Recombinant Pseudomonas aeruginosa UPF0114 protein PLES_49571 (PLES_49571)

How does PLES_49571 compare between different Pseudomonas aeruginosa strains?

While PLES_49571 is specifically named in the LESB58 strain, homologous proteins may exist in other P. aeruginosa strains under different locus identifiers. The Liverpool Epidemic Strain (LES) represents a highly transmissible lineage associated with cystic fibrosis infections.

When comparing with reference laboratory strains:

Researchers should note that genetic diversity between clinical isolates and laboratory strains can significantly impact experimental results and their clinical relevance .

What are the optimal conditions for recombinant expression of PLES_49571?

The recombinant PLES_49571 protein is typically expressed in E. coli expression systems with the following methodological considerations:

• Expression vector selection: Vectors containing strong inducible promoters (T7, tac) are recommended for membrane proteins

• Fusion tags: His-tag fusion has been successfully implemented for PLES_49571, facilitating purification via metal affinity chromatography

• Expression optimization:

  • Induction at lower temperatures (16-25°C) may improve protein folding

  • Use of E. coli strains optimized for membrane protein expression (C41, C43, or Lemo21)

  • Optimize induction time and inducer concentration through small-scale expression trials

Expression troubleshooting should include Western blot analysis to confirm expression and localization (membrane fraction vs. inclusion bodies) .

What are the recommended storage and reconstitution protocols for PLES_49571?

For optimal stability and activity, adhere to the following protocols:

Storage conditions:

• Store lyophilized protein at -20°C/-80°C

• Avoid repeated freeze-thaw cycles

• For working aliquots, store at 4°C for up to one week

Reconstitution protocol:

• Centrifuge vial briefly before opening to collect content at the bottom

• Reconstitute in deionized sterile water to a concentration of 0.1-1.0 mg/mL

• Add glycerol to 5-50% final concentration (recommended: 50%)

• Aliquot for long-term storage at -20°C/-80°C

Buffer composition:

• Tris/PBS-based buffer with 6% Trehalose, pH 8.0 has shown good stability results

• For membrane proteins, addition of mild detergents may be necessary for solubilization

How should I design experiments to investigate PLES_49571 function in Pseudomonas aeruginosa?

When investigating the function of PLES_49571, implement a multi-faceted experimental approach:

• Genetic approaches:

  • Create knockout mutants (ΔPles_49571) using allelic exchange or CRISPR-Cas9

  • Complement mutants with wild-type gene to confirm phenotype

  • Use conditional expression systems for essential genes

• Phenotypic characterization:

  • Compare growth curves under various conditions (carbon sources, stress)

  • Assess biofilm formation capacity

  • Evaluate antibiotic susceptibility profiles

• Protein-specific assays:

  • Membrane localization studies using fractionation and immunoblotting

  • Potential transport assays if transmembrane prediction is accurate

  • Protein-protein interaction studies (pull-down assays, bacterial two-hybrid)

Given the potential membrane localization of PLES_49571, experiments should include appropriate controls for membrane proteins and consider the influence of different growth conditions on expression .

What controls should be included when studying PLES_49571 in different experimental contexts?

Implementation of rigorous controls is essential for generating reliable data:

For P. aeruginosa experiments specifically, researchers should note that carbon source selection significantly impacts phenazine production and antibiotic tolerance, which could confound results when studying membrane proteins .

How can I investigate potential interactions between PLES_49571 and other proteins in Pseudomonas aeruginosa?

To explore the protein interaction network of PLES_49571:

• In vitro approaches:

  • Co-immunoprecipitation with tagged PLES_49571

  • Pull-down assays using recombinant protein as bait

  • Cross-linking mass spectrometry for membrane protein complexes

• In vivo approaches:

  • Bacterial two-hybrid or split-protein complementation assays

  • Proximity-dependent labeling (BioID, APEX)

  • Genetic suppressor screening

• Computational prediction:

  • Homology-based prediction from related UPF0114 family proteins

  • Co-expression analysis of transcriptomic data

  • Structural modeling of potential interaction interfaces

When designing interaction studies, consider the membrane localization of PLES_49571 and use appropriate detergents for solubilization without disrupting native interactions .

What approaches can be used to study PLES_49571 in the context of biofilm formation and antibiotic resistance?

Given P. aeruginosa's clinical relevance as a multidrug-resistant pathogen, investigating PLES_49571's role in biofilm formation and antibiotic resistance is valuable:

• Biofilm assays:

  • Static microtiter plate biofilm assays comparing wild-type and mutant strains

  • Flow cell biofilm systems for dynamic conditions

  • Confocal microscopy with fluorescent reporters to visualize biofilm architecture

• Antibiotic resistance studies:

  • Determine Minimum Inhibitory Concentrations (MICs) for various antibiotics

  • Analyze persister cell formation in ΔPles_49571 mutants

  • Assess tolerance under different carbon source conditions

• Combined approaches:

  • Antibiotic penetration assays in biofilms

  • Transcriptomic analysis under biofilm vs. planktonic conditions

  • In vivo infection models to assess virulence

For maximum clinical relevance, researchers should consider using both laboratory reference strains (PAO1, PA14) and clinical isolates, as laboratory strains may demonstrate significant deviations from observations in human infections .

How can I address solubility issues when working with recombinant PLES_49571?

Membrane proteins like PLES_49571 often present solubility challenges:

• Expression optimization strategies:

  • Reduce expression temperature to 16-20°C

  • Use lower inducer concentrations

  • Test different E. coli expression strains (BL21, C41/C43, Rosetta)

• Solubilization approaches:

  • Screen detergent panel (DDM, LMNG, OG) for optimal extraction

  • Test mixed micelle systems with lipids

  • Consider styrene maleic acid lipid particles (SMALPs) for native-like environment

• Alternative fusion strategies:

  • Solubility-enhancing tags (MBP, SUMO, Trx)

  • Periplasmic expression with signal sequences

  • Cell-free expression systems

For analytical methods, Blue-Native PAGE can help assess the quality of membrane protein preparations before proceeding to functional studies .

How should I interpret contradictory results when studying PLES_49571 across different Pseudomonas aeruginosa strains?

Strain-specific variations are common in P. aeruginosa research and require careful interpretation:

• Sources of variation:

  • Genetic background differences between clinical isolates and lab strains

  • Environmental adaptation (laboratory vs. clinical setting)

  • Presence of mobile genetic elements or pathogenicity islands

• Methodological approaches:

  • Perform comparative genomics to identify strain-specific gene content

  • Conduct complementation studies across strains

  • Use defined media to control for metabolic differences

• Interpretation framework:

  • Consider strain-specific adaptations to environment

  • Evaluate experimental conditions that may favor certain phenotypes

  • Assess data in context of strain virulence profiles

Research has shown that sub-lethal antibiotic exposures can induce different mutagenesis rates between laboratory strains (PAO1, PA14) and clinical isolates, highlighting the importance of strain selection in experimental design .