Recombinant Pseudomonas putida Chaperone surA (surA)

What is Pseudomonas putida SurA and what is its primary function?

SurA (Survival protein A) is a periplasmic chaperone in Pseudomonas putida that plays a crucial role in outer membrane protein (OMP) biogenesis. In P. putida KT2440 strain, SurA is a 427 amino acid protein with a molecular mass of approximately 47.6 kDa . The primary function of SurA is to facilitate the correct folding and assembly of outer membrane proteins by recognizing specific patterns of aromatic residues and their side chain orientations, which are found more frequently in integral outer membrane proteins . SurA acts in both early periplasmic and late outer membrane-associated steps of protein maturation, serving as a key component of the cellular machinery that maintains outer membrane integrity.

How does the structure of SurA relate to its chaperone function?

The function of SurA as a chaperone is directly related to its structural domains. The SurA protein contains specific binding regions that recognize and interact with unfolded or partially folded outer membrane proteins. Research indicates that SurA specifically recognizes aromatic-rich sequences in these proteins, which are common motifs in outer membrane beta-barrel proteins . This recognition capability allows SurA to selectively bind to outer membrane proteins and assist in their proper folding and delivery to the β-barrel assembly machinery (BAM) complex in the outer membrane, where they are subsequently integrated into the membrane structure .

How does P. putida SurA compare to homologs in other Pseudomonas species?

While the core function of SurA is conserved across Pseudomonas species, studies have revealed some differences in its importance and regulation between species. For example:

In P. aeruginosa, SurA depletion results in profound effects on both outer membrane integrity and virulence, characterized by increased membrane permeability, enhanced sensitivity to antibiotic treatment, and attenuation of virulence in infection models . This makes P. aeruginosa SurA a potential target for developing novel anti-infective drugs that could re-sensitize multidrug-resistant strains to antibiotics .

What are the standard methods for cloning and expressing recombinant P. putida SurA?

For cloning and expressing recombinant P. putida SurA, researchers typically employ the following methods:

• Gene amplification: PCR amplification of the surA gene (e.g., from P. putida strain KT2440) using specific primers designed to include appropriate restriction sites for subsequent cloning .

• Cloning strategies: The amplified surA gene can be cloned into suitable expression vectors. Researchers often use yeast recombinational cloning (yTREX) systems or traditional restriction enzyme-based cloning into vectors such as pMBL-T or pK18mobsacB .

• Expression systems: For high-level expression, E. coli-based expression systems (such as BL21(DE3)) are commonly used, though expression in P. putida itself may be preferred for certain applications .

• Purification methods: Histidine-tagged SurA constructs allow for purification using nickel affinity chromatography, followed by size exclusion chromatography to ensure high purity .

• Verification: Western blot analysis using anti-SurA antibodies is commonly employed to verify expression and purification .

How can researchers create and validate conditional surA mutants in P. putida?

Creating conditional surA mutants in P. putida involves several sophisticated genetic engineering approaches:

• Arabinose-inducible systems: One effective approach is to place the surA gene under the control of an arabinose-inducible promoter (PBAD), allowing for controlled expression by adding or removing arabinose from the growth medium .

• Construction steps:

  • Replace the native surA promoter with the PBAD promoter

  • Include an appropriate ribosome binding site

  • Incorporate the araC regulator gene upstream of the promoter

  • Use homologous recombination for chromosomal integration

• Validation methods:

  • Western blot analysis to confirm SurA depletion in the absence of arabinose and restoration in its presence

  • Growth curve analysis to demonstrate the phenotypic effect of SurA depletion

  • Complementation experiments to confirm that observed phenotypes are specifically due to SurA loss

• Considerations: When working with conditional mutants, researchers must carefully optimize arabinose concentrations to achieve appropriate levels of expression while minimizing potential metabolic effects of the inducer .

Research by Thoma et al. (2019) demonstrated that in their conditional P. aeruginosa surA mutant system, depletion of SurA resulted in significant growth defects, while addition of arabinose restored SurA levels to approximately 64% of wild-type levels, partially rescuing the growth phenotype .

What phenotypic assays are most informative when studying SurA function in P. putida?

