Recombinant Proteus mirabilis UPF0283 membrane protein PMI1371 (PMI1371)

What is PMI1371 and what is its significance in Proteus mirabilis?

PMI1371 is classified as a UPF0283 membrane protein found in Proteus mirabilis. It is a full-length protein consisting of 348 amino acids that functions as an integral membrane protein. The protein can be recombinantly expressed with a His-tag in E. coli expression systems, which facilitates its purification and subsequent biochemical characterization .

Proteus mirabilis is one of the most significant pathogens associated with catheter-associated urinary tract infections, and the emergence of multidrug-resistant (MDR) strains poses serious public health concerns . While the specific function of PMI1371 has not been fully characterized, membrane proteins in bacterial pathogens often play crucial roles in cellular processes including nutrient transport, signal transduction, and antibiotic resistance. Investigating PMI1371 may provide insights into P. mirabilis pathogenicity mechanisms and potential therapeutic targets.

Methodologically, researchers should approach PMI1371 studies by first confirming gene presence and expression levels across different P. mirabilis strains, particularly comparing clinical isolates with varying virulence profiles to establish correlations between protein expression and pathogenicity.

What expression systems are optimal for producing recombinant PMI1371?

For recombinant expression of PMI1371, E. coli systems have been successfully employed to produce His-tagged full-length protein . When selecting an expression system, researchers should consider:

• Bacterial expression systems: E. coli BL21(DE3) or similar strains are commonly used for membrane protein expression. These systems typically employ T7 promoter-based vectors with IPTG induction.

• Expression conditions: Optimization of temperature (often lowered to 16-20°C after induction), induction time, and IPTG concentration is critical for proper folding of membrane proteins.

• Solubilization strategies: Since PMI1371 is a membrane protein, effective solubilization using appropriate detergents (such as n-dodecyl-β-D-maltoside or CHAPS) is essential for extraction from the membrane fraction.

For methodological approaches, researchers should implement a staged screening process testing multiple expression constructs with varying tags (His6, MBP, SUMO) and optimize induction parameters through small-scale expression trials before scaling up.

How is the purity and functionality of recombinant PMI1371 assessed?

Assessment of purity and functionality for recombinant PMI1371 should follow established protocols for membrane proteins:

• Purity assessment:

  • SDS-PAGE analysis followed by Coomassie staining or Western blotting using anti-His antibodies

  • Size exclusion chromatography to evaluate monodispersity

  • Mass spectrometry for identity confirmation

• Functionality assessment:

  • Circular dichroism spectroscopy to confirm proper secondary structure

  • Thermal shift assays to evaluate protein stability

  • Ligand binding assays if potential binding partners are identified

Methodologically, researchers should implement quality control checkpoints throughout the purification process, with particular attention to detergent effects on protein structure and function, as inappropriate detergent selection can lead to protein denaturation or aggregation.

What is known about the structural characteristics of PMI1371?

As a UPF0283 family membrane protein, PMI1371 is presumed to contain transmembrane domains, though detailed structural information is limited. Researchers investigating its structure should consider:

• Bioinformatic analysis:

  • Transmembrane domain prediction using tools like TMHMM, Phobius, or TOPCONS

  • Secondary structure prediction using PSIPRED or JPred

  • Homology modeling based on structurally characterized related proteins

• Experimental approaches:

  • Limited proteolysis coupled with mass spectrometry to identify domain boundaries

  • Cross-linking studies to determine protein topology in membranes

  • X-ray crystallography or cryo-EM for high-resolution structure determination (requiring significant optimization of conditions)

For methodological implementation, researchers should begin with computational predictions to guide experimental design, particularly in optimizing constructs for structural studies by identifying and removing disordered regions while preserving functional domains.

How might PMI1371 contribute to virulence or antibiotic resistance in Proteus mirabilis?

While specific information about PMI1371's role in virulence or resistance is not directly established, research approaches should consider:

• Gene deletion/complementation studies:

  • Construction of PMI1371 knockout strains using CRISPR-Cas9 or homologous recombination

  • Phenotypic characterization of mutants for altered virulence properties

  • Complementation studies to confirm phenotype specificity

• Resistance mechanism investigations:

  • Determination of minimum inhibitory concentrations (MICs) in wild-type versus PMI1371 mutant strains

  • Membrane permeability assays to assess potential barrier functions

  • Efflux pump inhibitor studies if PMI1371 is hypothesized to function in efflux systems

Methodologically, researchers should employ experimental designs that control for potential compensatory mechanisms and polar effects of genetic manipulations. The prevalence of multidrug resistance in P. mirabilis often involves integrative and conjugative elements (ICEs) that carry resistance genes , so investigations should also examine potential associations between PMI1371 and these mobile genetic elements.

What experimental approaches are recommended for studying membrane protein interactions of PMI1371?

