Recombinant Pseudomonas putida Nucleoid-associated protein PP_0973 (PP_0973)

What is PP_0973 and which protein family does it belong to?

PP_0973 is a nucleoid-associated protein (NAP) from Pseudomonas putida strain ATCC 47054/DSM 6125/NCIMB 11950/KT2440. It belongs to the YejK family of nucleoid-associated proteins . These proteins are involved in bacterial chromosome organization and compaction, similar to histones in eukaryotes, but with additional regulatory functions. The protein is also known as NdpA (Nucleoid-associated protein A) in some annotation systems .

What are the basic physical properties of PP_0973?

PP_0973 is a protein consisting of 335 amino acids with a molecular mass of approximately 37.6 kDa . The protein has the following amino acid sequence:
MPIRHCIVHLIDKKPDGSPAVLHARDSELAASDAIENLLADLNDSYNAKQGKAWGFFHGESGAYPLSGWLKQYLDEEKDFTAFSRVAVEHLQKLMEESNLSTGGHILFAHYQQGMTEYLAIALLHHSEGVAVNAQLDVTPSRHLDLGQLHLAARINLSEWKNNQNSRQYISFIKGKNGKKVSDYFRDFIGCQEGVDGPGETRTLLKAFSDFVESEDLPEESAREKTQTLVEYATTQTKLGEPVTLEELSSLIDEDRPKAFYDHIRNKDYGLSPEIPADKRTLNQFRRFTGRAEGLSISFEAHLLGDKVEYDEAAGTLIIKGLPTTLVDQLKRRKD

Where is the PP_0973 gene located in the P. putida genome?

The PP_0973 gene is located in the Pseudomonas putida KT2440 genome. According to genome annotation data, it is positioned in the genomic region around position 965, with a G+C content of 49.70%, which is notably lower than the average for the P. putida genome (58.35-65.31%) . This relatively low G+C content might indicate that this region has distinct functional or evolutionary characteristics.

What is the general function of nucleoid-associated proteins like PP_0973?

Nucleoid-associated proteins like PP_0973 primarily function in chromosome organization and compaction in bacteria. Unlike eukaryotes that use histones, bacteria employ NAPs to organize their genomic DNA into a structure called the nucleoid. Beyond structural roles, NAPs often serve as global transcriptional regulators, influencing the expression of various genes in response to environmental conditions. As a member of the YejK family, PP_0973 likely participates in DNA binding, organization, and potentially regulation of gene expression.

Are there known orthologs of PP_0973 in other Pseudomonas species?

Yes, PP_0973 has identified orthologs in other Pseudomonas species. According to orthology databases, a homologous protein exists in Pseudomonas aeruginosa PACS2 (GI: 107103368, locus tag: PaerPA_01004438) . This conservation across Pseudomonas species suggests that the protein serves an important function in these bacteria. Comparative genomic studies of these orthologs might provide insights into the evolutionary conservation and functional importance of this nucleoid-associated protein.

What expression systems are most effective for producing recombinant PP_0973?

For recombinant expression of PP_0973, E. coli-based expression systems are typically most effective for laboratory-scale production. The protein can be expressed with a 6x His-tag for ease of purification, yielding protein with purity >90% as determined by SDS-PAGE . For optimal expression, consider using BL21(DE3) or similar strains with promoters like T7 or tac. Expression conditions should be optimized with induction at OD600 of 0.6-0.8 using IPTG concentrations between 0.1-1.0 mM, and growth temperatures of 16-30°C to minimize inclusion body formation.

What purification methods yield the highest purity of recombinant PP_0973?

The most effective purification strategy for His-tagged PP_0973 involves:

• Initial capture using immobilized metal affinity chromatography (IMAC) with Ni-NTA resin

• Intermediate purification with ion exchange chromatography (typically anion exchange using Q Sepharose)

• Final polishing with size exclusion chromatography (Superdex 75 or 200)

This three-step protocol consistently yields protein with >95% purity. For specialized applications requiring higher purity, additional chromatographic steps such as hydrophobic interaction chromatography might be necessary. Buffer optimization is critical, with typical conditions including 50 mM Tris-HCl pH 8.0, 300 mM NaCl, and 10% glycerol to maintain protein stability.

How can researchers verify the structural integrity of purified recombinant PP_0973?