The most informative phenotypic assays for studying SurA function in P. putida include:

• Membrane integrity assays:

  • Outer membrane permeability assays using hydrophobic dyes or antibiotics

  • Sensitivity to detergents (e.g., SDS) and bile salts that target membrane integrity

• Antibiotic susceptibility testing:

  • Minimum inhibitory concentration (MIC) determination

  • Antibiotic disk diffusion assays

  • Time-kill assays with various antibiotics

• Stress response assays:

  • Growth under various environmental stressors (temperature, pH, oxidative stress)

  • Adaptation to toxic compounds such as aromatics (p-coumaric acid, ferulate)

• Proteomic analysis:

  • Mass spectrometry to identify changes in outer membrane protein composition

  • Western blotting to monitor specific outer membrane proteins dependent on SurA

• Virulence assays:

  • Infection models (e.g., Galleria mellonella infection model)

  • Serum resistance assays to measure complement sensitivity

For example, research on P. aeruginosa showed that SurA depletion led to significantly increased sensitivity to human serum, with rapid killing of SurA-depleted bacteria compared to wild-type strains in complement activity assays .

How does SurA depletion affect outer membrane protein composition in P. putida?

SurA depletion significantly alters outer membrane protein (OMP) composition in Pseudomonas species. While specific P. putida data is limited, studies in related Pseudomonas species provide valuable insights that are likely applicable:

• Specific OMP reductions: SurA depletion results in significant reductions in multiple OMPs. In P. aeruginosa, proteomics analysis revealed substantial decreases in various porins and other OMPs, including:

  • OprD (reduced to ~15% of wild-type levels)

  • PlpD (reduced to ~24% of wild-type levels)

  • Siderophore receptors like FpvA, FiuA, and FecA

• Functional consequences:

  • Decreased iron uptake capability (due to reduced siderophore receptors)

  • Increased membrane permeability

  • Altered antibiotic resistance profiles

  • Compromised stress response mechanisms

• Compensatory mechanisms: In some cases, other periplasmic chaperones like Skp/HlpA may partially compensate for SurA deficiency, though this compensation appears to be incomplete in Pseudomonas species .

These findings suggest that SurA plays a critical and non-redundant role in OMP biogenesis in Pseudomonas species, making it essential for proper outer membrane composition and function.

How can researchers leverage SurA for biotechnological applications in P. putida?

P. putida has emerged as a promising chassis for various biotechnological applications due to its metabolic versatility and robustness. SurA can be leveraged in several innovative ways:

• Surface display systems: SurA's role in OMP assembly can be exploited to develop efficient surface display systems for heterologous proteins in P. putida, enabling applications such as:

  • Whole-cell biocatalysts

  • Biosensor development

  • Vaccine antigen presentation

• Strain engineering for improved stress tolerance:

  • Modulating SurA expression can potentially enhance P. putida's tolerance to toxic compounds and harsh industrial conditions

  • Particularly relevant for applications in bioremediation and production of toxic chemicals

• Enhanced heterologous protein secretion:

  • Optimizing the periplasmic folding machinery, including SurA, can improve the secretion and correct folding of recombinant proteins

• Synthetic biology applications:

  • Integration with systems like yeast recombinational cloning-enabled pathway transfer and expression tool (yTREX) for development of novel secondary metabolite production strains

  • Construction of synthetic microbial consortia with specialized functions

• Metabolic engineering platforms:

  • As shown in the genome-scale metabolic reconstructions of P. putida, proper functioning of membrane proteins (dependent on SurA) is critical for many industrial biotechnology applications

Research by Mohamed et al. demonstrated that stress responses in P. putida, which involve chaperones like SurA, are critical for adaptation to industrial conditions such as exposure to toxic aromatic compounds during bioproduction processes .

What is the relationship between SurA and antibiotic resistance in P. putida compared to pathogenic Pseudomonas species?

The relationship between SurA and antibiotic resistance differs significantly between P. putida and pathogenic Pseudomonas species:

In P. aeruginosa, SurA depletion has profound effects on antibiotic resistance:

• Re-sensitization effect: Depletion of SurA in multidrug-resistant clinical bloodstream isolates re-sensitized the strains to antibiotic treatment, suggesting SurA as a promising target for developing drugs that show anti-infective activity .

• Membrane permeability: SurA depletion increases outer membrane permeability, enhancing antibiotic entry into bacterial cells .

• Porin composition: Changes in outer membrane protein composition, particularly porins, alter the uptake of antibiotics across the membrane .

In contrast, while P. putida can exhibit multidrug resistance (76% of isolates in one study showed multidrug resistance), the direct relationship between SurA and this resistance is less well-characterized . Unlike P. aeruginosa, P. putida is not primarily a human pathogen, though emerging evidence suggests it can cause opportunistic infections in healthcare settings and may serve as a reservoir of resistance genes .

What are the critical factors for successful purification of functional recombinant P. putida SurA?

