Recombinant Pseudomonas syringae pv. tomato Probable rRNA maturation factor (PSPTO_4806)

What is PSPTO_4806 and what is its predicted function?

PSPTO_4806 is a gene from Pseudomonas syringae pv. tomato that encodes a probable rRNA maturation factor. Based on sequence homology and predicted structure, it likely plays a role in ribosomal RNA processing and maturation, similar to other rRNA maturation factors such as DIM2. These proteins are typically involved in the final steps of ribosomal subunit assembly, which is critical for proper translation and protein synthesis within the bacterial cell. The protein may function by interacting with rRNA precursors and facilitating their processing into mature, functional forms through mechanisms that potentially include protection of cleavage sites, recruitment of processing enzymes, or structural remodeling of pre-ribosomal particles .

What is the genomic context of PSPTO_4806 in Pseudomonas syringae pv. tomato?

PSPTO_4806 is found in the genome of Pseudomonas syringae pv. tomato, a Gram-negative, aerobic, rod-shaped bacterium belonging to the Pseudomonadaceae family. The genomic neighborhood of PSPTO_4806 likely contains other genes involved in ribosome assembly, RNA processing, or translation. Understanding this genomic context is essential for interpreting the protein's function, as co-regulated genes often participate in related cellular processes. P. syringae pv. tomato has been extensively studied as a model pathogen, particularly strain DC3000, which infects both tomato plants and the model plant Arabidopsis thaliana . This strain has a genome containing approximately 58-60 mol% GC content and encodes numerous pathogenicity and virulence factors that enable its lifestyle as a plant pathogen .

What are the recommended methods for expressing and purifying recombinant PSPTO_4806 protein?

Efficient expression and purification of recombinant PSPTO_4806 typically begins with codon optimization of the gene sequence for the expression host (commonly E. coli). For optimal expression, consider using a bacterial expression system with an inducible promoter (such as T7) and an affinity tag (His-tag or GST-tag) to facilitate purification. The expression construct should be transformed into an appropriate E. coli strain such as BL21(DE3) for protein production. After induction, cells should be lysed and the protein purified using affinity chromatography followed by size exclusion chromatography to ensure high purity. For structural studies, additional steps including tag removal via specific proteases may be necessary. Expression temperature, induction time, and buffer composition should be optimized empirically, as these factors significantly impact yield and solubility of recombinant proteins .

How can researchers design experiments to elucidate the specific role of PSPTO_4806 in rRNA maturation?

A comprehensive experimental approach should combine genetic, biochemical, and structural methods. Begin with gene knockout or depletion studies to observe phenotypic effects on bacterial growth, ribosome assembly, and rRNA processing patterns. RNA-seq and northern blot analyses can identify specific rRNA processing intermediates that accumulate in the absence of PSPTO_4806. To identify interaction partners, employ co-immunoprecipitation or bacterial two-hybrid assays, focusing on components of the ribosome assembly machinery. RNA-protein interactions can be characterized using RNA immunoprecipitation, electrophoretic mobility shift assays, or crosslinking techniques. For mechanistic insights, in vitro reconstitution of rRNA processing using purified components is valuable. A model-based design of experiments (MBDOE) approach can optimize these studies by identifying the most informative experimental conditions and measurements to distinguish between competing mechanistic hypotheses .

What modern structural biology techniques are most appropriate for studying PSPTO_4806?

Multiple complementary structural techniques should be employed to thoroughly characterize PSPTO_4806. X-ray crystallography can provide high-resolution structural information if well-diffracting crystals can be obtained. Cryo-electron microscopy (cryo-EM) is particularly powerful for visualizing PSPTO_4806 in complex with ribosomal subunits or other assembly factors, similar to studies of eukaryotic ribosome maturation factors . Nuclear magnetic resonance (NMR) spectroscopy can provide insights into dynamic regions and conformational changes. For a comprehensive understanding, these high-resolution techniques should be supplemented with small-angle X-ray scattering (SAXS) for solution-state structural information, hydrogen-deuterium exchange mass spectrometry (HDX-MS) to map protein-RNA interaction surfaces, and molecular dynamics simulations to predict functional movements. Cross-linking mass spectrometry can identify specific contact points between PSPTO_4806 and its binding partners within ribosomal assembly intermediates .

