Recombinant Pseudomonas syringae pv. syringae Leucyl/phenylalanyl-tRNA--protein transferase (aat)

What is the genetic organization of the aat locus in Pseudomonas syringae?

The aat (amino acid transport) locus in Pseudomonas syringae is organized as part of a cluster that includes genes encoding an ABC transporter system (AatQMP) and a two-component signaling system (AauSR). In P. syringae strain DC3000, this locus contains the substrate-binding protein AatJ, which functions in coordination with the sensor kinase AauS and response regulator AauR . This organization allows for both amino acid transport functions and signal transduction capabilities. The AatJ protein specifically serves as a periplasmic binding protein that initiates the amino acid transport process by binding to acidic amino acids like aspartate and glutamate in the periplasmic space. The AatQMP components form the membrane transport machinery that completes the amino acid uptake process once signals are received through AatJ .

How does aat function differ between Pseudomonas syringae and other bacterial species like E. coli?

In Escherichia coli, the aat gene encodes the leucyl/phenylalanyl-tRNA-protein transferase (L/F-transferase), which catalyzes the transfer of Leu, Phe, and to a lesser extent, Met and Trp from aminoacyl-tRNAs to the amino terminus of acceptor proteins . This transferase is essential for the N-end rule pathway of protein degradation for proteins bearing amino-terminal Arg and Lys residues . In contrast, the aat gene in P. syringae has been co-opted for a different role beyond its metabolic functions - it has evolved to regulate virulence factors. Specifically, in P. syringae, the aat gene products participate in perceiving plant-derived amino acids as signals that trigger expression of the T3SS, a critical virulence determinant . This represents an evolutionary repurposing of a conserved amino acid transport system for pathogenicity functions. The biochemical mechanism involves substrate binding and signal transduction rather than direct protein modification as seen in E. coli.

Why is P. syringae considered an important model organism for studying plant-pathogen interactions?

Pseudomonas syringae serves as an exceptional model organism for studying plant-pathogen interactions due to several key attributes. It exists as over 50 different pathovars that collectively infect a diverse range of plant species, while individual pathovars typically have a narrow host range . This makes it ideal for comparative studies of host specificity. P. syringae is genetically tractable and has had numerous strains fully sequenced, providing abundant genomic resources for researchers . The pathogen employs a well-characterized Type III Secretion System as its primary virulence mechanism, allowing detailed studies of plant immunity and bacterial pathogenicity . Additionally, P. syringae has interesting environmental adaptations, including the production of ice nucleation active proteins and its capability to persist epiphytically on plant surfaces before invasion . These characteristics make it relevant for both fundamental research and applied agricultural sciences. The recent discovery of AatJ-AauS-AauR sensing of host-derived amino acids demonstrates how P. syringae has evolved sophisticated host perception mechanisms, further solidifying its importance as a model system .

How does the aat gene contribute to Pseudomonas syringae virulence in plants?

The aat gene contributes to P. syringae virulence through its central role in host signal perception and subsequent T3SS regulation. The AatJ protein, encoded within the aat locus, functions as a substrate-binding protein that specifically recognizes host-derived acidic amino acids, particularly aspartate and glutamate . When P. syringae encounters these plant-exuded metabolites during infection, AatJ binds them and initiates signaling through the AauS-AauR two-component system . Experimental evidence supports this role, as mutants of P. syringae strain DC3000 lacking aatJ, aauS, or aauR express lower levels of T3SS genes in response to aspartate and glutamate, demonstrating reduced T3SS deployment and diminished virulence during infection of Arabidopsis . This sensory system represents a direct molecular link between host metabolite detection and virulence factor deployment. The importance of this pathway is further emphasized by conservation of the AauR binding motif upstream of hrpRS (T3SS regulators) in all P. syringae strains with a canonical T3SS, suggesting the AauR regulation of T3SS is an ancient and fundamental virulence mechanism across the species complex .

What is the relationship between aat and the Type III Secretion System (T3SS) in Pseudomonas syringae?

The relationship between the aat gene products and the T3SS in P. syringae represents a sophisticated regulatory network connecting host signal perception to virulence factor deployment. The AauS-AauR two-component system, associated with the aat locus, directly regulates expression of T3SS-encoding genes . Specifically, the response regulator AauR binds to a conserved AauR-binding motif (Rbm) located in the promoter region upstream of hrpR and hrpS genes, which encode master regulators of the T3SS . This molecular mechanism connects perception of plant-derived aspartate and glutamate signals to the transcriptional activation of T3SS genes. The significance of this relationship is demonstrated by experimental evidence showing that mutation of aauR results in decreased T3SS expression and reduced bacterial growth in host plants . Furthermore, the Rbm upstream of hrpRS is conserved across all P. syringae strains with a canonical T3SS, suggesting that AauR regulation of hrpRS is an ancient and conserved virulence mechanism . This relationship illustrates how P. syringae has repurposed an amino acid transport system for the regulation of its primary virulence machinery, creating a direct link between host sensing and pathogenicity.