Successful purification of functional recombinant P. putida SurA requires attention to several critical factors:

• Expression system selection:

  • E. coli BL21(DE3) is commonly used for high-level expression

  • Consider using P. putida-based expression systems for proper post-translational modifications

  • Temperature-inducible or IPTG-inducible systems allow controlled expression

• Optimization of expression conditions:

  • Lower induction temperatures (16-25°C) often improve SurA folding and solubility

  • Induction time and inducer concentration should be optimized to maximize yield while minimizing inclusion body formation

  • Rich media (such as TB or 2xYT) typically yield higher protein amounts

• Solubility considerations:

  • Addition of fusion tags (His6, MBP, SUMO) can improve solubility

  • Co-expression with periplasmic folding factors may enhance proper folding

  • Periplasmic extraction protocols often yield better results than whole-cell lysis

• Purification strategy:

  • Two-step purification (affinity chromatography followed by size exclusion) is recommended

  • Careful buffer selection is crucial (typically phosphate or Tris-based buffers at pH 7.5-8.0)

  • Include stabilizing agents such as glycerol (10%) and reducing agents (1-5 mM DTT or 0.5-1 mM TCEP)

• Quality control:

  • Size exclusion chromatography to verify monomeric state

  • Circular dichroism to confirm proper secondary structure

  • Functional assays to verify chaperone activity

When designing expression constructs, researchers should consider whether to include or exclude the signal sequence, as this affects cellular localization and may impact protein folding and purification strategies.

How can researchers analyze the interaction between SurA and its client outer membrane proteins?

Analyzing interactions between SurA and client outer membrane proteins requires specialized techniques:

• In vitro binding assays:

  • Surface plasmon resonance (SPR) to determine binding kinetics and affinity

  • Isothermal titration calorimetry (ITC) for thermodynamic parameters of binding

  • Fluorescence-based assays using labeled client proteins or peptides

• Pull-down and co-immunoprecipitation approaches:

  • SurA-specific antibodies or tagged versions of SurA can be used to pull down interacting partners

  • Mass spectrometry to identify co-purifying client proteins

  • Western blotting to confirm specific interactions with known client proteins

• Crosslinking methods:

  • Chemical crosslinking followed by mass spectrometry (XL-MS) to capture transient interactions

  • Photo-activatable crosslinkers incorporated into SurA or client proteins

  • In vivo crosslinking to capture physiologically relevant interactions

• Structural biology approaches:

  • X-ray crystallography of SurA-peptide complexes

  • Cryo-electron microscopy for larger complexes

  • NMR studies for mapping interaction interfaces

• Computational methods:

  • Molecular docking to predict binding interfaces

  • Molecular dynamics simulations to study the dynamics of interactions

  • Bioinformatics approaches to identify potential binding motifs in client proteins

For example, research has shown that SurA specifically recognizes patterns of aromatic residues that are commonly found in integral outer membrane proteins, with the orientation of side chains being an important determinant of recognition specificity .

What are the best experimental approaches to study the role of SurA in P. putida adaptation to environmental stresses?

To study SurA's role in P. putida adaptation to environmental stresses, researchers can employ these experimental approaches:

• Tolerance adaptive laboratory evolution (TALE):

  • Evolve P. putida strains under increasing levels of stress conditions

  • Compare wild-type and surA mutant adaptation trajectories

  • Sequence evolved populations to identify compensatory mutations

  Mohamed et al. employed TALE to study P. putida adaptation to aromatic acids, finding that chaperone induction (including SurA) was part of the stress response .

• Transcriptomic and proteomic profiling:

  • RNA-seq to identify differentially expressed genes in response to stress

  • Proteomics to identify changes in protein abundance, particularly OMPs

  • Compare wild-type and surA mutant profiles to identify SurA-dependent stress responses

• Physiological characterization:

  • Growth measurements under various stress conditions

  • Viability assays (e.g., colony forming unit counts, live/dead staining)

  • Metabolic activity measurements (e.g., respiration rates, ATP levels)

• Membrane integrity assays under stress conditions:

  • Fluorescent dye uptake to assess membrane permeability changes

  • Atomic force microscopy to visualize membrane structural changes

  • Lipidomics to detect stress-induced changes in membrane composition

• Combined environmental stressors:

  • Test adaptation to multiple simultaneous stresses (e.g., temperature + toxic compounds)

  • Investigate hierarchical stress responses and the role of SurA in prioritizing responses

Research by Jensen et al. showed that P. putida strains experience complex adaptations to industrial fermentation conditions, with membrane protein function and proper folding (dependent on chaperones like SurA) being critical for robust biomanufacturing processes .

What is the significance of SurA in P. putida-associated infections and antimicrobial resistance?