Is there evidence linking PSPTO_4806 to the virulence of Pseudomonas syringae pv. tomato?

While direct evidence specifically linking PSPTO_4806 to virulence is limited in the provided search results, proteins involved in fundamental cellular processes like ribosome assembly can indirectly affect virulence. In bacterial pathogens, proper ribosome function is essential for the timely production of virulence factors in response to host environmental cues. Disruption of rRNA maturation could potentially affect the expression of known virulence factors in P. syringae pv. tomato, which include toxins, extracellular proteins, polysaccharides, and proteins translocated into plant cells by the type III secretion system . By analogy to other bacterial systems, mutations affecting ribosome assembly often result in growth defects and reduced virulence. Future studies should investigate whether PSPTO_4806 is differentially regulated during infection or whether its mutation affects the expression of established virulence factors such as TvrR (PSPTO3576), a TetR-like transcriptional regulator known to be necessary for virulence in DC3000 .

How does PSPTO_4806 compare to other known virulence factors in Pseudomonas syringae pv. tomato?

Unlike classic virulence factors such as the type III secretion system components or effector proteins that directly manipulate host defenses, PSPTO_4806 likely contributes to virulence indirectly through its role in fundamental cellular processes. While dedicated virulence factors like TvrR (PSPTO3576) directly regulate pathogenicity genes , PSPTO_4806 as an rRNA maturation factor would primarily affect the bacterium's ability to synthesize proteins efficiently, including those virulence factors. This represents a different class of proteins that affect virulence through "fitness factors" rather than specific pathogenicity determinants. The contribution of such housekeeping genes to virulence can be substantial but is often overlooked in traditional virulence studies. Unlike the type III secretion system, which is highly regulated by environmental cues and the HrpL alternative sigma factor , PSPTO_4806 would likely be constitutively expressed with potential fine-tuning under certain stress conditions encountered during infection.

What experimental systems are best suited for studying the role of PSPTO_4806 in plant-pathogen interactions?

The ideal experimental system would utilize both the natural host plants of P. syringae pv. tomato and appropriate controls. Arabidopsis thaliana serves as an excellent model system due to its well-characterized genetics and susceptibility to P. syringae pv. tomato DC3000 . Tomato plants (Lycopersicon esculentum) represent the natural agricultural host. The experimental design should include:

• Comparison of wild-type and PSPTO_4806 mutant strains for their ability to:

  • Grow in planta (bacterial population counts)

  • Cause disease symptoms (quantitative disease scoring)

  • Suppress host immune responses (ROS burst, callose deposition)

• Complementation studies to verify phenotypes are specifically due to PSPTO_4806

• Transcriptomic and proteomic analyses to identify downstream effects on virulence factor expression

• Confocal microscopy with fluorescently tagged PSPTO_4806 to track its localization during infection

These experiments should be conducted under controlled environmental conditions that favor disease development, including appropriate temperature (13-25°C) and leaf wetness .

How might post-translational modifications affect the function of PSPTO_4806?

Post-translational modifications (PTMs) could significantly influence PSPTO_4806 function through several mechanisms. Phosphorylation, common in bacterial signaling systems, might regulate PSPTO_4806 activity in response to environmental cues encountered during infection. Sites of potential phosphorylation can be predicted computationally and verified using phospho-proteomics and site-directed mutagenesis. Other relevant PTMs might include acetylation, which can affect protein-nucleic acid interactions critical for rRNA maturation factors. PTMs could regulate PSPTO_4806 by altering its:

• Binding affinity for rRNA substrates

• Interactions with other ribosome assembly factors

• Cellular localization or stability

• Conformational dynamics

Research should employ mass spectrometry-based approaches to identify PTMs under different growth conditions, particularly comparing standard laboratory conditions with plant-mimicking environments. Functional consequences of identified modifications should be assessed through biochemical assays comparing wild-type PSPTO_4806 with proteins bearing mutations at modified residues .

What computational approaches can predict the structural features and functional domains of PSPTO_4806?

