Recombinant Pseudomonas putida Probable septum site-determining protein MinC (minC)

What is the MinC protein in Pseudomonas putida and how does it relate to bacterial cell division?

MinC is a critical cell division inhibitor in Pseudomonas putida that, together with MinD and MinE proteins, constitutes the Min system responsible for accurate selection of the division site at midcell. MinC functions by directly interacting with FtsZ, the bacterial tubulin homologue, to prevent inappropriate Z-ring formation at cell poles. The Min system ensures that division occurs precisely at the midcell position rather than at potential division sites near the cell poles .

In P. putida KT2440, the minCDE genes form a cluster in the PP_1732-PP_1734 locus, with the minC gene (encoding the septum site-determining protein) positioned as the last element in this sequence . The proper functioning of this system is essential for normal rod-shaped cell morphology and symmetric cell division.

How does the MinC protein in P. putida differ from MinC in model organisms like E. coli?

While the Min system is broadly conserved across bacterial species including both E. coli and P. putida, there are notable differences in protein behavior and structural characteristics. The fundamental function of preventing polar division remains consistent, but P. putida MinC exhibits unique characteristics aligned with this organism's environmental versatility and stress tolerance.

Studies have primarily focused on MinC from E. coli and Thermotoga maritima for structural analyses due to better protein behavior in vitro. T. maritima MinC has proven more amenable to crystallization and structural studies compared to the E. coli ortholog, which tends to behave poorly in structural investigations . Although specific P. putida MinC structural data is more limited, its functional properties appear to be conserved within the context of P. putida's robust cellular physiology and adaptability to various environmental stresses.

What genomic context surrounds the minC gene in P. putida and how is it regulated?

In P. putida KT2440, the minC gene is located within the minCDE operon (PP_1732-PP_1734), typical of the genomic organization in many Gram-negative bacteria. The minC gene specifically corresponds to locus PP_1734, with minD and minE preceding it in the operon . This genomic arrangement ensures coordinated expression of all three components of the Min system.

Regulation of the minCDE operon in P. putida likely involves multiple layers of control, including transcriptional regulation responsive to cell cycle cues and growth conditions. The precise regulatory mechanisms in P. putida have not been fully characterized compared to model organisms, presenting opportunities for further research into strain-specific regulatory patterns particularly relevant for recombinant expression systems.

What is the domain organization of MinC protein and how does it relate to function?

MinC protein consists of two distinct domains separated by a short linker region, each with specialized functions in cell division inhibition:

• N-terminal domain: Responsible for direct interaction with FtsZ and inhibition of FtsZ polymerization

• C-terminal domain: Forms a right-handed β-helix structure involved in dimerization and interaction with MinD

The crystal structure of MinC (determined from Thermotoga maritima) reveals that the protein exists as a tight dimer with an unusually large dimer interface approximately 32% of the C-terminal domain's surface area. This extensive interface contributes to the remarkable stability of the dimer even under conditions of extreme ionic strength . The structural flexibility between the two domains, demonstrated by different dimer conformations observed in crystal structures, likely facilitates the protein's ability to interact with both FtsZ and MinD simultaneously.

How can researchers effectively express and purify recombinant P. putida MinC for structural studies?

Expression and purification of recombinant P. putida MinC presents several challenges due to potential protein instability or poor solubility. Based on experiences with MinC from other bacterial species, the following methodological approach is recommended:

Expression strategy:

• Consider using a fusion protein approach (e.g., MalE-MinC fusion) as previously employed for E. coli MinC

• Express in a thermophilic host system such as T. maritima, which has proven more amenable to structural studies

• Optimize expression conditions using lower temperatures (16-20°C) during induction to enhance proper folding

Purification protocol:

• Employ immobilized metal affinity chromatography (IMAC) with a histidine tag

• Include size exclusion chromatography to separate dimeric from aggregated forms

• Maintain stabilizing buffer conditions (typically including 10% glycerol and reducing agents)

Researchers should note that MinC dimers demonstrate remarkable stability under various ionic strength conditions, which can be leveraged during purification protocols to maintain protein integrity.