To verify the structural integrity of purified recombinant PP_0973, researchers should employ multiple complementary techniques:

• Circular dichroism (CD) spectroscopy to assess secondary structure composition

• Thermal shift assays to evaluate protein stability

• Dynamic light scattering (DLS) to confirm monodispersity and absence of aggregation

• Limited proteolysis to verify proper folding (properly folded proteins typically have specific, limited cleavage patterns)

• Activity assays, particularly DNA binding assays, to confirm functional integrity

A properly folded nucleoid-associated protein should display characteristic alpha-helical content in CD spectra and demonstrate specific DNA-binding capabilities in electrophoretic mobility shift assays (EMSA) or fluorescence anisotropy measurements.

What DNA-binding assays are most appropriate for studying PP_0973 interactions with nucleic acids?

For studying PP_0973 interactions with nucleic acids, the following assays are particularly valuable:

• Electrophoretic Mobility Shift Assay (EMSA): Provides qualitative assessment of binding and can reveal multiple binding states or cooperativity.

• Fluorescence Anisotropy: Offers quantitative binding parameters (Kd values) and is suitable for equilibrium measurements.

• Microscale Thermophoresis (MST): Allows for determination of binding under various solution conditions with minimal sample consumption.

• Chromatin Immunoprecipitation (ChIP): For identifying genomic binding sites in vivo.

• DNase I Footprinting: To identify specific protected regions within a DNA sequence.

For nucleoid-associated proteins like PP_0973, it's important to test both sequence-specific and non-specific binding using different DNA substrates, including curved DNA segments that often interact preferentially with NAPs.

How can researchers accurately determine the oligomeric state of PP_0973 in solution?

Determining the oligomeric state of PP_0973 requires a multi-method approach:

For a comprehensive analysis, SEC-MALS combined with AUC provides the most reliable determination of the oligomeric state under physiological conditions. When studying NAPs like PP_0973, it's important to assess oligomerization both in the presence and absence of DNA, as DNA binding can induce changes in quaternary structure.

How does PP_0973 compare with other nucleoid-associated proteins in terms of genomic binding patterns?

PP_0973, as a member of the YejK family, likely exhibits distinct genomic binding patterns compared to other well-characterized NAPs such as H-NS, Fis, HU, and IHF. While classical NAPs often show preference for AT-rich regions or specific DNA structures, YejK family proteins may have unique binding preferences that remain to be fully characterized.

To compare binding patterns, researchers should consider:

• Performing ChIP-seq analysis of PP_0973 and other NAPs in parallel

• Comparing binding motifs using MEME or similar motif discovery tools

• Analyzing binding sites in relation to genomic features (promoters, transcription start sites)

• Examining binding dynamics during different growth phases and environmental conditions

• Correlating binding patterns with transcriptomic data to identify regulatory roles

Unlike H-NS, which primarily forms filaments along DNA and often silences horizontally acquired genes, or Fis, which targets specific sequences often associated with highly transcribed genes, PP_0973 may have specialized functions related to particular genomic regions or cellular processes in Pseudomonas.

What genomic regions in P. putida show altered properties in PP_0973 knockout or overexpression strains?

In PP_0973 knockout or overexpression strains, the following genomic regions would be of particular interest:

• Regions with unusual G+C content: Given that PP_0973 is located in a region with lower G+C content (49.70%) than the genomic average , it may preferentially interact with other low G+C regions.

• Mobile genetic elements: Many NAPs regulate horizontally acquired DNA and mobile genetic elements.

• Stress response genes: Expression of genes involved in environmental adaptation may be affected.

• Regions containing genes with atypical codon usage: These often represent horizontally acquired genes that might be regulated by NAPs.

• Regions containing highly conserved or species-specific genes: To understand evolutionary roles.

What other proteins interact with PP_0973 and how do these interactions affect its function?

To identify proteins that interact with PP_0973, researchers should employ multiple complementary approaches:

• Pull-down assays with tagged recombinant PP_0973

• Bacterial two-hybrid screens

• Co-immunoprecipitation followed by mass spectrometry

• Proximity labeling approaches (BioID or APEX)

• Crosslinking mass spectrometry (XL-MS)

Potential interaction partners may include:

• Other nucleoid-associated proteins

• Components of the transcription machinery

• Replication and repair proteins

• Stress response regulators

• Cell division proteins

Functional characterization of identified interactions should include:

• Verification by independent methods (FRET, SPR, etc.)

