Recombinant Pseudomonas syringae pv. tomato tRNA-dihydrouridine synthase B (dusB)

What is the function of tRNA-dihydrouridine synthase B (dusB) in Pseudomonas syringae pv. tomato DC3000?

tRNA-dihydrouridine synthase B (dusB) in Pseudomonas syringae pv. tomato DC3000 catalyzes the reduction of specific uridine residues in tRNA molecules to dihydrouridine. This post-transcriptional modification contributes to tRNA stability and proper folding, which is essential for efficient translation processes. Dihydrouridine is one of the most abundant modified nucleosides in bacterial tRNAs, particularly in those functioning under low-temperature conditions. In P. syringae pv. tomato, which experiences temperature fluctuations during plant colonization, dusB likely plays a critical role in adapting translation machinery to changing environmental conditions .

How does dusB differ structurally and functionally from other dihydrouridine synthases in Pseudomonas species?

While specific structural data for P. syringae pv. tomato dusB is limited, comparative analyses with other Pseudomonas species show that dihydrouridine synthases share a conserved core structure with distinct substrate recognition domains. Based on studies of DusA from P. aeruginosa, which contains 43% α-helices and 16% β-strands with a Tm value of 46.2°C and Cm of 2.7 M for urea , we can infer that dusB likely has similar structural characteristics but with distinct substrate specificity patterns.

The Dus family in bacteria typically includes three to four members (DusA, DusB, DusC, and sometimes DusD), each responsible for modifying specific positions in tRNA. While DusA tends to modify positions 21 and 22 in the D-loop of tRNA, dusB typically targets different positions, contributing to the complete modification profile necessary for optimal tRNA functioning. The specific substrate recognition mechanisms of dusB involve interactions between the enzyme's recognition domain and the three-dimensional structure of tRNA molecules.

How does dusB contribute to the virulence of Pseudomonas syringae pv. tomato DC3000?

The role of dusB in P. syringae pv. tomato pathogenicity appears to be indirect but significant. As a tRNA modification enzyme, dusB maintains translation efficiency under stress conditions that bacteria encounter during plant infection. Research suggests that proper tRNA modification is crucial for bacterial adaptation to host environments, particularly when faced with plant defense responses that include oxidative stress, pH changes, and antimicrobial compounds.

During infection, P. syringae pv. tomato manipulates host hormone pathways, including abscisic acid (ABA) signaling . The efficient translation of virulence factors, including type III secretion system (T3SS) components and effector proteins, depends on properly modified tRNAs. When dusB function is compromised, the resulting translational inefficiencies may impair the expression of key virulence factors, potentially reducing bacterial fitness in planta.

Additionally, the ability of P. syringae to adapt to the plant apoplast environment, where pH and nutrient conditions differ significantly from the external environment, may be partially dependent on dusB-mediated tRNA modifications that maintain efficient protein synthesis under these specialized conditions.

What is the relationship between dusB activity and type III secretion system function in plant pathogenic bacteria?

While direct experimental evidence linking dusB to T3SS function is limited, the relationship likely centers on translational efficiency. The T3SS machinery in P. syringae pv. tomato DC3000 is composed of multiple proteins encoded by the hrp/hrc gene cluster , and their coordinated expression is essential for successful pathogenesis.

The expression of T3SS components is governed by the HrpL sigma factor, which regulates numerous genes during infection . Optimal translation of these genes requires properly modified tRNAs. dusB, by ensuring the correct modification of tRNAs, likely supports the efficient translation of T3SS components and effectors, particularly under the stress conditions encountered during infection.

Experimental evidence from other bacterial systems suggests that defects in tRNA modification can lead to reduced expression of virulence factors. In the context of P. syringae pv. tomato, the relationship between dusB and T3SS function could be investigated by:

• Creating dusB deletion mutants and assessing T3SS protein levels

• Measuring effector translocation efficiency in dusB mutants

• Examining hrp gene expression and translation rates in the absence of functional dusB

What are the key structural features of recombinant dusB from P. syringae pv. tomato, and how do they relate to its catalytic function?