A multi-faceted computational approach can provide valuable insights into PSPTO_4806 structure and function. Begin with advanced sequence analysis using profile hidden Markov models (HMMs) to identify conserved domains and distant homologs with known functions. Modern protein structure prediction tools like AlphaFold2 can generate highly accurate structural models, particularly valuable if experimental structures are unavailable. These models can be refined through molecular dynamics simulations to explore conformational flexibility. Functional analysis should include:

• Identification of putative RNA-binding residues using machine learning approaches

• Prediction of protein-protein interaction sites through conservation mapping and surface analysis

• Molecular docking simulations with predicted rRNA substrates

• Comparative analysis with known rRNA maturation factors like DIM2

The predicted functional sites should guide experimental validation through site-directed mutagenesis. Additionally, coevolution analysis can identify residues that have evolved together, potentially indicating functional coupling or physical interaction within the protein or with partners .

How can researchers distinguish between direct and indirect effects when studying PSPTO_4806 function?

Distinguishing direct from indirect effects is a significant challenge when studying proteins involved in fundamental cellular processes like ribosome assembly. A comprehensive strategy should include:

• In vitro reconstitution systems: Purified components allow direct assessment of PSPTO_4806 activity on defined rRNA substrates without cellular complexity.

• Rapid depletion approaches: Systems allowing quick protein depletion (e.g., degron tags) help separate immediate direct effects from downstream consequences.

• Separation of function mutations: Engineered variants affecting specific activities while preserving protein structure can isolate particular functions.

• Temporal analysis: Time-course experiments tracking cellular changes following PSPTO_4806 depletion can separate primary from secondary effects.

• Compensatory mutations: If a specific rRNA interaction is proposed, complementary mutations in both PSPTO_4806 and its rRNA target that restore function provide strong evidence for direct interaction.

• Chemical crosslinking: Capturing transient interactions through crosslinking followed by mass spectrometry identifies direct binding partners.

The experimental design should incorporate appropriate controls and utilize statistical methods to quantify the confidence in observed effects .

What are the common challenges in expressing recombinant PSPTO_4806 and how can they be addressed?

Researchers frequently encounter several challenges when expressing recombinant PSPTO_4806:

For particularly challenging cases, consider alternative expression systems such as cell-free protein synthesis, which can produce proteins toxic to living cells. Additionally, co-expression with potential binding partners may improve solubility and stability .

How can researchers optimize experimental conditions for studying PSPTO_4806-RNA interactions?

Optimizing experimental conditions for PSPTO_4806-RNA interaction studies requires careful consideration of multiple factors:

• Buffer composition: Screen various conditions including:

  • Monovalent salt concentration (50-300 mM KCl or NaCl)

  • Divalent cations (1-10 mM Mg²⁺) critical for RNA structure

  • pH range (6.8-8.0)

  • Reducing agents to maintain protein activity

• RNA substrate preparation:

  • In vitro transcribed RNA requires careful purification

  • Consider native purification from bacterial cells

  • Test both full-length rRNA precursors and defined fragments

• Detection methods:

  • Fluorescence anisotropy for quantitative binding measurements

  • Filter binding assays for rapid screening

  • Electrophoretic mobility shift assays for complex visualization

• Experimental controls:

  • Include non-specific RNA controls

  • Test mutated versions of PSPTO_4806

  • Competition assays to confirm specificity

A model-based design of experiments (MBDOE) approach can systematically identify optimal experimental conditions by exploring the parameter space efficiently, determining which combination of factors yields the most informative results about the interaction dynamics .

What strategies can overcome data inconsistencies when studying PSPTO_4806 in different experimental systems?

When confronting data inconsistencies across different experimental systems, researchers should implement the following strategies:

• Standardize experimental conditions: Develop consistent protocols for cell growth, protein expression, and activity assays that can be replicated across laboratories.

• Use multiple complementary approaches: Combine genetic, biochemical, and structural methods to build a more robust understanding, as each approach has inherent limitations.

• Control for strain-specific effects: Verify findings in multiple P. syringae pv. tomato strains beyond DC3000, as strain-specific genetic backgrounds can influence results .

• Address context-dependency: Systematically vary experimental conditions to identify factors that explain observed inconsistencies, such as:

  • Growth phase effects

  • Media composition differences

  • Temperature variations

  • Host plant genotype influence

• Quantitative analysis: Apply statistical methods and mathematical modeling to quantify uncertainty and distinguish significant from artifactual variations.

• Collaborative validation: Engage multiple laboratories to independently verify key findings using standardized protocols and reagents.

By implementing these approaches, researchers can develop a more coherent understanding of PSPTO_4806 function despite initial data inconsistencies .