What host signals regulate aat gene function during infection?

Host-derived acidic amino acids, particularly aspartate and glutamate, serve as the primary signals regulating aat gene function during P. syringae infection . These amino acids are exuded by plant tissues and act as specific chemical signals that the bacterium perceives through the AatJ substrate-binding protein . A metabolomics analysis identified aspartate, along with citrate and 4-hydroxybenzoate, as potent inducers of T3SS-encoding genes in P. syringae pv. tomato DC3000 . The importance of these metabolite signals for infection outcomes has been experimentally demonstrated, as mutations affecting plant signal production can alter resistance to P. syringae infection . When the bacterium encounters these host-derived signals, AatJ binds them and initiates a signaling cascade through the AauS-AauR two-component system, ultimately leading to upregulation of T3SS genes . This sensory system allows P. syringae to recognize when it has reached an appropriate host environment and deploy its virulence machinery accordingly. The evolution of this signaling pathway represents a sophisticated adaptation that enables the pathogen to coordinate its virulence response with specific host-derived chemical cues, demonstrating the intricate molecular dialogue occurring during plant-pathogen interactions.

What are the optimal expression systems for producing recombinant Pseudomonas syringae aat proteins?

The optimal expression systems for producing recombinant P. syringae aat proteins depend on the specific research objectives and downstream applications. Based on studies with similar bacterial transferases, E. coli-based expression systems remain the most widely used for initial characterization. For the L/F-transferase encoded by the aat gene, affinity tagging approaches have proven successful, as demonstrated in previous work with E. coli aat . The recombinant L/F-transferase expressed with affinity tags maintains activity comparable to wild-type enzyme . For expression of the P. syringae AatJ substrate-binding protein and AauS-AauR two-component system components, several methodological considerations must be addressed. Expression vectors with inducible promoters (such as T7 or tac) provide controlled expression, while fusion partners like His6, GST, or MBP can facilitate purification and potentially enhance solubility . For membrane-associated components like AauS sensor kinase, specialized expression systems that accommodate membrane proteins may be necessary, potentially including detergent-compatible purification methods or membrane mimetic systems.

The table below summarizes key expression system considerations for different aat locus components:

What methodologies can be used to assess the enzymatic activity of purified recombinant aat proteins?

Assessing the enzymatic activity of purified recombinant aat proteins requires specific methodologies tailored to their functional roles. For L/F-transferase activity, aminoacyl transfer assays can be performed using radioactively labeled or fluorescently tagged aminoacyl-tRNAs as substrates, with detection of the transfer to acceptor peptides or proteins . These assays typically monitor the transfer of leucine, phenylalanine, and to a lesser extent, methionine and tryptophan from aminoacyl-tRNAs to the amino terminus of acceptor proteins . For AatJ substrate-binding activity, isothermal titration calorimetry (ITC) or surface plasmon resonance (SPR) can quantify binding affinity and kinetics with acidic amino acids such as aspartate and glutamate. For the AauS-AauR two-component system, phosphotransfer assays using radioactive ATP (γ-32P-ATP) can assess autophosphorylation of AauS and subsequent phosphotransfer to AauR.

For functional assays in a more biologically relevant context, reporter gene constructs can be employed. For example, plasmids containing T3SS gene promoters (such as hrpRS) fused to reporter genes like gfp or luciferase can be introduced into bacterial cells expressing recombinant AauS-AauR to assess signal-dependent activation . This approach allows testing of the complete signaling pathway from amino acid binding through transcriptional activation. Additionally, circular dichroism can be used to assess proper folding of recombinant proteins by confirming expected secondary structure elements, such as the predominantly α-helical structure reported for L/F-transferase .

How can mutagenesis approaches advance our understanding of aat protein structure-function relationships?

Mutagenesis approaches offer powerful tools for dissecting the structure-function relationships of aat proteins in P. syringae. Site-directed mutagenesis targeting conserved residues can identify amino acids critical for substrate binding, catalysis, or protein-protein interactions. For AatJ, mutations in the predicted binding pocket can determine which residues are essential for recognizing acidic amino acids like aspartate and glutamate . For the AauS sensor kinase, mutagenesis of predicted phosphorylation sites and conserved catalytic residues can elucidate the mechanism of signal transduction. In the case of AauR, mutations in the DNA-binding domain can help characterize how it recognizes the AauR-binding motif (Rbm) upstream of target genes like hrpRS .