The structural features of dusB from P. syringae pv. tomato can be inferred from studies of related dihydrouridine synthases. Based on research on DusA from P. aeruginosa, dusB likely contains:

• A flavin-binding domain containing conserved residues that coordinate with the flavin cofactor

• A tRNA-binding domain with positively charged regions that interact with the negatively charged tRNA backbone

• A catalytic domain with residues directly involved in the reduction of uridine to dihydrouridine

The enzyme contains a significant proportion of α-helical structures (approximately 40-45%) and β-strands (15-20%), creating a fold that enables both substrate recognition and catalysis . The catalytic mechanism involves NADPH-dependent reduction of the C5-C6 double bond in the uracil ring of specific uridine residues in tRNA, a reaction catalyzed by the bound flavin cofactor.

The thermal stability of these enzymes is moderate, with melting temperatures typically in the range of 45-50°C, reflecting their adaptation to mesophilic bacterial growth conditions. This stability profile may be particularly important for P. syringae pv. tomato, which must function across varying temperatures in the plant environment.

What methodologies are most effective for purifying and characterizing recombinant dusB from P. syringae pv. tomato?

Based on successful approaches with related Dus enzymes, the following methodologies are recommended for purifying and characterizing recombinant dusB:

Purification Protocol:

• Cloning and Expression:

  • Clone the dusB gene from P. syringae pv. tomato DC3000 genomic DNA using PCR with specific primers

  • Insert the gene into an expression vector (e.g., pET series) with an N- or C-terminal His-tag

  • Transform into an E. coli expression strain (BL21(DE3) or derivatives)

  • Induce expression with IPTG (typically 0.1-0.5 mM) at lower temperatures (16-25°C) to enhance solubility

• Purification Steps:

  • Lyse cells using sonication or French press in a buffer containing 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10% glycerol, and protease inhibitors

  • Perform Ni-NTA affinity chromatography with imidazole gradient elution

  • Further purify using size exclusion chromatography (Superdex 200)

  • Verify purity by SDS-PAGE and Western blotting

Characterization Methods:

• Structural Analysis:

  • Circular Dichroism (CD) spectroscopy to determine secondary structure content

  • Thermal stability assessment through temperature-induced unfolding monitored by CD

  • X-ray crystallography for high-resolution structural determination

• Functional Analysis:

  • In vitro activity assays using synthetic or purified tRNAs as substrates

  • Quantification of dihydrouridine formation using HPLC or mass spectrometry

  • Determination of kinetic parameters (Km, kcat) for different tRNA substrates

  • Cofactor binding studies using flavin fluorescence quenching

• Substrate Specificity:

  • Site-directed mutagenesis of potential substrate recognition residues

  • Comparison of activity toward different tRNA molecules

  • In vitro modification assays combined with primer extension analysis to map modified positions

How can I effectively generate and validate dusB knockout mutants in P. syringae pv. tomato?

Generating and validating dusB knockout mutants in P. syringae pv. tomato requires careful approach due to the potentially essential nature of tRNA modifications. The following methodology is recommended:

Generation of dusB Knockout Mutants:

• Allelic Exchange Method:

  • Construct a suicide vector containing 500-1000 bp homology regions flanking the dusB gene, with an antibiotic resistance marker between them

  • Introduce the vector into P. syringae pv. tomato via electroporation or conjugation

  • Select for integrants on appropriate antibiotic-containing media

  • Counter-select for resolution of the integrated plasmid using sucrose sensitivity (if using sacB-based vectors)

  • Screen colonies for deletion of dusB

• RecTE-based Recombineering:

  • Utilize the RecTE recombination system from P. syringae, which has been shown to effectively promote recombination of linear DNA in Pseudomonas species

  • Design PCR products with antibiotic resistance cassettes flanked by 50-100 bp homology regions to dusB

  • Express RecTE from a plasmid in P. syringae pv. tomato prior to transformation with the knockout construct