What advanced biophysical techniques are most informative for studying P. putida MinC structural dynamics?

Understanding the structural dynamics of MinC requires a combination of complementary biophysical approaches:

These techniques provide complementary insights into MinC's structural adaptability. The crystal structure of MinC from T. maritima revealed two different MinC dimers in the same crystal, highlighting flexibility in the linker region between domains . This structural plasticity likely plays a functional role in MinC's ability to regulate cell division through interactions with both MinD and FtsZ.

How does MinC interact with FtsZ to inhibit inappropriate cell division?

MinC inhibits cell division through direct interaction with FtsZ, but unlike other FtsZ inhibitors such as SulA, it does not target the GTPase activity of FtsZ. Instead, MinC appears to select for the FtsZ polymer rather than the monomer form . The inhibitory mechanism involves:

• N-terminal domain of MinC binds to the C-terminal tail of FtsZ

• This interaction prevents lateral interactions between FtsZ protofilaments

• MinC, when complexed with MinD, becomes concentrated at the cell membrane

• The MinCD complex destabilizes FtsZ polymers at the membrane surface, preventing Z-ring formation at inappropriate locations

This selective targeting of FtsZ polymers rather than monomers allows MinC to regulate Z-ring positioning without completely preventing FtsZ assembly, thereby ensuring that division occurs only at the proper midcell location.

What phenotypic changes occur in P. putida when MinC function is disrupted?

Disruption of MinC function in P. putida leads to characteristic phenotypic alterations in cell morphology and division patterns:

• Formation of minicells - achromosomal, nanosized (100-400 nm diameter) cells resulting from polar division events

• Asymmetrical cell division with variable cell sizes

• Occasional filamentation due to complete inhibition of cell division

• Irregular placement of division septa throughout the cell length

While specific disruption of MinC has not been extensively characterized in P. putida, studies with MinD disruption in P. putida SEM1.3 demonstrate that interference with the Min system leads to asymmetrical, abortive division resulting in minicell formation . These minicells, though achromosomal, retain plasmid DNA and metabolic activity, making them potentially useful for biotechnological applications.

How do environmental conditions affect MinC function in P. putida compared to other bacteria?

P. putida's native adaptation to diverse environmental conditions may influence MinC function in ways distinct from model organisms. Key considerations include:

• Temperature adaptations - P. putida thrives in moderate temperatures compared to thermophiles like T. maritima or mesophiles like E. coli

• Stress response integration - P. putida's remarkable stress tolerance may involve modified Min system dynamics

• Metabolic versatility - The opportunistic and undemanding nutritional capabilities of P. putida may affect cell division regulation under different nutrient conditions

P. putida's inherent resilience to harsh environments, including polluted sites with toxic chemicals , suggests its cell division machinery, including the Min system, may have evolved specific adaptations to maintain function under stress conditions. These adaptations could manifest as altered protein stability, interaction dynamics, or regulatory mechanisms compared to MinC in other bacterial species.

What are the most effective genetic tools for manipulating minC in P. putida?

Several advanced genetic engineering approaches have been developed for P. putida that can be effectively applied to minC manipulation:

• SEVA platform vectors - The Standard European Vector Architecture provides modular vectors specifically optimized for P. putida that facilitate flexible genetic constructs for minC studies

• Transposon-based systems:

  • Mini-Tn5 vectors for random insertion

  • Mini-Tn7 vectors for site-specific insertion into the genome

• Homologous recombination techniques:

  • The pEMG system using I-SceI homing endonuclease for precise genomic modifications

  • Self-curing helper plasmids to streamline the process

• CRISPR/Cas9 technologies:

  • CRISPR interference (CRISPRi) for transcriptional repression of minC

  • Direct genome editing for precise modifications

Recent innovations in P. putida genetic engineering include thermoinducible recombineering systems and RecET-based markerless recombineering for large gene cluster manipulations . These advanced tools allow for precise control over minC expression, localization, and function.

How can researchers design effective fluorescent protein fusions to study MinC localization dynamics?

