Recombinant Haemophilus influenzae Electron transport complex protein RnfB (rnfB)

What is the Rnf complex in Haemophilus influenzae and what role does RnfB play?

The Rnf (Rhodobacter nitrogen fixation) complex is a membrane-bound enzyme system widely distributed among anaerobic bacteria, including H. influenzae. This respiratory enzyme complex couples the oxidation of reduced ferredoxin with the reduction of NAD+, generating an ion gradient (Na+ or H+) across the cytoplasmic membrane for energy conservation .

Within this complex, RnfB functions as the ferredoxin-binding subunit, serving as the entry point for electrons from reduced ferredoxin. Research on the Rnf complex from Acetobacterium woodii demonstrates that RnfB contains approximately 25 mol iron and 26 mol sulfur per mol of protein, consistent with the presence of six [4Fe-4S] centers that facilitate electron transfer . These iron-sulfur clusters create an electron transfer pathway that allows electrons to flow from reduced ferredoxin through RnfB into the other components of the Rnf complex.

The Rnf complex is critical for energy conservation in anaerobic environments, allowing H. influenzae to generate energy during colonization of host tissues where oxygen may be limited .

How is the Rnf complex involved in bacterial energy metabolism?

The Rnf complex serves as a simplified respiratory chain in many anaerobic bacteria, contributing to energy conservation through multiple mechanisms:

• Ion gradient generation: The complex functions as a primary ion pump, translocating either Na+ or H+ across the cytoplasmic membrane during electron transfer . In Thermotoga maritima, for example, the Rnf complex has been demonstrated to function as a Na+ pump that, together with a Na+-F1FO ATP synthase, constitutes a simple, two-component respiratory chain .

• Redox balancing: By coupling ferredoxin oxidation with NAD+ reduction, the Rnf complex helps maintain redox homeostasis within bacterial cells.

• Reverse electron transport: In some bacteria, the Rnf complex can operate bidirectionally, using the ion gradient to drive the energetically unfavorable reduction of ferredoxin with NADH as electron donor .

This versatility makes the Rnf complex crucial for metabolic flexibility, particularly in environments with fluctuating oxygen levels such as those encountered by H. influenzae during host colonization and infection.

What are the predicted structural features of H. influenzae RnfB?

Based on studies of RnfB from related bacteria, H. influenzae RnfB likely possesses several key structural features:

• Multiple iron-sulfur clusters: The protein contains approximately six [4Fe-4S] centers, requiring around 24-25 iron atoms and a similar number of sulfur atoms per molecule .

• Conserved cysteine motifs: These motifs (typically CX2CX2CX3CP) coordinate the iron-sulfur clusters and are essential for electron transfer function.

• Ferredoxin-binding domain: A specific region that recognizes and binds ferredoxin, facilitating efficient electron transfer.

• Membrane association domain: While primarily a cytoplasmic protein, RnfB likely has regions that interact with membrane-embedded components of the Rnf complex.

• Potential signal sequence: As with other proteins destined for membrane complexes, RnfB may contain signal sequences directing its proper localization.

The arrangement of these structural elements creates an electron transfer pathway that enables electrons to flow from ferredoxin through the iron-sulfur clusters of RnfB and into the membrane components of the Rnf complex.

What are effective strategies for expressing recombinant H. influenzae RnfB?

Expression of functional RnfB presents significant challenges due to its iron-sulfur cluster content. Based on experiences with similar proteins, the following strategies are recommended:

• Fusion protein approaches: RnfB from Acetobacterium woodii could only be successfully produced as a fusion protein with maltose-binding protein (MBP) . Similar approaches may be effective for H. influenzae RnfB, with potential fusion partners including:

  • Maltose-binding protein (MBP)

  • Thioredoxin (Trx)

  • Small ubiquitin-like modifier (SUMO)

  • Glutathione S-transferase (GST)

• Expression host optimization: While E. coli is commonly used, specialized strains that enhance iron-sulfur protein expression may improve yields. Alternative hosts including yeast, baculovirus, or mammalian cell systems could be considered for particularly challenging cases .

• Co-expression with iron-sulfur cluster assembly machinery: Including genes for iron-sulfur cluster biogenesis (such as isc or suf operons) can improve incorporation of these essential cofactors.

• Expression conditions optimization: Key parameters include:

What purification protocols maintain RnfB stability and function?

