Recombinant Pseudomonas aeruginosa Preprotein translocase subunit SecE (secE)

What is the function of SecE in the Pseudomonas aeruginosa Sec pathway?

SecE functions as an essential component of the SecYEG translocon complex in the Sec pathway of P. aeruginosa. This pathway serves as a general export mechanism involved in the transport of periplasmic and outer membrane proteins . The SecYEG complex forms a protein-conducting channel across the inner membrane that facilitates the passage of unfolded proteins. SecE specifically provides structural stability to the translocon complex, preventing SecY degradation and maintaining the integrity of the channel pore. The Sec pathway is particularly notable because it handles the majority of protein translocation across the inner membrane in P. aeruginosa, which is crucial for bacterial survival and virulence .

How does SecE integrate with other components of the Sec translocation machinery?

SecE integrates with SecY and SecG to form the core translocon complex (SecYEG) that spans the inner membrane of P. aeruginosa. This complex works in conjunction with the SecA ATPase, which provides the energy for protein translocation through ATP hydrolysis . SecA from P. aeruginosa (PaSecA) has been shown to possess intrinsic ATPase activity that increases when stimulated by lipids . The SecYEG complex recognizes proteins with N-terminal signal peptides that have been targeted for export. During translocation, SecE stabilizes the complex as SecA pushes the unfolded protein substrate through the channel in a stepwise manner. The interaction between SecE and SecY is particularly critical, as SecE prevents the degradation of SecY when not engaged in translocation, thus maintaining the translocation-competent state of the machinery .

What structural features characterize P. aeruginosa SecE?

P. aeruginosa SecE is a relatively small but essential membrane protein characterized by:

While the search results don't provide specific structural data on P. aeruginosa SecE, comparative analysis with homologous proteins suggests that the transmembrane domain of SecE interacts with specific transmembrane segments of SecY, forming a lateral gate through which signal sequences and transmembrane segments of translocating proteins can exit into the lipid bilayer. This arrangement is crucial for both protein secretion across the membrane and the integration of membrane proteins into the lipid bilayer .

How does the Sec pathway in P. aeruginosa differ from other secretion systems?

The Sec pathway in P. aeruginosa represents a two-step secretion mechanism, distinguishing it from single-step secretion systems:

The Sec pathway handles a large volume of proteins destined for the periplasm or outer membrane, whereas other systems like the type III secretion system (exemplified by PopB/PopD translocators) are specialized for injecting effector proteins directly into host cells . Unlike the Tat pathway, which translocates fully folded proteins, the Sec pathway translocates proteins in an unfolded state .

What are the current methodologies for expressing and purifying recombinant P. aeruginosa SecE?

Current methodologies for expressing and purifying recombinant P. aeruginosa SecE can be adapted from approaches used for other Sec components and membrane proteins:

• Expression system selection:

  • E. coli BL21(DE3) derivatives are commonly used for expression of P. aeruginosa proteins, as demonstrated with SecA and PE38KDEL .

  • For membrane proteins like SecE, specialized strains like C41(DE3) or C43(DE3) may provide better expression.

• Vector design:

  • pET-based vectors under T7 promoter control have proven successful for P. aeruginosa protein expression .

  • Inclusion of affinity tags (His6 or Strep-tag) facilitates purification while minimizing interference with function.

• Expression optimization:

  • Induction at OD600 of 0.6 with 0.5 mM IPTG at 37°C for 6 hours has been effective for other P. aeruginosa recombinant proteins .

  • For membrane proteins like SecE, lower temperatures (16-30°C) during induction may improve proper membrane integration.

• Membrane protein extraction:

  • Gentle cell lysis using French press or sonication in buffer containing glycerol and protease inhibitors.

  • Membrane fraction isolation via ultracentrifugation followed by detergent solubilization (typically DDM, LDAO, or C12E8).

• Purification strategy:

  • Affinity chromatography as the initial capture step.

  • Ion exchange chromatography (such as SP-Sepharose) has been effective for PaSecA purification with >98% purity .

  • Size exclusion chromatography as a final polishing step to ensure homogeneity and remove aggregates.

When implementing these methods, it's critical to verify the functional state of SecE by assessing its ability to interact with SecY and form a stable complex. Co-expression with SecY may improve stability and yield of functionally relevant SecE protein .

How can researchers effectively incorporate recombinant SecE into functional reconstitution experiments?

Functional reconstitution of recombinant SecE requires careful experimental design to maintain the protein's native activity:

• Complex reconstitution approach:

  • Co-purification with SecY and SecG partners is often necessary as SecE alone may be unstable.