Designing fluorescent protein fusions to study MinC localization dynamics requires careful consideration of several factors:

Fusion design considerations:

• Terminal selection - C-terminal fusions typically preserve MinC function better than N-terminal fusions due to critical FtsZ-interacting regions in the N-terminal domain

• Linker optimization - Flexible linkers (e.g., GGGGS repeats) help maintain independent folding and function of both domains

• Fluorescent protein selection - msfGFP or mNeonGreen provide superior brightness and photostability in bacterial systems

Expression strategy:

• Chromosomal integration at the native locus preserves physiological expression levels

• Inducible systems allow titration of expression to avoid artifacts from overexpression

• Dual-color imaging with FtsZ-RFP provides valuable colocalization data

To validate functionality of MinC fusions, researchers should confirm that cell morphology, division patterns, and growth rates remain comparable to wild-type cells. Time-lapse microscopy with optimized acquisition parameters (minimizing phototoxicity while maintaining temporal resolution) is essential for capturing the dynamic oscillation patterns of MinC.

What strategies can be employed to create conditional minC mutants in P. putida?

Creating conditional minC mutants in P. putida is critical for studying essential gene functions without lethal consequences. Several sophisticated approaches are available:

• Inducible expression systems:

  • The XylS/Pm expression system responsive to m-toluic acid

  • Tetracycline-responsive systems adapted for P. putida

  • Thermoinducible systems for temperature-controlled expression

• Degron-based approaches:

  • N-terminal degron tags for proteolysis control

  • Auxin-inducible degron systems adapted for bacterial use

• CRISPRi-based transcriptional control:

  • Target minC promoter or early coding sequence

  • Use dCas9 without nuclease activity to block transcription

  • Employ tunable inducible promoters for dCas9 expression

• Protein complementation approaches:

  • Split protein systems where function is restored upon addition of a small molecule

  • Chemical rescue of inactive point mutants

When designing conditional systems, researchers should carefully validate the degree of MinC depletion using quantitative proteomics or western blotting, and characterize the resulting phenotypes across different growth conditions relevant to P. putida's natural physiology.

How can engineered MinC variants be used to generate P. putida minicells for biotechnological applications?

Engineered MinC variants offer precise control over minicell formation in P. putida, enabling the development of specialized biotechnological applications:

The strategic manipulation of MinC function, particularly in conjunction with MinD, allows for the production of minicells - achromosomal, nanosized bacterial derivatives that retain metabolic activity but cannot grow or duplicate . These minicells have several advantageous properties:

• Retention of plasmid DNA content (~901 ± 19 ng/mg CDW, approximately 20% higher than parental cells)

• Ability to perform various cellular processes including ATP synthesis and protein translation

• Enhanced tolerance to toxic compounds compared to whole cells

• Capacity for bioproduction of short-chain ketones and other compounds

Experimental approaches for minicell production involve either MinC mutation/deletion or transcriptional interference of minD using CRISPRi systems. The resulting minicells can be harvested through a three-step sequential centrifugation protocol, yielding approximately 0.25 g cell dry weight per liter of minicells from a 1.5 g/L culture .

What insights can MinC research provide for understanding bacterial stress responses in P. putida?

MinC research intersects with broader understanding of bacterial stress responses in P. putida through several mechanistic connections:

• Cell division-stress coordination:

  • MinC function may be modulated during stress to adjust division timing

  • Stress-induced changes in membrane composition potentially affect MinCD membrane binding

• Metabolic state integration:

  • P. putida's versatile metabolism may signal to cell division machinery

  • Energy status (ATP/ADP ratios) could impact MinD function and consequently MinC localization

• Environmental adaptation mechanisms:

  • P. putida's remarkable tolerance to harsh environments and toxic chemicals may involve specialized adaptations in its cell division machinery

  • MinC dynamics might differ under various stress conditions (oxidative stress, solvent exposure)

Studying MinC under different stress conditions relevant to P. putida's environmental niches can provide fundamental insights into how cell division regulation is integrated with stress adaptation mechanisms. This understanding is particularly relevant given P. putida's applications in bioremediation of polluted environments and industrial biotechnology.