Maintaining the integrity of iron-sulfur clusters during purification is critical for obtaining functional RnfB. A comprehensive purification protocol should include:

• Buffer composition optimizations:

  • Include reducing agents (1-5 mM DTT or β-mercaptoethanol)

  • Maintain anaerobic conditions throughout purification

  • Add glycerol (10-20%) to stabilize protein structure

  • Include protease inhibitors to prevent degradation

• Purification sequence:

  • Initial capture using affinity chromatography (based on fusion tags)

  • Intermediate purification via ion exchange chromatography

  • Polishing step using size exclusion chromatography

• Quality control checks:

  • Iron and sulfur content determination (expected ~25 mol Fe and ~26 mol S per mol RnfB)

  • UV-visible spectroscopy to verify iron-sulfur cluster integrity

  • Activity assays measuring electron transfer from ferredoxin

• Storage considerations:

  • Flash-freeze in liquid nitrogen

  • Store at -80°C in single-use aliquots

  • Include cryoprotectants (glycerol 20-30%)

  • Maintain anaerobic conditions

These stringent purification conditions are essential for maintaining the integrity of the iron-sulfur clusters that are crucial for RnfB function.

How can researchers verify proper incorporation of iron-sulfur clusters in purified RnfB?

Verification of proper iron-sulfur cluster incorporation requires multiple complementary approaches:

• Quantitative elemental analysis:

  • Measure iron content using atomic absorption spectroscopy or inductively coupled plasma mass spectrometry

  • Determine sulfur content using similar methods

  • Expect approximately 25 mol iron and 26 mol sulfur per mol of RnfB protein

• Spectroscopic characterization:

  • UV-visible spectroscopy: Iron-sulfur proteins typically show characteristic absorption peaks at 320-450 nm

  • Electron Paramagnetic Resonance (EPR): Provides definitive characterization of [4Fe-4S] centers

  • Circular Dichroism (CD): Can reveal both protein folding and iron-sulfur cluster incorporation

• Functional validation:

  • Measure electron transfer from reduced ferredoxin to RnfB

  • Monitor reduction of RnfB iron-sulfur clusters using spectroscopic methods

• Structural integrity assessment:

  • Size exclusion chromatography to confirm monomeric state

  • Thermal shift assays to evaluate stability

Comparative analysis with well-characterized iron-sulfur proteins can provide benchmarks for these measurements, helping researchers determine if their purified RnfB contains properly incorporated clusters.

What experimental approaches can demonstrate the ferredoxin-binding activity of RnfB?

Multiple complementary techniques can characterize the interaction between RnfB and ferredoxin:

• Biophysical interaction analyses:

  • Surface Plasmon Resonance (SPR): Provides real-time binding kinetics and affinity constants

  • Isothermal Titration Calorimetry (ITC): Measures thermodynamic parameters of binding

  • Microscale Thermophoresis (MST): Determines binding affinities in solution

  • Bio-Layer Interferometry (BLI): Offers label-free, real-time measurement of interactions

• Biochemical assays:

  • Pull-down assays using immobilized RnfB or ferredoxin

  • Co-immunoprecipitation with antibodies against either protein

  • Far-western blotting to visualize interactions

• Spectroscopic approaches:

  • Changes in RnfB spectral properties upon ferredoxin binding

  • Fluorescence anisotropy if either protein can be fluorescently labeled

  • Nuclear Magnetic Resonance (NMR) for detailed interaction mapping

• Functional electron transfer assays:

  • Monitor reduction of RnfB iron-sulfur clusters using ferredoxin as electron donor

  • Measure electron transfer rates under varying conditions

These techniques should be performed under anaerobic conditions to maintain the integrity of the iron-sulfur clusters in both RnfB and ferredoxin.

How can researchers measure electron transfer between ferredoxin and RnfB?