• Mapping interaction domains through truncation and mutation studies

• Assessment of how interactions change under different physiological conditions

• Determination of how interactions affect DNA binding and other functions

How does post-translational modification affect the activity of PP_0973?

Although specific post-translational modifications (PTMs) of PP_0973 have not been extensively characterized in the provided search results, NAPs are often subject to various modifications that regulate their activity. To study potential PTMs of PP_0973, researchers should:

• Perform mass spectrometry analysis of the protein isolated under different growth conditions to identify modifications

• Create site-directed mutants of potential modification sites to assess functional consequences

• Use phosphoproteomic approaches to identify potential phosphorylation events

• Examine acetylation patterns, which often regulate DNA-binding proteins

Common PTMs that might regulate PP_0973 include:

• Phosphorylation (affecting DNA binding affinity)

• Acetylation (modulating interaction with DNA and other proteins)

• Methylation (altering regulatory functions)

• Oxidation (potentially serving as a redox sensor)

The presence and abundance of these modifications likely change in response to environmental conditions, growth phase, or stress, providing a mechanism for dynamic regulation of nucleoid structure and gene expression.

How can CRISPR-Cas9 genome editing be applied to study PP_0973 function in P. putida?

CRISPR-Cas9 genome editing provides powerful approaches for studying PP_0973 function in P. putida. Researchers can implement the following strategies:

• Gene knockout: Complete deletion of PP_0973 to assess loss-of-function phenotypes

• Point mutations: Introduction of specific amino acid changes to study structure-function relationships

• Domain swapping: Replacing domains with those from other NAPs to understand functional specificity

• Promoter modifications: Altering expression levels or patterns

• Fluorescent protein tagging: Fusing fluorescent proteins for localization studies

• CRISPRi: Using catalytically inactive Cas9 for transient repression

For P. putida specifically, the protocol should include:

• Optimized sgRNA design with high specificity for the PP_0973 locus

• Efficient delivery methods (electroporation or conjugation)

• Temperature-controlled expression (typically 30°C for P. putida)

• Appropriate selection markers (typically kanamycin or gentamicin resistance)

• Verification by sequencing and RT-qPCR

Phenotypic characterization should examine growth rates, stress responses, biofilm formation, and genome-wide expression changes to comprehensively understand PP_0973 function.

What high-throughput approaches can identify genome-wide binding sites of PP_0973?

Several high-throughput approaches can identify genome-wide binding sites of PP_0973:

For NAPs with potential broad binding patterns like PP_0973, it's crucial to analyze data with algorithms that can detect both sharp peaks (site-specific binding) and broader enrichment regions (cooperative or non-specific binding). Integration with RNA-seq, DNase-seq, and other genomic data provides context for interpreting binding patterns and their functional consequences.

What are the most common issues when expressing recombinant PP_0973 and how can they be resolved?

Researchers often encounter several challenges when expressing recombinant PP_0973:

• Inclusion body formation: As a DNA-binding protein, PP_0973 may aggregate when overexpressed.

  • Solution: Lower expression temperature (16-20°C), reduce inducer concentration, use specialized strains like Arctic Express, or include solubility-enhancing fusion tags like SUMO or MBP.

• Proteolytic degradation: NAPs can be susceptible to proteolysis.

  • Solution: Include protease inhibitors throughout purification, use protease-deficient expression strains, optimize buffer conditions, and minimize time between cell lysis and initial purification steps.

• Co-purification with bacterial DNA: DNA-binding proteins often retain bound nucleic acids.

  • Solution: Include DNase treatment during lysis, incorporate high-salt washes (0.5-1.0 M NaCl) during purification, and consider polyethyleneimine precipitation to remove nucleic acids.

• Poor yield: Expression levels may be low due to toxicity.

  • Solution: Use tightly controlled expression systems, consider auto-induction media, optimize codon usage for the expression host, or try different promoter systems.

• Loss of activity: The recombinant protein may fold incorrectly or lose activity during purification.

  • Solution: Include stabilizing agents (glycerol, specific ions), optimize buffer composition based on thermal shift assays, and validate activity immediately after purification.

How can researchers overcome challenges in studying protein-DNA interactions for nucleoid-associated proteins like PP_0973?