How does MinC function relate to antimicrobial resistance mechanisms in P. putida?

The relationship between MinC function and antimicrobial resistance in P. putida presents a complex and largely unexplored research frontier:

P. putida strains serve as environmental reservoirs of antimicrobial resistance, particularly to β-lactamic antibiotics . The connection between cell division proteins like MinC and antimicrobial resistance mechanisms may involve:

• Cell wall synthesis coordination:

  • MinC's role in division site selection indirectly affects localization of cell wall synthesis machinery

  • β-lactams target cell wall synthesis, creating potential functional interactions

• Stress response pathways:

  • Antimicrobial exposure triggers stress responses that may affect MinC regulation

  • Altered MinC function could contribute to adaptive responses

• Physiological state determination:

  • Division rate changes mediated by MinC may influence susceptibility to antibiotics

  • Different growth states show varying resistance profiles

Research examining MinC function in P. putida isolates with different antimicrobial resistance profiles could reveal unexpected connections between fundamental cell division machinery and acquired resistance mechanisms. Particularly interesting would be comparative studies between environmental isolates from farms (46% acquired resistance to cefotaxime) versus fluvial strains (22% acquired resistance) .

What imaging techniques are most effective for visualizing MinC dynamics in living P. putida cells?

Advanced microscopy approaches provide powerful tools for investigating MinC dynamics in living P. putida cells:

For optimal results when imaging MinC in P. putida:

• Use microfluidic chambers to maintain constant nutrient flow and precise environmental control

• Implement accurate temperature control systems calibrated to P. putida's preferred growth conditions

• Apply deconvolution algorithms specific to rod-shaped bacteria for improved signal-to-noise ratio

• Consider photoactivatable fluorescent proteins for pulse-chase experiments tracking protein turnover

The oscillatory behavior of MinC typically manifests on the timescale of seconds to minutes, necessitating appropriate temporal resolution balanced with minimizing phototoxicity during long-term imaging.

What biochemical assays can be used to characterize MinC interactions with FtsZ in P. putida?

Multiple biochemical approaches can characterize the interactions between MinC and FtsZ in P. putida:

In vitro interaction assays:

• Surface Plasmon Resonance (SPR) - Determines binding kinetics (association/dissociation rates)

• Isothermal Titration Calorimetry (ITC) - Provides thermodynamic parameters of interaction

• Microscale Thermophoresis (MST) - Measures interactions in solution with minimal protein consumption

• Size Exclusion Chromatography with Multi-Angle Light Scattering (SEC-MALS) - Characterizes complex formation and stoichiometry

Functional assays:

• FtsZ polymerization assays (light scattering or sedimentation) - Measures MinC's effect on FtsZ assembly

• GTPase activity assays - Tests whether MinC affects FtsZ's enzymatic function (unlike SulA)

• Electron microscopy of FtsZ filaments - Visualizes structural effects of MinC on FtsZ polymers

• TIRF microscopy with fluorescent FtsZ - Directly observes MinC's effect on FtsZ dynamics on supported lipid bilayers

When designing these experiments, it's important to account for the specific properties of P. putida proteins, which may differ from model organisms. Controls should include comparisons with well-characterized MinC variants from E. coli or T. maritima to benchmark assay performance.

How can computational approaches enhance our understanding of MinC function in P. putida?

Computational methods provide powerful tools for investigating MinC structure, dynamics, and functional interactions in P. putida:

Structural bioinformatics:

• Homology modeling based on T. maritima MinC crystal structure

• Molecular dynamics simulations to explore conformational flexibility

• Protein-protein docking to predict interaction interfaces with FtsZ and MinD

• Coevolutionary analysis to identify functionally coupled residues

Systems biology approaches:

• Gene regulatory network modeling of the Min system in the context of P. putida-specific pathways

• Integration of transcriptomic and proteomic data to identify condition-specific regulation

• Agent-based modeling of MinC oscillation patterns in rod-shaped bacteria

• Flux balance analysis incorporating cell division constraints

Machine learning applications:

• Prediction of MinC variants with altered FtsZ binding specificity

• Classification of cellular phenotypes resulting from Min system perturbations

• Integration of microscopy data for automated tracking of MinC dynamics

These computational approaches are particularly valuable given the experimental challenges of working with MinC proteins and can guide targeted experimental designs to test specific hypotheses about P. putida MinC function.