  • Transform the knockout construct and select on appropriate antibiotics

Validation Methods:

• Genetic Verification:

  • PCR amplification across the deletion junction

  • Whole-genome sequencing to confirm the deletion and rule out off-target effects

  • RT-PCR to confirm the absence of dusB transcript

• Functional Verification:

  • Analysis of tRNA modifications using LC-MS/MS to confirm reduction in dihydrouridine content

  • Complementation studies by reintroducing dusB on a plasmid to restore wild-type phenotypes

• Phenotypic Characterization:

  • Growth curve analysis under different conditions (temperature, pH, oxidative stress)

  • Virulence assays on host plants

  • Protein synthesis rate measurements

What are the most appropriate in vitro assays for measuring dusB enzymatic activity and substrate specificity?

In Vitro Enzymatic Activity Assays:

• Radioisotope-Based Assays:

  • Incubate purified recombinant dusB with [³H]-labeled uridine-containing tRNA substrates

  • Quantify the incorporation of tritium into dihydrouridine by scintillation counting

  • Calculate enzyme kinetics based on time-course measurements

• LC-MS/MS Analysis:

  • React purified dusB with tRNA substrates in the presence of NADPH and flavin

  • Digest tRNA into nucleosides using nuclease P1 and alkaline phosphatase

  • Analyze modified nucleosides by LC-MS/MS, quantifying dihydrouridine formation

  • Compare reaction products with synthetic standards

• Spectrophotometric Assays:

  • Monitor NADPH oxidation at 340 nm as an indirect measure of dusB activity

  • Calculate reaction rates based on the decrease in NADPH absorbance

  • Determine kinetic parameters using varying concentrations of substrate tRNA

Substrate Specificity Analysis:

• Comparative tRNA Modification:

  • Test activity of dusB against different tRNA isoacceptors

  • Use primer extension with reverse transcriptase to map modification sites (reverse transcriptase pauses or misincorporates nucleotides at modified positions)

  • Create a substrate preference profile based on relative activity measurements

• Structure-Activity Relationships:

  • Generate tRNA variants with mutations at potential modification sites

  • Assess the impact on dusB activity to identify critical recognition elements

  • Use tRNA fragments to determine minimum substrate requirements

Note: These values are representative based on similar enzymes; specific values for P. syringae pv. tomato dusB would need experimental verification.

How does environmental stress affect dusB expression and activity in P. syringae pv. tomato during plant infection?

Environmental stresses encountered during plant infection significantly influence dusB expression and activity. Research indicates that various stress conditions relevant to plant colonization alter the requirement for tRNA modifications:

• Temperature Fluctuations:

  • dusB expression is typically upregulated at lower temperatures (18-20°C), corresponding with increased dihydrouridine content in tRNAs

  • This modification helps maintain tRNA flexibility at lower temperatures, compensating for reduced molecular motion

  • During day/night temperature cycling experienced in plant environments, dusB activity may show corresponding cyclical patterns

• Oxidative Stress Response:

  • Plant defense responses generate reactive oxygen species (ROS)

  • P. syringae pv. tomato experiences oxidative stress during infection, particularly through the plant oxidative burst

  • Research suggests a connection between oxidative stress sensing and bacterial motility regulation through proteins like ChrR (PSPTO_1042)

  • dusB may be regulated as part of this stress response network to maintain translation fidelity under oxidative conditions

• pH Adaptation:

  • The plant apoplast typically has an acidic pH (5.5-6.5)

  • P. syringae activates specific gene expression programs under low pH conditions

  • tRNA modifications appear to be critical for optimal translation under pH stress

  • dusB activity may be modulated in response to the acidic environment encountered during infection

Methodological Approaches for Investigation:

• Transcriptional Profiling:

  • RT-qPCR analysis of dusB expression under various stress conditions

  • RNA-seq to position dusB within stress-responsive regulons

  • Promoter-reporter fusions to monitor dusB expression dynamics in real-time during infection

• Proteomics Approaches:

  • Quantitative proteomics to measure dusB protein levels under stress

  • Post-translational modification analysis to identify regulatory modifications

  • Protein-protein interaction studies to map dusB within stress response networks

• tRNA Modification Analysis:

  • LC-MS/MS quantification of dihydrouridine levels in tRNAs isolated from bacteria exposed to different stresses

  • Correlation of modification patterns with stress adaptation and virulence

What is the interplay between dusB and other tRNA modification enzymes in regulating translation during P. syringae pv. tomato pathogenesis?

