Study Notes on Bdellovibrio bacteriovorus and tdTomato Expression

Visualizing Bdellovibrio bacteriovorus

Introduction

  • Bdellovibrio bacteriovorus: A Gram-negative bacterium in the delta subgroup of proteobacteria characterized by a predatory life cycle.
  • Recent work indicates its potential to control bacteria and biofilms.
  • Traditionally, predation dynamics were monitored through prey cell reduction.

Study Overview

  • Introduced pMQ414: A plasmid expressing tdTomato fluorescent reporter protein into both host-independent (HI) and host-dependent strains of B. bacteriovorus 109J.
  • This construct allowed real-time monitoring of predator proliferation under various growth conditions and interactions.
  • Verified through traditional methods and new observations of predator behavior in different environments.

Predation Mechanism

  • Bdellovibrio bacteriovorus targets other Gram-negative bacteria.
    • Attaches to prey cells, enters periplasm, forms bdelloplast, grows in periplasmic space, and eventually releases progeny to continue predation cycle.
  • Recent advancements in genetic tools and understanding of predatory bacteria have promoted new therapeutic interests due to antibiotic resistance.

Methods

Bacterial Strains and Growth Conditions

  • Predatory strains:
    • B. bacteriovorus 109J (ATCC 43826) and HI-A variant.
  • Prey strains included:
    • Escherichia coli S17-1, and others like Acinetobacter baumannii, Pseudomonas aeruginosa, Klebsiella pneumoniae, Enterococcus faecalis, Staphylococcus epidermidis, Staphylococcus aureus, Candida albicans.
  • Cultured prey in lysogeny broth (LB) or yeast mold broth (for yeasts).
Predator Stock Lysates Preparation
  • Predators harvested by coculturing prey cells with dilute nutrient broth (1:10 dilution).
  • Cultures incubated at 30°C.
  • Stock lysates filtered through a 0.45-µm pore-size filter before use.
  • Predatory strains cultured in PYE medium with nutrient broth and divalent ions for optimal growth.

Predation Experiments

  • Cocultures formed by adding corresponding predator and prey cells in nutrient medium (DNB).
  • Measurement methods:
    • Dilution plating and CFU enumeration for prey cells.
    • PFU enumeration for predatory bacteria.
  • Spectrophotometry at optical density 600 nm (OD600) and microplate readers for fluorescence measurement.

Construction of B. bacteriovorus Expressing tdTomato

  • The IncQ family RSF1010 replicon was used for plasmid amplification, providing autonomous replication in B. bacteriovorus.
  • Constructed plasmid pMQ414 contained:
    • tdTomato gene under the PnptII promoter and genetic tools for selection in B. bacteriovorus.
  • Verified through PCR and confocal microscopy.

Interaction Visualization

  • Cocultured BbpMQ with prey cells for microscopy.
    • Observed predator growth and predation through fluorescence microscopy using confocal laser scanning microscopy.

Results

Fluorescent Expression and Monitoring

  • BbHIpMQ expressed tdTomato clearly in broth culture, with fluorescence intensity correlating with prey growth dynamics.
  • Temperature influence on growth was documented, with optimal growth at 30°C.

Data Confirmation

  • Microscopy confirmed predator presence within bdelloplasts of prey cells.
  • Growth patterns across multiple conditions showed similar trends in predator proliferation and prey reduction.
  • Notable results indicated reduced efficacy on heat-killed prey:
    • K. pneumoniae showed decreased predation capability when heat-killed, demonstrating direct relation to their metabolic state.

Predation on Different Host Cells

  • Prey dynamics among multiple Gram-negative bacterial species evaluated:
    • K. pneumoniae showed the highest predator fluorescence, while minimal interaction with P. aeruginosa suggested host resistance.
  • The study highlighted the role of nonhost cells (decoys) on predatory efficiency.

Interaction with Human Cells

  • Investigated predation on human corneal limbal epithelial cells (HCLE).
    • Very low attachment rates of B. bacteriovorus to HCLE were noted at a multiplicity of infection (MOI) of 3 × 10^6, raising critical questions for therapeutic applicability in vivo.

Discussion

  • Recent explorations into predatory bacteria indicate their potential for therapeutic applications, particularly for antibiotic-resistant strains.
  • The methodology using fluorescent markers presents a crucial advancement for real-time predation monitoring, thereby enhancing study efficiency.
  • Data indicate that ecological interactions can significantly impact predation dynamics, emphasizing the complexity of predator-prey relationships.

Future Applications

  • The fluorescent construct pMQ414 shall facilitate further understanding of predatory mechanisms and predator-prey dynamics in various environments.
  • Potential for broader applications in other Gram-negative predatory bacteria to study interactions in situ, thus shedding light on microbial ecology.

Acknowledgments

  • The research benefitted from various grants and institutional support, recognizing contributions to fluorescence microscopy and research to combat antibiotic resistance.