  • Cell-free expression systems can provide advantages for direct incorporation into liposomes.

  • Sequential addition protocol: first incorporate SecY, followed by SecE and SecG, may improve complex formation efficiency.

• Liposome preparation:

  • Use of E. coli polar lipid extract or synthetic lipid mixtures (POPE:POPG:cardiolipin at 7:2:1 ratio).

  • Liposome size control (100-200 nm) via extrusion through polycarbonate filters.

  • Protein:lipid ratios typically between 1:1000 to 1:4000 (w/w) depending on experimental goals.

• Functional verification methods:

  • ATPase assays measuring SecA stimulation in the presence of the reconstituted complex.

  • Translocation assays using fluorescently labeled substrate proteins.

  • Patch-clamp experiments to measure channel activity of the reconstituted SecYEG complex.

• Interaction with signal sequences:

  • Crosslinking experiments with photoactivatable analogs of signal peptides.

  • Fluorescence resonance energy transfer (FRET) between labeled SecE and substrate proteins.

Successful reconstitution can be verified by comparing the ATPase activities of the SecA component when stimulated by liposomes containing the reconstituted complex, similar to the approach used for PaSecA characterization, which showed liposome-stimulated ATPase activity at approximately 50% of the level observed with E. coli SecA .

What genetic approaches are most effective for studying SecE function in P. aeruginosa?

Since SecE is essential for viability, genetic approaches must be carefully designed to study its function without compromising bacterial survival:

• Conditional expression systems:

  • Replaceable promoter systems where native secE is placed under control of an inducible promoter.

  • Dual plasmid systems: depletion of chromosomal SecE while expressing mutant variants from a plasmid.

  • CRISPR interference (CRISPRi) for tunable repression of secE expression.

• Site-directed mutagenesis strategies:

  • Alanine-scanning mutagenesis of conserved residues to identify functional domains.

  • Introduction of cysteine residues for subsequent crosslinking or labeling experiments.

  • Targeted mutations based on sequence conservation analysis across species.

• Domain swap experiments:

  • Replacement of P. aeruginosa SecE domains with homologous regions from other species to identify species-specific functions.

  • Creation of chimeric proteins to isolate functional regions.

• Reporter fusion constructs:

  • C-terminal fusions with fluorescent proteins to study localization patterns.

  • Split-protein complementation assays to study protein-protein interactions in vivo.

• Suppressor screens:

  • Isolation of intragenic or extragenic suppressors of conditional secE mutations.

  • Identification of compensatory mutations in interacting partners like SecY.

When implementing these approaches, researchers should consider the stochastic expression patterns observed with other P. aeruginosa secretion components, such as those seen with the reb cluster genes, where fluorescence microscopy revealed expression in only 0.26% of the cell population . Similar expression patterns might occur with secE under certain conditions, necessitating single-cell analysis approaches.

How does the structure-function relationship of SecE contribute to antibiotic resistance mechanisms in P. aeruginosa?

The structure-function relationship of SecE has several implications for antibiotic resistance in P. aeruginosa:

• Role in membrane protein insertion:

  • SecE contributes to the insertion of multidrug efflux pumps into the membrane.

  • Alterations in SecE function can affect the assembly and activity of these pumps.

  • The SecYEG complex facilitates the insertion of membrane proteins that modify lipopolysaccharide structure, affecting permeability to antibiotics.

• Secretion of resistance determinants:

  • The Sec pathway translocates β-lactamases and other hydrolytic enzymes to the periplasm.

  • Efficiency of SecE function directly impacts the export of these resistance proteins.

• Stress response coupling:

  • SecE functionality is linked to envelope stress responses that upregulate under antibiotic pressure.

  • Mutations affecting SecE can alter these stress responses, potentially enhancing resistance.

• Biofilm formation contributions:

  • The Sec pathway is involved in secreting proteins important for biofilm formation.

  • SecE function affects export of adhesins and matrix components that contribute to the antibiotic-resistant biofilm lifestyle of P. aeruginosa.

• Interaction with two-component systems:

  • SecE-dependent protein export influences signaling through two-component systems that regulate resistance gene expression.

Research examining these relationships would benefit from approaches similar to those used to study the PopB/PopD translocators, where purified proteins were used to examine molecular interactions and conformational changes in membrane environments . Such methodologies could reveal how SecE structure influences the translocation of specific resistance determinants.

What are the critical parameters for optimizing expression of soluble vs. membrane-integrated forms of recombinant SecE?