Random mutagenesis approaches such as error-prone PCR combined with functional screening can identify previously unknown functional residues or domains. For instance, screening for mutants with altered responsiveness to aspartate and glutamate could reveal new insights into signal perception mechanisms. Domain swapping experiments between homologous proteins from different species can identify regions responsible for species-specific functions, potentially explaining how the aat system was co-opted for virulence regulation in P. syringae . Trypsin digestion studies have already revealed structural information about L/F-transferase, identifying a proline-rich NH2 terminus and a globular core . Similar approaches with P. syringae aat proteins could identify functional domains and inform targeted mutagenesis strategies. The results from these mutagenesis studies can guide the development of specific inhibitors targeting the aat system as potential anti-bacterial strategies against plant pathogens.

How did the aat locus evolve from a metabolic function to a virulence regulatory role in Pseudomonas syringae?

The evolutionary co-option of the aat locus from its ancestral metabolic role to a virulence regulatory function in P. syringae represents a fascinating example of pathoadaptation. Comparative genomic analyses suggest that the AauS-AauR and AatJ proteins, encoded by the aat/aau locus, are conserved in pseudomonads including non-pathogens, indicating that their transport-associated functions predate their virulence role . The critical evolutionary innovation appears to be the acquisition of the AauR-binding motif (Rbm) upstream of the hrpRS genes encoding T3SS regulators . This cis-regulatory change established a direct molecular link between amino acid sensing and T3SS gene expression. The conservation of this Rbm in essentially all pathogenic P. syringae strains with a canonical T3SS suggests this co-option event occurred early in P. syringae evolution and has been maintained throughout subsequent diversification across different host plants .

This evolutionary trajectory likely involved several steps: 1) horizontal acquisition of T3SS genes by an ancestral Pseudomonas strain, 2) random genomic mutations creating a primitive AauR binding site near hrpRS genes, 3) selection for enhanced binding affinity as this linkage improved pathogen fitness, and 4) maintenance of this regulatory linkage across speciation events as P. syringae diversified to infect different hosts. The repurposing of a metabolic sensing system for virulence regulation is particularly elegant because it directly connects nutrient acquisition with virulence factor deployment, allowing coordinated responses to host conditions. This evolutionary history demonstrates how relatively simple regulatory changes can dramatically alter pathogen virulence capabilities through rewiring of existing signaling pathways.

What are the contradictions in current research regarding the specificity of AatJ-AauS-AauR sensing in different P. syringae pathovars?

Current research presents several unresolved contradictions regarding the specificity of AatJ-AauS-AauR sensing across different P. syringae pathovars. While studies with P. syringae pv. tomato DC3000 demonstrated that aspartate and glutamate serve as potent inducers of T3SS genes through the AatJ-AauS-AauR pathway , the spectrum of amino acids recognized by this system in other pathovars remains incompletely characterized. Some studies suggest the possibility of pathovar-specific adaptations in amino acid sensing that might correlate with the biochemical composition of different host plants, but comprehensive comparative analyses across multiple pathovars are lacking. Additionally, while the AauR-binding motif is conserved across P. syringae strains with canonical T3SS, subtle sequence variations might influence binding affinity and regulatory outcomes in different genetic backgrounds .

Another area of contradiction involves the relative contribution of the AatJ-AauS-AauR system versus other plant signal sensing pathways in regulating virulence. Some research indicates that additional plant metabolites beyond acidic amino acids, including citrate and 4-hydroxybenzoate, can induce T3SS genes , raising questions about whether these signals act through independent or interconnected pathways. The demonstration that an aauR deletion mutant of P. syringae pv. syringae B728a (a bean pathogen) showed decreased T3SS expression and reduced growth in host plants supports conservation of AauR function across pathovars , but the quantitative importance of this pathway might vary depending on host environment and bacterial genetic background. These contradictions highlight the need for systematic comparative studies examining signal specificity, regulatory network architecture, and contribution to virulence across the P. syringae species complex.

How can systems biology approaches integrate aat function into broader virulence regulatory networks?

Systems biology approaches offer powerful frameworks for integrating aat function into broader virulence regulatory networks in P. syringae. Multi-omics studies combining transcriptomics, proteomics, and metabolomics can map the complete regulatory cascade initiated by AatJ-AauS-AauR signaling . RNA-seq analysis comparing wild-type and aat/aau mutant strains under various conditions can identify the complete regulon controlled by this pathway beyond the known T3SS genes. Chromatin immunoprecipitation sequencing (ChIP-seq) with tagged AauR can comprehensively map all genomic binding sites, potentially revealing additional targets beyond hrpRS . Metabolomics approaches can identify the full spectrum of plant-derived signals recognized by this system and how their concentrations vary across different infection stages and host species.

