targeting ComO to block natural transformation

  • Reduce the spread of antibiotic resistance genes in H. influenzae

  • Develop combination therapies targeting transformation and conventional targets

  • Create screening assays for transformation inhibitors using ComO as a target

• Biotechnology Applications:

  • Engineer improved transformation systems for difficult-to-transform bacteria

  • Develop controllable gene delivery systems based on natural competence

  • Create biosensors using ComO-based DNA binding domains

  • Optimize DNA uptake for synthetic biology applications

• Diagnostic Tools:

  • Develop detection methods for naturally competent pathogenic strains

  • Create rapid tests for transformation-proficient H. influenzae isolates

  • Design molecular tools to track horizontal gene transfer in clinical settings

  • Implement surveillance systems for monitoring competence in pathogen populations

• Vaccine Development:

  • Evaluate ComO as a potential vaccine antigen

  • Create attenuated strains with controlled competence for live vaccines

  • Design DNA vaccines optimized for uptake via the natural competence machinery

  • Develop adjuvants that target competence-related immune responses

• Evolutionary Studies:

  • Investigate the role of ComO-mediated transformation in bacterial adaptation

  • Study host-pathogen co-evolution through competence-mediated gene transfer

  • Model the impact of transformation on population dynamics

  • Analyze the contribution of horizontal gene transfer to virulence acquisition

These applications highlight the potential translational impact of fundamental research on ComO function, extending beyond basic understanding to practical applications in medicine, biotechnology, and public health.