What are common pitfalls in recombinant expression of P. putida MinC and how can they be overcome?

Researchers frequently encounter several challenges when working with recombinant P. putida MinC:

Challenge 1: Poor solubility

• Solution: Optimize expression conditions using lower temperatures (16-20°C) and mild induction

• Solution: Test fusion partners that enhance solubility (MBP, SUMO, or thioredoxin)

• Solution: Explore P. putida-derived expression systems that may better support proper folding

Challenge 2: Protein instability

• Solution: Include stabilizing agents in buffers (glycerol, reducing agents, specific ions)

• Solution: Explore structural insights from T. maritima MinC to identify stabilizing mutations

• Solution: Consider co-expression with interaction partners (MinD) to stabilize the protein

Challenge 3: Functional verification

• Solution: Develop robust activity assays based on FtsZ interaction

• Solution: Use complementation of minC mutants to verify function in vivo

• Solution: Implement fluorescence-based assays to monitor interaction dynamics

Challenge 4: Protein purification difficulties

• Solution: Exploit the dimeric nature of MinC for size-based separation techniques

• Solution: Leverage the unusually stable dimer interface (32% of C-terminal domain surface)

• Solution: Consider on-column refolding protocols if inclusion bodies form

Researchers should note that previous studies with E. coli MinC used MalE-MinC fusion proteins to overcome similar challenges , suggesting that fusion protein approaches may be particularly effective for P. putida MinC.

How can researchers address variability in MinC-mediated phenotypes across different P. putida strains?

Addressing strain-to-strain variability in MinC-mediated phenotypes requires systematic approaches:

• Genomic characterization:

  • Sequence the complete minCDE operon across strains

  • Identify single nucleotide polymorphisms or structural variations

  • Analyze promoter regions for regulatory differences

• Expression profiling:

  • Quantify minC transcript levels under standardized conditions

  • Measure protein abundance using quantitative proteomics

  • Characterize expression dynamics throughout growth phases

• Standardized phenotyping:

  • Develop consistent microscopy protocols for morphological analysis

  • Implement automated image analysis for objective quantification

  • Use controlled growth conditions to minimize environmental variables

• Genetic complementation:

  • Create a standard expression construct for cross-strain testing

  • Perform reciprocal complementation experiments

  • Identify strain-specific genetic modifiers

P. putida's remarkable genomic plasticity and adaptation to diverse ecological niches likely contributes to strain-specific variations in cell division regulation. Researchers should consider the evolutionary context of different P. putida isolates when interpreting experimental results.

What considerations are important when transferring MinC research findings between P. putida and other bacterial species?

Translating MinC research findings between P. putida and other bacterial species requires careful consideration of several factors:

• Evolutionary context:

  • P. putida belongs to γ-proteobacteria, affecting evolutionary distance to model organisms

  • Consider horizontal gene transfer history when interpreting functional conservation

  • Analyze synteny of the min locus and associated genes across species

• Physiological differences:

  • P. putida's versatile metabolism and stress tolerance may influence MinC regulation

  • Temperature adaptations differ between mesophiles, thermophiles, and psychrophiles

  • Growth rate differences affect the relative timing of cell division events

• Experimental standardization:

  • Normalize findings to cell cycle stage rather than absolute time

  • Account for differences in cellular dimensions and morphology

  • Consider species-specific optimal growth conditions

• Molecular adaptations:

  • Protein sequence conservation doesn't always reflect functional conservation

  • Codon optimization may be necessary for heterologous expression

  • Post-translational modifications may differ between species

While the fundamental role of MinC in preventing polar division appears conserved across bacterial species, the specific molecular mechanisms, protein dynamics, and regulatory circuits may have evolved distinctive features in P. putida related to its environmental adaptability and metabolic versatility .