Electron transfer between ferredoxin and RnfB can be measured using several methodologies:

• Spectrophotometric assays:

  • Directly monitor reduction/oxidation of iron-sulfur clusters via absorbance changes at characteristic wavelengths

  • Use artificial electron acceptors with easily measurable spectral changes (e.g., methyl viologen)

  • Employ stopped-flow spectroscopy for rapid kinetic measurements

• Electrochemical methods:

  • Protein film voltammetry to determine redox potentials and electron transfer rates

  • Mediated electrochemistry using appropriate redox mediators

  • Biofuel cell approaches to measure electron flow through immobilized proteins

• EPR spectroscopy:

  • Monitor changes in EPR spectra as iron-sulfur clusters change oxidation states

  • Freeze-quench experiments to capture transient intermediates

• Reconstitution experiments:

  • Incorporate RnfB into proteoliposomes with other Rnf components

  • Measure complete electron transfer pathway from ferredoxin through the complex

What complementary techniques should be combined to fully characterize RnfB function?

A comprehensive characterization of RnfB requires integration of multiple techniques:

• Structural characterization:

  • X-ray crystallography or cryo-EM for high-resolution structure

  • Small-angle X-ray scattering (SAXS) for solution structure

  • Hydrogen-deuterium exchange mass spectrometry (HDX-MS) for dynamics

• Spectroscopic analyses:

  • UV-visible spectroscopy for iron-sulfur cluster characterization

  • EPR spectroscopy for detailed electronic structure

  • Mössbauer spectroscopy for iron oxidation states

• Protein-protein interaction studies:

  • Cross-linking coupled with mass spectrometry

  • Co-immunoprecipitation with other Rnf complex components

  • Bacterial two-hybrid screening for interaction partners

• Functional assays:

  • Ferredoxin binding and electron transfer measurements

  • Integration with other Rnf components in reconstituted systems

  • Ion translocation assays in proteoliposomes

• In vivo validation:

  • Genetic complementation of rnfB mutants

  • Site-directed mutagenesis of key residues

  • Phenotypic analysis of mutants under relevant conditions

This integrated approach provides a comprehensive understanding of RnfB's structural properties, interactions, and functional mechanisms within the broader context of the Rnf complex and H. influenzae energy metabolism.

How might RnfB contribute to H. influenzae pathogenesis and adaptation to host environments?

The contribution of RnfB to H. influenzae pathogenesis likely centers on energy metabolism during host colonization and infection:

• Adaptation to microaerobic/anaerobic host niches:

  • H. influenzae encounters varying oxygen levels in different host tissues

  • The Rnf complex provides alternative energy conservation mechanisms when oxygen is limited

  • This may be particularly important during colonization of the middle ear, sinuses, and lower respiratory tract

• Metabolic flexibility during infection:

  • Different carbon and energy sources are available in different host environments

  • The Rnf complex may allow utilization of diverse electron donors and acceptors

  • This flexibility could contribute to persistence during chronic infections

• Biofilm formation and persistence:

  • Biofilms contain microenvironments with varying oxygen concentrations

  • RnfB-dependent energy conservation may be crucial in biofilm microenvironments

  • This could contribute to antibiotic tolerance and persistence

• Potential synergy with virulence factors:

  • H. influenzae produces adhesins like HMW1 that contribute to intracellular invasion

  • RnfB-dependent metabolism might provide energy required for expression of these virulence factors

  • Alternatively, it may support bacterial survival after invasion

These potential contributions could be investigated through comparative analyses of rnfB mutants and wild-type strains in relevant infection models or under conditions mimicking host environments.

What approaches can reveal the structural integration of RnfB within the complete Rnf complex?

Understanding how RnfB integrates into the complete Rnf complex requires specialized structural biology approaches:

• Crosslinking coupled with mass spectrometry (XL-MS):

  • Chemical crosslinkers can capture transient protein-protein interactions

  • MS analysis identifies crosslinked peptides, revealing proximity relationships

  • This provides distance constraints for modeling the complex architecture

• Cryo-electron microscopy (cryo-EM):

  • Can determine structures of large membrane protein complexes

  • Particularly valuable for the complete Rnf complex with RnfB in place

  • Sub-nanometer resolution can reveal protein-protein interfaces

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS):

  • Identifies regions protected from solvent upon complex formation

  • Can map interaction interfaces between RnfB and other Rnf components

  • Provides dynamic information not available from static structures

• Site-directed mutagenesis with functional readouts:

  • Systematic mutagenesis of predicted interface residues

  • Assay effects on complex assembly and function

  • Can validate structural models and identify critical interactions

• Native mass spectrometry:

  • Determines subunit stoichiometry within intact complexes

  • Can distinguish different assembly states

  • Provides molecular weight confirmation of the complete complex

• In situ structural techniques:

  • Cellular tomography to visualize complexes in their native environment

  • Correlative light and electron microscopy (CLEM)

  • In-cell NMR for dynamic information

Integration of data from these complementary approaches would provide a comprehensive structural model of RnfB's position and orientation within the complete Rnf complex.