Studying protein-DNA interactions for nucleoid-associated proteins presents unique challenges that can be addressed through specific strategies:

• Distinguishing specific from non-specific binding:

  • Use competition assays with specific and non-specific competitors

  • Perform salt titration experiments (specific interactions typically resist higher salt)

  • Employ multiple techniques (EMSA, footprinting, fluorescence-based methods) to cross-validate

• Determining binding parameters when cooperativity is present:

  • Use appropriate mathematical models that account for cooperativity

  • Perform binding assays at various protein and DNA concentrations

  • Consider single-molecule techniques to observe binding events directly

• Identifying physiologically relevant binding sites:

  • Compare in vitro binding with in vivo ChIP-seq data

  • Correlate binding with transcriptional effects

  • Consider the native chromosomal context and DNA topology

• Accounting for effects of DNA topology:

  • Include experiments with supercoiled DNA when possible

  • Consider how nucleoid-associated proteins both respond to and influence DNA topology

  • Use techniques like atomic force microscopy to visualize structural changes

• Addressing redundancy among nucleoid-associated proteins:

  • Create multiple knockout strains

  • Study binding in the absence of other major NAPs

  • Investigate condition-specific roles through varied experimental conditions

What bioinformatic approaches are most effective for analyzing PP_0973 binding patterns in ChIP-seq data?

For effective analysis of PP_0973 ChIP-seq data, researchers should consider the following bioinformatic approaches:

• Peak calling optimization:

  • Traditional peak callers (MACS2, HOMER) may need parameter adjustment for broad binding patterns

  • Consider specialized algorithms for nucleoid-associated proteins that may have different binding profiles than transcription factors

  • Use multiple peak calling methods and compare results

• Motif discovery and analysis:

  • Apply de novo motif discovery (MEME, HOMER) to identify potential binding preferences

  • Consider discriminative motif discovery comparing bound vs. unbound regions

  • Analyze motif distribution relative to peak centers to distinguish direct from indirect binding

• Integration with other genomic data:

  • Correlate binding with gene expression (RNA-seq)

  • Compare with DNA accessibility data (ATAC-seq, DNase-seq)

  • Analyze binding in relation to genomic features (promoters, terminators, etc.)

  • Incorporate DNA shape predictions to identify structural preferences

• Comparative genomics:

  • Compare binding sites across related Pseudomonas species

  • Analyze conservation of bound sequences

  • Examine binding relative to horizontally acquired regions vs. core genome

• Network analysis:

  • Construct regulatory networks by integrating multiple NAPs' binding data

  • Identify co-regulated gene clusters

  • Perform Gene Ontology enrichment on bound regions to identify functional categories

These approaches should be adapted based on the specific binding characteristics of PP_0973, which may differ from better-studied NAPs like H-NS or Fis.

Current State of Knowledge and Research Gaps

Our current understanding of PP_0973 from Pseudomonas putida is still developing. While we know its basic structural properties (335 amino acids, 37.6 kDa) and genomic context , many aspects of its function remain to be fully elucidated. As a nucleoid-associated protein, PP_0973 likely plays important roles in chromosome organization and potentially gene regulation, but the specific mechanisms, binding preferences, and regulatory networks involved require further investigation. The presence of orthologs in other Pseudomonas species suggests evolutionary conservation of function, making this an interesting protein for comparative studies.

Major research gaps include: detailed structural characterization, genome-wide binding profiles under different conditions, identification of interacting proteins, and phenotypic consequences of gene deletion or overexpression. Addressing these gaps will require interdisciplinary approaches combining molecular biology, genomics, structural biology, and systems biology. The YejK family of proteins, to which PP_0973 belongs, is less well-characterized than other NAP families, providing opportunities for novel discoveries about bacterial chromosome organization and gene regulation mechanisms.

Future Research Directions

Future research on PP_0973 should focus on several promising directions:

• Structural characterization through X-ray crystallography or cryo-electron microscopy, particularly in complex with DNA, to understand binding mechanisms.

• Genome-wide binding studies under diverse environmental conditions to map dynamic changes in binding patterns and identify condition-specific functions.

• Creation of knockout and complementation strains to characterize phenotypic consequences and perform transcriptomic analyses.

• Comparative studies with orthologs from other Pseudomonas species to understand evolutionary conservation and specialization.

• Investigation of potential roles in specific cellular processes such as stress response, biofilm formation, or virulence (in pathogenic Pseudomonas species).

• Integration of multi-omics data to position PP_0973 within the broader regulatory networks of Pseudomonas putida.

• Development of PP_0973 as a potential biotechnological tool, leveraging its DNA-binding properties for synthetic biology applications.