The coordination between dusB and other tRNA modification enzymes represents a sophisticated regulatory network affecting translation during pathogenesis. This interplay is particularly significant in P. syringae pv. tomato, which must adapt its translation machinery to various microenvironments within the plant:

• Coordinated tRNA Modification:

  • Multiple modification enzymes, including TruA (pseudouridine synthase) and RluA, act on the same tRNA molecules

  • The specific arrangement of modifications creates a "modification signature" that influences tRNA function

  • These combined modifications affect tRNA stability, aminoacylation efficiency, and codon recognition

• Differential Regulation Under Stress:

  • Different tRNA modification enzymes respond to distinct stress signals

  • During infection, the relative activity of these enzymes likely shifts based on environmental cues

  • The resulting changes in tRNA modification patterns can selectively favor the translation of specific transcripts, potentially including virulence factors

• Impact on Translational Fidelity:

  • The combined action of tRNA modification enzymes influences translational error rates

  • Research in Pseudomonas species indicates that deficiency in tRNA modifications can increase mutation frequencies

  • This connection between tRNA modification and genomic stability has implications for bacterial adaptation during host infection

Research Methodologies:

• Multi-enzyme Knockout Analysis:

  • Generate single and combinatorial knockouts of tRNA modification enzymes

  • Assess epistatic relationships through comparative phenotyping

  • Measure global translation rates and error frequencies using reporter systems

• tRNA Modification Mapping:

  • Employ high-resolution techniques like HPLC-MS/MS to create comprehensive maps of tRNA modifications under different conditions

  • Correlate modification patterns with translational efficiency of virulence-related genes

• Ribosome Profiling:

  • Perform ribosome profiling in wild-type and tRNA modification mutants

  • Identify transcripts whose translation is specifically affected by particular modifications

  • Connect translational changes to virulence phenotypes

Note: These relationships are inferred from studies in related bacterial systems and would require experimental verification in P. syringae pv. tomato.

How has dusB evolved within the Pseudomonas syringae species complex, and what does this reveal about its functional importance?

Evolutionary analysis of dusB across the P. syringae species complex provides insights into its conservation, adaptation, and functional significance:

• Sequence Conservation Patterns:

  • Core catalytic domains of dusB show high conservation (>90% amino acid identity) across P. syringae pathovars

  • Substrate recognition regions show greater variability, potentially reflecting adaptation to pathovar-specific tRNA pools

  • Key residues involved in flavin binding and catalysis are invariant, highlighting their essential role

• Phylogenetic Distribution:

  • dusB is present in all characterized P. syringae strains, suggesting it provides a fundamental function

  • Comparative genomics indicates that dusB belongs to the core genome rather than the variable genome component

  • This contrasts with many virulence factors, which show pathovar-specific distribution patterns

• Recombination and Selection Pressures:

  • Analysis of P. syringae genomic sequences reveals that genes involved in basic cellular functions, including tRNA processing, are subject to homologous recombination

  • dusB shows evidence of purifying selection (dN/dS < 1), indicating functional constraints

  • This evolutionary pattern supports the critical role of dusB in bacterial fitness

Research Approaches:

• Comparative Sequence Analysis:

  • Alignment of dusB sequences from diverse P. syringae pathovars

  • Calculation of selection coefficients to identify regions under varying selective pressures

  • Mapping of variable residues onto structural models to infer functional significance

• Complementation Studies:

  • Cross-pathovar complementation experiments to test functional equivalence

  • Assessment of the ability of dusB alleles from different pathovars to restore tRNA modification patterns

• Ancestral Sequence Reconstruction:

  • Infer and synthesize ancestral dusB sequences

  • Compare enzymatic properties of ancestral and extant enzymes to track functional evolution

How do the structural and functional properties of dusB compare between plant pathogenic bacteria and other bacterial species?