How can systems biology approaches integrate RnfB function into models of H. influenzae metabolism?

Systems biology offers powerful frameworks for understanding RnfB's role in the broader context of H. influenzae metabolism:

• Genome-scale metabolic modeling:

  • Incorporate RnfB-catalyzed reactions into existing H. influenzae metabolic models

  • Perform flux balance analysis to predict phenotypic consequences of rnfB mutations

  • Identify metabolic pathways dependent on RnfB function

• Multi-omics integration:

  • Combine transcriptomics, proteomics, and metabolomics data from wild-type and rnfB mutants

  • Identify regulatory networks affected by RnfB activity

  • Map metabolic adaptations to rnfB deletion or mutation

• Protein-protein interaction networks:

  • Map interactions between RnfB and other proteins using high-throughput methods

  • Position RnfB within the broader cellular interactome

  • Identify unexpected functional connections

• In silico modeling of electron transfer pathways:

  • Model electron flow from various metabolic processes through RnfB

  • Predict energy yields under different environmental conditions

  • Identify rate-limiting steps in energy conservation

• Host-pathogen interaction models:

  • Integrate RnfB function into models of H. influenzae adaptation to host environments

  • Predict metabolic adaptations during infection

  • Identify potential vulnerabilities for therapeutic targeting

What genetic strategies are most effective for creating and complementing rnfB mutations in H. influenzae?

H. influenzae's natural competence facilitates several genetic approaches for studying rnfB:

• Natural transformation-based gene replacement:

  • H. influenzae readily takes up and recombines homologous DNA

  • Construct allelic replacement cassettes with antibiotic resistance markers flanked by homologous regions

  • This approach allows precise deletion or modification of rnfB with minimal disruption to surrounding genes

• Markerless mutation strategies:

  • Two-step process using counterselectable markers (e.g., sacB)

  • First integration of the entire construct, then resolution to leave only the desired mutation

  • Useful for creating point mutations or in-frame deletions without polar effects

• Complementation approaches:

  • Chromosomal integration at neutral sites using natural transformation

  • Plasmid-based expression systems (though limited options exist for H. influenzae)

  • Use of native promoters to maintain physiological expression levels

• Transformed recombinant enrichment profiling (TREP):

  • Generate pools of recombinants with varying alleles of rnfB

  • Select for specific phenotypes related to RnfB function

  • Sequence to identify genetic variants associated with functional changes

• Conditional expression systems:

  • Implement inducible or repressible promoters to control rnfB expression

  • Particularly valuable if rnfB deletion is lethal under certain conditions

  • Allows titration of expression levels to study threshold effects

When implementing these strategies, researchers should consider potential polar effects on downstream genes in the rnf operon, as well as the possibility that rnfB may be essential under certain growth conditions.

How can phenotypic analyses definitively link rnfB mutations to specific metabolic defects?

A comprehensive phenotypic analysis should include:

• Growth kinetics under varying conditions:

  • Aerobic vs. anaerobic vs. microaerobic environments

  • Different carbon sources requiring varying redox balancing

  • Chemical inhibitors of specific metabolic pathways

  • Growth in presence of oxidative stress agents

• Metabolite profiling:

  • Measure NAD+/NADH ratios as indicators of redox state

  • Quantify fermentation end products to identify metabolic shifts

  • Monitor intracellular ATP levels as indicators of energy status

• Membrane potential and ion gradient measurements:

  • Use fluorescent probes to measure membrane potential

  • Assess Na+ and H+ gradients across membranes

  • Compare wild-type, mutant, and complemented strains

• Electron transfer chain activity:

  • Measure oxidation rates of various electron donors

  • Assess reduction rates of terminal electron acceptors

  • Compare activities in membrane fractions vs. whole cells

• In vivo infection models:

  • Colonization efficiency in animal models

  • Persistence during experimental infection

  • Competition assays between wild-type and mutant strains

To definitively link phenotypes to rnfB mutation, researchers should demonstrate that:

• The phenotype is consistently observed in independently constructed mutants

• Complementation with wild-type rnfB restores normal phenotype

• Point mutations in key functional domains produce predictable phenotypic changes

• The severity of phenotype correlates with biochemical measurements of RnfB activity

What approaches can determine if RnfB is essential for H. influenzae under specific environmental conditions?