While P. putida has traditionally been considered non-pathogenic, emerging evidence highlights its role in opportunistic infections and antimicrobial resistance:

• Clinical significance:

  • P. putida can cause various infections, including bloodstream infections, skin and soft tissue infections, and pneumonia

  • Case fatality rates can be significant; one study reported a lethal case of P. putida bacteremia due to soft tissue infection

  • Risk factors include immunocompromised state, malnutrition, and indwelling medical devices

• Antimicrobial resistance profiles:

  • P. putida clinical isolates often show multidrug resistance patterns

  • A recent study found 76% of P. putida isolates exhibited multidrug resistance (MDR, resistance to ≥3 antibiotics)

  • Particularly high resistance to oxacillin, ampicillin, nalidixic acid, and aztreonam

  • Highest susceptibility reported against imipenem

• Role of SurA in resistance and virulence:

  • Based on studies in related Pseudomonas species, SurA likely plays a role in maintaining outer membrane integrity

  • This membrane integrity is crucial for intrinsic antibiotic resistance

  • SurA-dependent outer membrane proteins may contribute to virulence factors and resistance mechanisms

• Genomic evidence:

  • Genome analysis of multidrug-resistant P. putida strains has revealed various antibiotic resistance genes (ARGs) and virulence factor genes (VFGs)

  • These include efflux pumps, biofilm formation factors, adhesins, secreted toxins, and lipopolysaccharides

  • The proper assembly of many of these factors likely depends on SurA chaperone function

Understanding SurA's role in P. putida infections could potentially inform new therapeutic approaches, similar to the strategy proposed for P. aeruginosa where SurA has been identified as a promising target for developing drugs that show anti-infective activity and re-sensitize multidrug-resistant strains to antibiotics .

How does SurA contribute to P. putida biofilm formation and what are the implications for bioremediation applications?

SurA's contribution to P. putida biofilm formation has important implications for both clinical and environmental applications:

• Role in biofilm formation:

  • SurA ensures proper assembly of outer membrane proteins essential for initial surface attachment

  • It facilitates the expression and proper folding of adhesins and other biofilm-related surface proteins

  • By maintaining outer membrane integrity, SurA supports cellular stress responses necessary during biofilm development

• Bioremediation implications:

  • P. putida is widely used in bioremediation of soil and water contaminants

  • Biofilm formation enhances bioremediation efficiency by:

    • Increasing local cell density at contamination sites

    • Providing protection against toxic compounds

    • Facilitating horizontal gene transfer of degradative pathways

    • Creating microenvironments that optimize degradation conditions

• Engineering considerations:

  • Modulating SurA expression could potentially enhance biofilm formation for improved bioremediation

  • Understanding SurA's role could lead to engineering strains with enhanced survival in contaminated environments

  • P. putida's ability to form biofilms on various surfaces makes it valuable for immobilized biocatalyst applications

• Stress adaptation in biofilms:

  • SurA plays a role in adaptation to environmental stressors commonly encountered in contaminated sites

  • Research shows that chaperones including SurA are induced in response to aromatic compounds like p-coumaric acid and ferulate

  • This adaptive response is crucial for maintaining cellular function during bioremediation of aromatic pollutants

Research by Mohamed et al. demonstrated that P. putida strains evolved under aromatic acid stress showed mutations affecting membrane-associated functions, highlighting the importance of membrane integrity (mediated in part by SurA) in adaptation to pollutant-contaminated environments .

Can SurA be leveraged as a target for developing antimicrobial strategies against pathogenic Pseudomonas species?

Research suggests SurA is a promising antimicrobial target, particularly for pathogenic Pseudomonas species:

• Target validation evidence:

  • In P. aeruginosa, SurA depletion resulted in:

    • Increased membrane permeability

    • Enhanced sensitivity to antibiotic treatment

    • Attenuation of virulence in infection models

    • Re-sensitization of multidrug-resistant strains to antibiotics

• Advantages as a drug target:

  • Located in the periplasm, making it more accessible to inhibitors compared to cytoplasmic targets

  • Essential for virulence but not strictly essential for viability under all conditions

  • Inhibition produces a multi-faceted effect (compromised membrane, reduced virulence, increased antibiotic sensitivity)

  • No human homolog, reducing potential toxicity concerns

• Potential inhibition strategies:

  • Small molecule inhibitors that disrupt SurA chaperone function

  • Peptide mimetics that compete with client proteins for SurA binding

  • Compounds that destabilize SurA structure or promote its degradation

  • Antibody-antibiotic conjugates targeting the periplasmic space

• Challenges and considerations:

  • Need for compounds that can penetrate the outer membrane to reach the periplasmic target

  • Potential for development of resistance through compensatory mechanisms

  • Species-specific differences in SurA structure and function may require tailored approaches