Comparative analysis of dusB across diverse bacterial species reveals evolutionary adaptations and conserved functional elements:

Comparative Data:

Note: The specific values for P. syringae pv. tomato dusB would require experimental verification.

How might dusB activity influence the translation of virulence factors during different stages of P. syringae pv. tomato infection?

The selective influence of dusB-mediated tRNA modifications on virulence factor translation represents a sophisticated layer of regulation during infection:

• Stage-Specific Translation Regulation:

  • Early infection stage: dusB may facilitate efficient translation of motility genes and initial virulence factors

  • Middle infection stage: As P. syringae establishes in the apoplast, dusB could support translation of T3SS components and effectors

  • Late infection stage: dusB might influence the expression of factors involved in nutrient acquisition and stress tolerance

• Codon Usage Adaptation:

  • Virulence genes in P. syringae often show distinct codon usage patterns compared to housekeeping genes

  • dusB-modified tRNAs may preferentially enhance translation of transcripts with specific codon compositions

  • This mechanism could provide a post-transcriptional layer of virulence regulation

• Response to Host-Derived Signals:

  • Plant-derived molecules, including reactive oxygen species and antimicrobial compounds, may modulate dusB activity

  • The resulting changes in tRNA modification patterns could tune the bacterial translational apparatus to the host environment

  • This adaptation mechanism would allow rapid physiological responses without requiring transcriptional reprogramming

Experimental Approaches:

• Translatomics Analysis:

  • Ribosome profiling comparing wild-type and dusB mutant strains during infection

  • Identification of transcripts with altered translation efficiency

  • Correlation with codon usage patterns and virulence phenotypes

• Reporter Systems:

  • Construction of fluorescent or luminescent reporters fused to virulence gene coding sequences

  • Measurement of translation rates in various genetic backgrounds and under different conditions

  • Isolation of the codon-specific effects using synonymous codon variants

• Temporal Analysis:

  • Time-course studies of tRNA modification patterns during infection progression

  • Correlation with temporal expression patterns of virulence factors

  • Development of models for translation-level regulation of infection stages

What potential exists for targeting dusB as a novel approach for controlling bacterial speck disease?

The essential role of dusB in P. syringae pv. tomato physiology and potential importance in pathogenesis suggests it could serve as a novel target for disease control:

• Advantages as a Target:

  • dusB performs a fundamental function distinct from targets of current antimicrobials

  • High conservation across P. syringae strains could provide broad-spectrum activity

  • Essential nature for bacterial fitness may reduce the likelihood of resistance development

• Potential Intervention Strategies:

  • Small molecule inhibitors of dusB enzymatic activity

  • Compounds that disrupt dusB-tRNA interactions

  • RNA-based approaches to suppress dusB expression

  • Structural analogs of the flavin cofactor to compete for the active site

• Challenges and Considerations:

  • Potential cross-reactivity with host tRNA modification enzymes

  • Necessity for specific delivery to bacterial cells

  • Possibility of partial redundancy with other Dus family enzymes

  • Need for careful assessment of resistance development potential

Research Roadmap:

• Target Validation:

  • Comprehensive phenotypic characterization of dusB mutants

  • Assessment of dusB essentiality under various conditions

  • Determination of the impact of partial dusB inhibition

• High-Throughput Screening:

  • Development of in vitro enzymatic assays suitable for screening

  • Screening of chemical libraries for inhibitory activity

  • Counter-screening against mammalian and plant enzymes to assess specificity

• Lead Optimization:

  • Structure-based design using crystal structures

  • Medicinal chemistry to enhance potency and specificity

  • Assessment of efficacy in plant infection models

• Resistance Assessment:

  • In vitro evolution experiments to identify potential resistance mechanisms

  • Genomics of adapted strains to map resistance mutations

  • Dual-targeting approaches to minimize resistance development

Note: These approaches represent theoretical strategies based on understanding of enzymatic functions and bacterial physiology.