Determining conditional essentiality of rnfB requires sophisticated genetic and phenotypic approaches:

• Conditional knockout strategies:

  • Place rnfB under control of inducible promoters

  • Withdraw inducer and monitor viability under different conditions

  • Identify conditions where depletion leads to growth arrest or death

• High-density transposon mutagenesis:

  • Generate saturating transposon libraries under permissive conditions

  • Transfer libraries to various selective conditions

  • Use deep sequencing to identify conditions where rnfB insertions are depleted

• Synthetic lethality screening:

  • Identify gene pairs where individual deletions are tolerated but double mutations are lethal

  • Can reveal pathways that become essential when rnfB is absent

• Growth rescue experiments:

  • Attempt to rescue rnfB mutant growth defects with metabolic supplements

  • Test alternative electron donors/acceptors

  • Identify bypasses of RnfB-dependent processes

• Comparative genomics approaches:

  • Examine conservation of rnfB across H. influenzae strains adapted to different niches

  • Compare with related species to identify correlations between gene presence and lifestyle

• Adaptive laboratory evolution:

  • Subject conditional rnfB mutants to prolonged growth under selective conditions

  • Sequence evolved strains to identify compensatory mutations

  • Can reveal alternative pathways that can substitute for RnfB function

These approaches can identify specific conditions where RnfB becomes essential, providing insights into its critical physiological roles during H. influenzae adaptation to various environments, including those encountered during infection.

How does H. influenzae RnfB compare structurally and functionally to homologs in other bacterial species?

Comparative analysis reveals both conservation and diversity in RnfB across bacterial species:

Understanding these comparative differences provides insights into the evolutionary adaptation of RnfB to different bacterial lifestyles and metabolic requirements.

What alternative energy conservation systems might compensate for RnfB deficiency in H. influenzae?

Several alternative systems might partially compensate for RnfB deficiency:

• Nqr (Na⁺-translocating NADH:quinone oxidoreductase) complex:

  • Can establish Na⁺ gradients using NADH as electron donor

  • Functionally analogous to the Rnf complex but with different electron input

• Substrate-level phosphorylation pathways:

  • Direct ATP generation through metabolic reactions

  • Independent of membrane potential or ion gradients

  • May be upregulated when Rnf-dependent energy conservation is compromised

• Alternative respiratory chains:

  • H. influenzae possesses other respiratory components for aerobic and anaerobic respiration

  • Cytochrome bd oxidase functions under microaerobic conditions

  • Terminal reductases for alternative electron acceptors may become more important

• Proton-translocating transhydrogenase:

  • Can couple proton translocation with interconversion of NADH and NADPH

  • Provides an alternative mechanism for generating proton gradients

• Fermentation pathways:

  • Shift metabolism toward fermentative end products

  • Maintain redox balance through alternative mechanisms

  • May be essential survival strategy when RnfB is absent

The effectiveness of these compensatory mechanisms likely depends on specific environmental conditions, explaining why rnfB may be essential in some environments but dispensable in others.

How might research on the Rnf complex in other bacteria inform therapeutic strategies targeting H. influenzae energy metabolism?

Research on the Rnf complex across bacterial species suggests several potential therapeutic approaches:

• Selective inhibition opportunities:

  • Structural differences between bacterial and human proteins offer selectivity

  • The absence of Rnf complexes in humans makes them attractive antimicrobial targets

  • Compounds that disrupt RnfB-ferredoxin interaction could specifically impair anaerobic energy conservation

• Combination therapy strategies:

  • Simultaneously targeting Rnf complex and alternative energy conservation pathways

  • This approach could prevent metabolic adaptation and resistance development

  • Particularly effective during chronic infections where bacteria rely on diverse energy sources

• Environmental niche disruption:

  • Altering the host environment to make RnfB function more critical

  • Changing redox conditions to increase dependency on Rnf-mediated energy conservation

  • Could enhance efficacy of existing antibiotics by restricting metabolic flexibility

• Vaccine development considerations:

  • Though membrane-associated, portions of RnfB may be surface-exposed

  • Conservation across H. influenzae strains could provide broad protection

  • Targeting multiple components of the Rnf complex could enhance vaccine efficacy

• Diagnostic applications:

  • Monitoring expression of rnfB as a biomarker for metabolic state during infection

  • Could indicate transition to persistent, treatment-resistant growth modes

  • Potential for targeted treatment based on metabolic profiling

These therapeutic strategies would be particularly relevant for chronic H. influenzae infections where bacteria establish persistent populations in microaerobic or anaerobic host environments, such as in chronic obstructive pulmonary disease or otitis media.