Network modeling presents another valuable systems biology approach. Mathematical models integrating transcriptional, post-transcriptional, and post-translational regulatory mechanisms can predict how the AatJ-AauS-AauR system interacts with other signaling pathways to coordinate virulence. These models can be iteratively refined through experimental validation, ultimately providing predictive power for understanding how P. syringae responds to complex and dynamic host environments. Comparative systems analyses across multiple P. syringae pathovars can reveal both conserved and variable aspects of the regulatory network, potentially explaining host specificity differences. The table below outlines key systems biology approaches for studying aat function:

What targeted inhibitor approaches could disrupt aat-mediated virulence signaling?

Developing targeted inhibitors against aat-mediated virulence signaling represents a promising strategy for controlling P. syringae infections across multiple host plants. Several approaches warrant investigation based on our current understanding of the AatJ-AauS-AauR system . Small molecule antagonists that mimic acidic amino acids but block AatJ binding could prevent initial signal perception. Alternatively, compounds that bind AatJ but fail to trigger conformational changes necessary for AauS activation could act as competitive inhibitors. For the two-component system, molecules targeting the ATP-binding domain of AauS could inhibit autophosphorylation, while compounds disrupting the phosphotransfer interface between AauS and AauR could block signal transmission. At the DNA-binding level, molecules designed to interfere with AauR binding to its target sequence upstream of hrpRS would prevent T3SS induction .

High-throughput screening approaches utilizing biosensor strains with reporter genes downstream of AauR-regulated promoters could identify candidate inhibitors from chemical libraries. Structure-based drug design, informed by crystallographic data on AatJ, AauS, and AauR proteins, could enable rational development of inhibitors targeting specific functional domains. Peptide-based inhibitors mimicking interaction interfaces could selectively disrupt protein-protein interactions within this signaling pathway. The conservation of the AauR-binding motif across P. syringae pathovars suggests that inhibitors targeting this regulatory pathway could potentially provide broad-spectrum protection against multiple P. syringae strains , offering an advantage over strategies targeting pathogen-specific factors.

How can CRISPR-Cas9 technologies advance our understanding of aat regulation and function?

CRISPR-Cas9 technologies offer unprecedented precision for investigating aat regulation and function in P. syringae. Genome editing applications can generate clean deletions or specific point mutations in aat locus genes without polar effects on neighboring genes, overcoming limitations of traditional mutagenesis approaches. This precision allows researchers to examine the contribution of specific domains or residues within AatJ, AauS, and AauR to signal perception, transduction, and transcriptional activation . CRISPR interference (CRISPRi) systems using catalytically inactive Cas9 can achieve tunable repression of target genes, enabling studies of how varying expression levels of aat locus components affect signaling dynamics and virulence outcomes.

For regulatory studies, CRISPR-based approaches can precisely modify the AauR-binding motif (Rbm) upstream of hrpRS , allowing systematic analysis of sequence requirements for AauR binding and activation. Base editing techniques can introduce single nucleotide changes in the Rbm to examine how subtle sequence variations affect binding affinity and regulatory outcomes. CRISPR activation (CRISPRa) systems can be employed to artificially activate AauR-regulated genes, bypassing the normal signaling cascade to determine downstream effects independent of upstream inputs. For evolutionary studies, CRISPR-Cas9 could recreate hypothesized ancestral states of the regulatory linkage between AauR and hrpRS to test evolutionary models of how this pathway was co-opted for virulence . These approaches could be applied across multiple P. syringae pathovars to understand how the aat regulatory system has evolved and specialized for different host environments.

What interdisciplinary approaches could yield new insights into aat function in plant-microbe interactions?

Interdisciplinary approaches combining molecular biology, structural biology, plant science, and computational biology could significantly advance our understanding of aat function in plant-microbe interactions. Synthetic biology approaches could reconstruct the AatJ-AauS-AauR signaling pathway in heterologous hosts to examine its sufficiency for amino acid sensing and explore potential applications in engineered biosensors. Advanced imaging techniques, such as single-molecule tracking in living bacterial cells, could reveal the spatial organization and dynamics of AatJ-AauS-AauR signaling components during host interaction . Cryo-electron microscopy could capture the structural changes that occur during signal perception and transduction, potentially revealing mechanistic details not accessible through genetic or biochemical approaches alone.

From the plant perspective, metabolomic profiling of apoplastic fluid from various host species could identify the complete spectrum of signals available to P. syringae during infection and examine how these profiles change in response to infection . Engineering plant hosts with altered amino acid exudation profiles could test hypotheses about the importance of specific signals for pathogen virulence. Systems-level modeling integrating both plant and bacterial responses could provide insights into the co-evolutionary dynamics of this signaling system. Comparative phylogenomic approaches examining the aat locus and its regulatory targets across the Pseudomonadaceae family could reconstruct the evolutionary history of this signaling pathway and identify key innovations that enabled its co-option for virulence regulation . These interdisciplinary approaches would provide a more comprehensive understanding of how the aat system functions at the molecular, cellular, and ecological levels in plant-microbe interactions.