For investigating protein-protein or protein-ligand interactions of PMI1371, researchers should consider:

• Co-immunoprecipitation studies:

  • Using anti-His antibodies to pull down PMI1371 complexes

  • Mass spectrometry identification of co-precipitated proteins

  • Confirmation with reverse co-IP using antibodies against identified partners

• Bacterial two-hybrid systems:

  • Modified bacterial two-hybrid systems designed for membrane proteins

  • Split-ubiquitin yeast two-hybrid as an alternative for membrane protein interactions

• Cross-linking mass spectrometry:

  • In vivo cross-linking followed by MS/MS analysis

  • Identification of specific interaction sites through cross-linked peptides

Methodologically, controls are crucial when studying membrane protein interactions. Researchers should include non-specific binding controls and validate interactions through multiple independent techniques. The experimental design should account for the membrane environment's influence on protein interactions, potentially using nanodiscs or liposomes to maintain native-like conditions .

How can researchers address solubility and stability challenges when working with recombinant PMI1371?

Membrane proteins like PMI1371 present significant challenges in maintenance of solubility and stability. Advanced approaches include:

• Detergent screening:

  • Systematic testing of detergents from different classes (maltoside, glucoside, fos-choline)

  • Assessment of protein stability in each detergent using size exclusion chromatography

  • Thermal stability assays to identify optimal detergent conditions

• Membrane mimetic systems:

  • Reconstitution into nanodiscs or SMALPs (Styrene-Maleic Acid Lipid Particles)

  • Incorporation into liposomes of varying lipid compositions

  • Use of amphipols for long-term stability

• Construct optimization:

  • Design of truncated constructs based on domain predictions

  • Fusion with solubility-enhancing partners (MBP, SUMO)

  • Surface entropy reduction through site-directed mutagenesis

Methodologically, researchers should implement a systematic workflow beginning with expression screening, followed by detergent optimization, and finally reconstitution into appropriate membrane mimetics based on downstream applications .

What bioinformatic approaches can predict functional domains and evolutionary relationships of PMI1371?

Advanced bioinformatic analyses provide valuable insights into potential functions and conservation of PMI1371:

• Sequence-based analyses:

  • Multiple sequence alignment with UPF0283 family members

  • Identification of conserved motifs using MEME or similar tools

  • Phylogenetic analysis to determine evolutionary relationships

• Structure-based predictions:

  • Ab initio structure prediction using AlphaFold2 or RoseTTAFold

  • Functional site prediction through ConSurf or similar conservation mapping

  • Molecular docking studies if potential ligands are identified

• Genomic context analysis:

  • Examination of gene neighborhood conservation across bacterial species

  • Identification of co-evolved gene pairs suggesting functional relationships

  • Analysis of regulatory elements controlling PMI1371 expression

The table below summarizes bioinformatic tools for PMI1371 analysis:

Methodologically, researchers should integrate multiple prediction methods and evaluate consensus findings rather than relying on single algorithms, as membrane protein predictions often contain higher uncertainty than soluble proteins.

How does the experimental design affect research outcomes when studying PMI1371 function?

The choice of experimental design significantly impacts the validity and interpretation of results when studying PMI1371:

• Repeated Measures Design:

  • Valuable for comparing PMI1371 behavior under different conditions using the same protein preparation

  • Reduces variability from protein batch differences

  • Limitations include potential time-dependent stability issues with membrane proteins

• Independent Groups Design:

  • Appropriate for comparing wild-type versus mutant forms of PMI1371

  • Eliminates order effects but introduces batch-to-batch variability

  • Requires careful standardization of expression and purification protocols

• Matched Pairs Design:

  • Useful for comparing PMI1371 from different bacterial strains

  • Controls for experimental conditions while testing strain-specific differences

  • Requires matching based on relevant parameters (expression level, etc.)

Methodologically, researchers should carefully consider statistical power requirements for each design type, implementing appropriate controls for membrane protein-specific challenges such as detergent effects, lipid environment influences, and time-dependent stability factors.

How might PMI1371 relate to the pathogenicity mechanisms in multidrug-resistant P. mirabilis strains?

Research on multidrug-resistant P. mirabilis strains has identified several important pathogenicity and resistance mechanisms that provide context for investigating PMI1371:

• Relationship to mobile genetic elements:

  • SXT/R391 integrative and conjugative elements (ICEs) commonly carry resistance genes in P. mirabilis

  • These ICEs integrate into the chromosome and can mobilize through site-specific recombination

  • Investigation of PMI1371 expression in strains with different ICE profiles

• Potential involvement in antimicrobial resistance:

  • P. mirabilis strains carrying multiple antimicrobial resistance genes (ARGs) show varying phenotype-genotype correlations

  • Expression studies comparing PMI1371 levels in susceptible versus resistant isolates

  • Assessment of membrane permeability in relation to PMI1371 expression levels

• Virulence factor associations:

  • P. mirabilis causes catheter-associated UTIs through multiple virulence mechanisms

  • Potential co-regulation between PMI1371 and known virulence factors

  • Biofilm formation studies comparing wild-type and PMI1371 mutant strains

The table below summarizes P. mirabilis resistance mechanisms that could be examined in relation to PMI1371:

Methodologically, researchers should implement comparative genomics approaches combined with functional studies to establish connections between PMI1371 and pathogenicity mechanisms, particularly focusing on membrane-associated functions that could influence resistance phenotypes .