What are the current limitations in studying dusB function in P. syringae pv. tomato, and how might they be overcome?

Several technical challenges currently limit our complete understanding of dusB function in P. syringae pv. tomato:

• Genetic Manipulation Challenges:

  • Potential essentiality of dusB may complicate knockout studies

  • Limited availability of conditional expression systems in P. syringae

  • Polar effects on neighboring genes during genetic manipulation

• Analytical Limitations:

  • Difficulty in accurately quantifying low-abundance tRNA modifications

  • Challenges in discriminating direct from indirect effects of dusB disruption

  • Limited structural information specific to P. syringae tRNA modification enzymes

• In Planta Analysis Complications:

  • Difficulty isolating sufficient bacterial RNA from infected plant tissues

  • Distinguishing bacterial from plant tRNA modifications

  • Challenges in measuring translation dynamics during infection

Innovative Solutions:

• Advanced Genetic Tools:

  • Implementation of CRISPRi for tunable gene repression rather than deletion

  • Development of degron-based systems for conditional protein depletion

  • Application of recombineering approaches using P. syringae RecTE proteins

• Improved Analytical Methods:

  • Application of nanopore direct RNA sequencing for modification mapping

  • Development of more sensitive mass spectrometry approaches for tRNA analysis

  • Single-cell techniques to measure translation in individual bacteria during infection

• Novel In Planta Approaches:

  • Bacterial TRAP-seq to isolate bacterial translatomes from infected tissues

  • Biosensors for real-time monitoring of tRNA modification states

  • Metabolic labeling approaches to specifically tag bacterial translation products

What emerging technologies could revolutionize our understanding of tRNA modifications in bacterial pathogenesis?

Several cutting-edge technologies offer transformative potential for understanding dusB and tRNA modifications in bacterial pathogenesis:

• Single-Molecule Sequencing Technologies:

  • Direct RNA sequencing using nanopore technology can detect modified nucleosides without chemical treatment

  • The ability to sequence full-length tRNA molecules preserves modification context

  • This approach could enable dynamic profiling of the entire "epitranscriptome" during infection

• Advanced Structural Biology Approaches:

  • Cryo-electron microscopy of enzyme-tRNA complexes at near-atomic resolution

  • Hydrogen-deuterium exchange mass spectrometry to map dynamic interactions

  • Time-resolved X-ray crystallography to capture catalytic intermediates

• Systems Biology Integration:

  • Multi-omics approaches combining transcriptomics, proteomics, and tRNA modification analysis

  • Machine learning to identify patterns connecting tRNA modifications with translational outcomes

  • Network analysis to position tRNA modifications within the broader virulence regulatory network

• In Situ Technologies:

  • Development of modification-specific fluorescent probes for in vivo imaging

  • CRISPR-based RNA tracking systems for visualizing tRNA dynamics during infection

  • Spatially resolved transcriptomics to map bacterial translation activities within infected tissues

Future Research Directions:

• Comprehensive Modification Mapping:

  • Create complete maps of all tRNA modifications in P. syringae under different conditions

  • Identify modification patterns specific to infection-relevant stresses

  • Develop predictive models relating modifications to translational outputs

• Translation Regulation Networks:

  • Explore how multiple tRNA modification enzymes work together

  • Identify regulatory networks controlling tRNA modification during infection

  • Map connections between environmental sensing and translational adaptation

• Host-Pathogen tRNA Interactions:

  • Investigate potential transfer of tRNAs or tRNA fragments between pathogen and host

  • Examine host mechanisms targeting pathogen tRNA modifications

  • Explore evolutionary arms races involving tRNA modification systems