JW - Mutations and diversification of biofilms Flashcards
What are some characteristics of biofilms? (3)
- Highly heterogeneous structures
- Consist of microbial communities embedded in a self-produced extracellular polymeric substance (EPS) matrix
- Heterogeneity occurs at multiple levels, making biofilms resilient and difficult to eradicate
How do biofilms contribute to antimicrobial tolerance? (5)
- Matrix impedance – The EPS matrix acts as a physical barrier that hinders antibiotic penetration (e.g., gentamicin)
- Enzymes – Bacteria within biofilms produce enzymes like β-lactamase, which break down antibiotics
- Biochemical changes – Biofilm bacteria undergo physiological changes, such as producing periplasmic glucans, reducing antibiotic effectiveness
- Metabolic gradients – Nutrient and oxygen limitations create metabolic gradients, making antibiotics like Ciprofloxacin (replication inhibitor) and Tobramycin (translation inhibitor) only effective against metabolically active outer layers
- physiological subpopulations - Biofilms contain diverse subpopulations, including “persister” cells that are slow-growing/dormant.
Why is understanding biofilm development important? (2)
- Identifying gene systems involved in biofilm formation can help develop control strategies
- Biofilm physiology may be manipulated to enhance or inhibit development
What is a biofilm flow cell?
- A laboratory device used to study biofilm formation under controlled flow conditions
- Mimics natural environments like medical implants, pipelines, and aquatic systems
What are key features of a biofilm flow cell? (4)
- Continuous Fluid Flow – Supplies nutrients in a steady flow, simulating real-world conditions
- Transparent Chamber – Allows real-time microscopic observation
- Controlled Environment – Adjustable parameters (temperature, flow rate, shear stress)
- Removable Surfaces – Biofilms grow on materials (glass, metal, polymers) that can be removed for analysis
Only studying biofilms as planktonic cells would be washed out of the system
How does biofilm-associated growth impact genetics?
- Induces genome-wide heritable genetic changes
- Genetic heterogeneity and increased mutation rates contribute to biofilm development
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Example: Phenotype arrays show different substrate-utilization profiles of CF P. aeruginosa isolates:
- wild type (WT) and SCV1 strains (A Small Colony Variant that was isolated from a biofilm) had different metabolic profiles
- Role of genetics within the biofilm context remains largely unexplored.
How do P. aeruginosa mutator strains impact microcolony growth? (3)
Deletion of mutS/mutL error repair genes - mutation frequency 100x more than WT:
- Increased biofilm biovolume
- Increased microcolony size
- Enhanced microcolony development due to deletion of DNA error repair genes
What was the microcolony initiation experiment? (4)
- Robert Hancock Laboratories (Vancouver, Canada).
- Investigated how disrupting DNA repair mechanisms affects early biofilm formation
- Used P. aeruginosa strain PAO1
- Method: Tn5 insertion mutant library targeting DNA repair genes
What DNA repair genes were studied in the microcolony initiation experiment? (10)
- himA – Integration host factor
- micA – Mismatch repair protein
- mutL – Mismatch repair protein
- recN - DNA repair protein
- recQ - DNA helicase
- recR – Recombination protein
- sss – Site-specific recombinase
- ung – Uracil-DNA glycosylase
- uvrC – Excinuclease subunit
- xseA – Exodeoxyribonuclease
How was mutation frequency linked to microcolony growth in the microcolony initiation experiment? (2)
- Rifampicin Resistance Assay – Used to measure mutation rate
- A strong positive correlation between mutation frequency and microcolony growth in P. aeruginosa DNA repair mutants
What approach was used to observe genome evolution in P. aeruginosa biofilms? (7)
Deep sequencing
- Harvest cells of 8/9 day -old PA01 biofilms
- Extract total DNA from cell pellet
- High throughput PYROSEQUENCING – 50x coverage
- Use alignment software to crossmatch against Pseudomonas reference genome
- Automated detection and manual check of differences
- Mutations compared to predicted gene functions to predict impact
What types of mutations were found in biofilm deep sequencing? (3)
- 115 SNPs identified
- A Pf4-like phage region had higher sequencing coverage (~75x), suggesting multiple copies
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Only point mutations were observed:
- No recombinations
- No large deletions/insertions
- Single nucleotide polymorphisms (SNPs) in coding/non-coding regions
Which genes were affected by non-synonymous mutations? (4)
- Regulatory proteins – Genes controlling gene expression
- Energy generation – Genes in metabolic pathways
- Transport proteins – Genes moving molecules in/out of cells
- Phage-associated genes – Mostly hypothetical proteins
No particular pattern of mutations
What is the GFP-based mutation detection system? (2)
- A reporter system using GFP mut2, a +1 frameshift mutation that disrupts GFP fluorescence
- Frameshift reversion events (from subsequent mutation) restores fluorescence, allowing real-time mutation detection via epifluorescence or confocal microscopy
How do Darwinian processes contribute to biofilm development? (2)
- Microcolonies act as foci for genetic mutation and evolution
- Microcolony growth may involve mutation selection
What is the model for microcolony development? (3)
- Primary Mutation – A single cell mutates, gaining a selective advantage
- Clonal Expansion – The mutated cell proliferates, forming a microcolony
- Secondary Mutation – Further mutations arise, enhancing survival or differentiation
Similar to tumour development