What key questions about H. influenzae RnfB remain to be addressed?

Despite progress in understanding the Rnf complex, several critical questions about H. influenzae RnfB remain unanswered:

• Structural details:

  • High-resolution structure of H. influenzae RnfB remains undetermined

  • Exact arrangement of iron-sulfur clusters and electron transfer pathways

  • Structural basis of ferredoxin recognition and binding

• Physiological electron donors:

  • Identity of the specific ferredoxin(s) that interact with RnfB in H. influenzae

  • Whether RnfB can accept electrons from alternative donor proteins

  • Redox potential matching between physiological partners

• Regulatory mechanisms:

  • How rnfB expression is regulated in response to environmental conditions

  • Post-translational modifications that might affect RnfB function

  • Protein-protein interactions that modulate activity

• Directionality of electron flow:

  • Whether H. influenzae RnfB functions bidirectionally like some bacterial homologs

  • Conditions that might favor reverse electron transport

  • Structural features that determine directionality

• Role in pathogenesis:

  • Direct contribution of RnfB to survival in specific host environments

  • Interaction with other virulence mechanisms

  • Potential as a therapeutic target

Addressing these questions will require integrative approaches combining structural biology, biochemistry, genetics, and infection models.

What emerging technologies could advance our understanding of RnfB structure and function?

Several cutting-edge technologies show particular promise for RnfB research:

• Cryo-electron microscopy advances:

  • Single-particle analysis for high-resolution structures of the complete Rnf complex

  • Tomography of intact bacterial membranes to visualize native complexes

  • Time-resolved cryo-EM to capture different functional states

• Advanced genetic tools:

  • CRISPR-Cas systems adapted for H. influenzae genome editing

  • Inducible CRISPR interference for controlled gene repression

  • Base editors for precise introduction of point mutations

• Single-molecule techniques:

  • FRET-based approaches to measure conformational changes during electron transfer

  • Single-molecule force spectroscopy to characterize protein-protein interactions

  • Nanopore analysis for studying membrane protein complexes

• Advanced imaging:

  • Super-resolution microscopy to visualize Rnf complex distribution in bacterial cells

  • Correlative light and electron microscopy for linking localization with structure

  • Expansion microscopy to enhance visualization of protein complexes

• Computational approaches:

  • Machine learning for predicting protein-protein interactions

  • Molecular dynamics simulations of electron transfer processes

  • Quantum mechanical calculations of iron-sulfur cluster properties

These technologies promise to overcome current technical limitations in studying membrane-associated iron-sulfur proteins and their complexes.

How might understanding RnfB contribute to broader knowledge of bacterial bioenergetics and pathogenesis?

Research on H. influenzae RnfB has far-reaching implications:

• Evolutionary insights:

  • The Rnf complex represents an ancient mechanism of energy conservation

  • Understanding RnfB can illuminate the evolution of respiratory chains

  • May provide insights into early bioenergetic systems on Earth

• Host-pathogen interaction principles:

  • Reveals how pathogens adapt their energy metabolism to host environments

  • Illustrates metabolic flexibility as a virulence strategy

  • Demonstrates how energy conservation systems integrate with pathogenesis

• Antimicrobial resistance connections:

  • Metabolic flexibility contributes to tolerance and persistence

  • Alternative energy conservation pathways may support survival during antibiotic exposure

  • Targeting energy conservation could provide new therapeutic approaches

• Synthetic biology applications:

  • Engineering electron transport systems for biotechnological applications

  • Creating artificial systems for biological electron transfer

  • Developing biosensors based on electron transfer proteins

• Ecological understanding:

  • Insights into how bacteria adapt to environmental redox gradients

  • Mechanisms of survival in polymicrobial communities

  • Contribution to biofilm formation and maintenance