Optimizing expression of SecE requires different approaches depending on whether the soluble domain or the complete membrane-integrated protein is desired:

• For soluble domain expression:

  • Express only the cytoplasmic and/or periplasmic domains without the transmembrane segment.

  • Fusion partners that enhance solubility (MBP, SUMO, or thioredoxin) significantly improve yield.

  • Lower temperatures (16-20°C) and reduced inducer concentrations (0.1-0.2 mM IPTG) minimize inclusion body formation.

  • Rich media (Terrific Broth or Super Broth) with glucose supplementation improves soluble yield.

  • Lysis buffers containing 5-10% glycerol and mild detergents (0.1% Triton X-100) enhance recovery.

• For membrane-integrated SecE:

  • Specialized E. coli strains (C41/C43, Lemo21) provide better membrane protein expression.

  • Slower induction protocols: grow to higher density (OD600 ≥0.8) before adding reduced IPTG concentrations (0.1-0.5 mM).

  • Post-induction temperature reduction to 20-30°C improves membrane integration.

  • Addition of membrane-fluidizing agents (benzyl alcohol at 10 mM) can improve membrane protein yield.

  • Extraction requires careful detergent selection: initial screening of DDM, LDAO, and C12E8 at concentrations just above CMC.

What analytical techniques are most informative for assessing SecE interactions with SecY and SecA?

Multiple complementary techniques provide comprehensive insights into SecE interactions:

• In vitro biochemical approaches:

  • Co-immunoprecipitation with antibodies against specific components

  • Pull-down assays using differentially tagged components

  • Size exclusion chromatography to analyze complex formation

  • Surface plasmon resonance for binding kinetics determination

  • Isothermal titration calorimetry for thermodynamic parameters

  • Blue native PAGE for native complex visualization

• Structural biology techniques:

  • Cryo-electron microscopy of purified complexes

  • X-ray crystallography (challenging but potentially informative)

  • NMR spectroscopy for dynamics studies of soluble domains

  • Hydrogen-deuterium exchange mass spectrometry to map interaction interfaces

• Biophysical techniques:

  • Site-directed fluorescence labeling with environment-sensitive probes (similar to the approach used with PopD)

  • Fluorescence resonance energy transfer (FRET) between labeled components

  • Crosslinking coupled with mass spectrometry (XL-MS) to map interaction sites

  • Single-molecule techniques to observe dynamic assembly/disassembly

• In vivo approaches:

  • Bacterial two-hybrid systems

  • Split fluorescent protein complementation

  • FRET in live cells with fluorescently tagged components

  • In vivo crosslinking using photo-activatable unnatural amino acids

When implementing these methods, researchers should consider the approaches used to study PopB/PopD interactions, where site-directed fluorescence labeling with environment-sensitive probes revealed conformational changes upon membrane insertion . Similar techniques could elucidate how SecE conformation changes when interacting with SecY and SecA in membrane environments.

How can researchers effectively design experiments to study the role of SecE in virulence factor secretion?

Designing experiments to study SecE's role in virulence factor secretion requires multi-layered approaches:

• Genetic manipulation strategies:

  • Construction of conditional secE mutants using inducible promoters

  • Site-directed mutagenesis targeting conserved residues

  • Creation of SecE variants with altered interaction capabilities

  • Development of reporter strains where secreted virulence factors are tagged

• Secretome analysis protocols:

  • Quantitative proteomics comparing wild-type vs. SecE-depleted conditions

  • Pulse-chase experiments with radioisotope labeling to track specific virulence factors

  • Western blot analysis of specific marker proteins in cellular vs. secreted fractions

  • Activity assays for secreted enzymes (e.g., elastase, lipase, alkaline phosphatase)

• In vitro reconstitution approaches:

  • Purification of SecYEG complexes containing wild-type or mutant SecE

  • Reconstitution into liposomes or nanodiscs

  • In vitro translocation assays with fluorescently labeled virulence factor precursors

  • Direct measurement of translocation efficiency for specific virulence factors

• Infection model designs:

  • Cell culture infection models with SecE-depleted P. aeruginosa

  • Selective inhibition of SecE during different infection stages

  • Tracking of specific virulence factor delivery using fluorescent tags

  • Quantification of host cell responses to infection under varying SecE conditions

These methodologies should be integrated with approaches similar to those used to study other P. aeruginosa secretion systems. For example, researchers studying the type III secretion system used fluorescence microscopy to track translocator proteins in host cell membranes , and similar approaches could be adapted to study Sec-dependent virulence factors. Additionally, methods used to study reb gene expression during host colonization could be applied to study secE expression patterns during infection .

What computational tools and databases are most valuable for P. aeruginosa SecE research?

Researchers studying P. aeruginosa SecE can leverage multiple computational resources:

• Sequence analysis tools:

  • Pseudomonas Genome Database (pseudomonas.com) - comprehensive genomic information including secE conservation across 241+ P. aeruginosa strains

  • BLAST and HMMER for homology searches

  • Multiple sequence alignment tools (MUSCLE, T-Coffee, MAFFT) for evolutionary analysis

  • ConSurf for conservation mapping onto structural models

  • SignalP and TMHMM for signal sequence and transmembrane domain prediction

• Structural analysis resources:

  • AlphaFold and RoseTTAFold for protein structure prediction

  • SWISS-MODEL for homology modeling based on crystallized SecE homologs

  • Molecular dynamics simulation packages (GROMACS, NAMD) for membrane protein dynamics

  • HADDOCK or ClusPro for protein-protein docking predictions

  • Molecular visualization tools (PyMOL, UCSF Chimera) for structural analysis

• Expression and purification design tools:

  • OptimumGene™ for codon optimization

  • ExPASy tools for calculating physical and chemical parameters

  • PROSO II for solubility prediction

  • ProtParam for sequence-based property calculation

• Systems biology resources:

  • STRING database for protein-protein interaction networks

  • KEGG for metabolic pathway analysis

  • Virtual Footprint for promoter analysis and regulation prediction

  • BioCyc for P. aeruginosa pathway information

• Specialized Pseudomonas databases:

  • Pseudomonas Genome Database for strain variation analysis

  • Pseudomonas aeruginosa Secretome Database (currently not available publicly)

  • PseudomonasNet for gene expression data integration

These resources can be particularly valuable when analyzing strain-dependent variations in secE, similar to the approach used to analyze the strain-dependent variation in reb cluster genes, which were found in 147 out of 241 P. aeruginosa complete strain genomes in the Pseudomonas Genome Database .

What are the future research directions for P. aeruginosa SecE studies?

Several promising research directions will advance our understanding of P. aeruginosa SecE:

• Structural biology advances:

  • Cryo-EM structures of the complete P. aeruginosa SecYEG translocon

  • Capturing different conformational states during the translocation cycle

  • Structural determination of species-specific features distinguishing P. aeruginosa SecE from homologs

• Systems-level understanding:

  • Comprehensive secretome analysis under varying conditions

  • Global mapping of SecE genetic interactions through transposon sequencing

  • Quantitative models of secretion capacity and its impact on virulence

• Translational applications:

  • Development of SecE-targeting antimicrobial peptides

  • Small molecule inhibitors specific to P. aeruginosa SecYEG

  • Vaccines targeting surface-exposed domains of SecE

• Host-pathogen interface:

  • SecE role in immune response evasion

  • Impact of host factors on Sec-dependent secretion

  • Temporal dynamics of SecE activity during infection progression

• Technological innovations:

  • Single-molecule tracking of SecE in living bacteria

  • Optogenetic control of SecE function

  • CRISPR-based screening for SecE functional domains

These directions build upon current understanding of P. aeruginosa secretion systems while incorporating emerging technologies. They parallel the research trajectory seen with other secretion components such as PopB/PopD translocators, where detailed molecular mechanisms were elucidated through increasingly sophisticated structural and functional analyses .

How does SecE contribute to the broader understanding of bacterial protein secretion mechanisms?

SecE's contribution to bacterial protein secretion extends beyond its specific role in P. aeruginosa:

• Evolutionary perspectives:

  • SecE represents one of the most conserved components of bacterial protein export

  • Comparison of P. aeruginosa SecE with homologs provides insights into adaptation strategies

  • Analysis of co-evolution patterns between SecE and other Sec components reveals functional constraints

• Mechanistic insights:

  • P. aeruginosa SecE studies help elucidate the fundamental principles of protein translocation

  • The interface between SecE and SecY forms a critical lateral gate for membrane protein insertion

  • SecE stabilization of the translocon represents a universal feature that prevents premature channel collapse

• Bacterial physiology implications:

  • SecE functionality connects protein export to broader cellular processes including stress responses

  • The essential nature of SecE highlights the central importance of protein secretion to bacterial survival

  • Integration of SecE activity with other secretion pathways demonstrates the interconnected nature of bacterial protein export

• Comparative secretion system biology:

  • SecE studies provide contrast to specialized secretion systems like the T3SS

  • Understanding the two-step secretion process involving SecE helps differentiate it from single-step systems

  • The universality of SecE across bacteria versus the specialized distribution of systems like T3SS highlights evolutionary strategies