Recombinant Pseudomonas aeruginosa Oligoribonuclease (orn)

What is Pseudomonas aeruginosa Oligoribonuclease (orn) and what is its primary function?

Oligoribonuclease (orn) is a highly conserved bacterial 3′ to 5′ exonuclease that specifically degrades small RNA oligomers consisting of 2-5 nucleotides, known as nanoRNAs, converting them to mononucleotides. Orn plays two critical roles in P. aeruginosa: (1) it participates in RNA turnover by degrading nanoRNAs, which can otherwise function as primers for transcription initiation and alter global gene expression, and (2) it serves a crucial function in c-di-GMP signaling by degrading 5′-phosphoguanylyl-(3′,5′)-guanosine (pGpG), the intermediate product of c-di-GMP degradation by EAL domain-containing phosphodiesterases (PDEs) .

How is orn structurally and functionally conserved across bacterial species?

Orn is highly conserved among Actinobacteria and various Proteobacteria, including Beta- and Delta-proteobacteria, suggesting its fundamental importance to bacterial physiology. The E. coli ortholog of Orn can alleviate pGpG accumulation and restore wild-type phenotypes to a P. aeruginosa Δorn mutant, indicating functional conservation across bacterial species . This conservation extends to the enzymatic properties, as both E. coli and P. aeruginosa Orn are manganese (Mn²⁺)-dependent 3′→5′ exonucleases that produce 5′-phosphorylated ribonucleotide monomers from polyribonucleotides .

What are the biochemical properties of recombinant P. aeruginosa orn?

Recombinant P. aeruginosa Orn functions as a manganese-dependent 3′→5′ exonuclease that efficiently degrades small RNA oligomers. The enzyme shows a preference for purine-rich nanoRNA substrates and can readily degrade pGpG to GMP with an initial reaction rate of approximately 10⁻⁷ mol/min under laboratory conditions . Importantly, Orn exhibits no enzymatic activity against c-di-GMP itself, but rather acts specifically on the pGpG intermediate . The ribonuclease activity of Orn is calcium-insensitive, distinguishing it from some other nucleases .

What are the recommended methods for expressing and purifying recombinant P. aeruginosa orn?

For recombinant expression and purification of P. aeruginosa Orn, researchers typically use an E. coli expression system with a His-tag fusion for affinity purification. The purification workflow generally involves the following steps:

• Cloning the orn gene from P. aeruginosa into an expression vector with an appropriate tag (His-tag is commonly used)

• Transforming the construct into an E. coli expression strain (BL21(DE3) or similar)

• Inducing protein expression with IPTG at optimal temperature and duration

• Lysing cells using sonication or pressure homogenization

• Purifying the protein using nickel affinity chromatography

• Further purification by size exclusion chromatography

• Confirming purity by SDS-PAGE and activity by enzymatic assays

The resulting recombinant protein should be stored with manganese ions (Mn²⁺) as cofactors to maintain enzymatic activity.

How can researchers assess orn enzymatic activity in vitro?

Orn enzymatic activity can be assessed through multiple complementary approaches:

• Substrate degradation assays: Using synthetic RNA oligomers of 2-5 nucleotides as substrates and monitoring their degradation using HPLC or capillary electrophoresis.

• pGpG degradation assay: Incubating purified orn with pGpG and monitoring conversion to GMP using liquid chromatography-tandem mass spectrometry (LC-MS/MS) .

• Radiolabeled substrate assays: Using ³²P-labeled RNA oligomers and analyzing the products by polyacrylamide gel electrophoresis.

• Fluorescent substrate assays: Employing fluorescently labeled RNA substrates and measuring changes in fluorescence as degradation occurs.

For optimal activity, assays should be conducted in buffer containing Mn²⁺ ions (typically 1-5 mM), at pH 7.5-8.0, and at temperatures of 25-37°C .

What genetic approaches are used to study orn function in P. aeruginosa?

Researchers employ several genetic approaches to study orn function in P. aeruginosa:

• Gene deletion: Construction of precise Δorn deletion mutants using suicide plasmid homologous recombination methods . This approach allows researchers to study the consequences of complete orn loss on bacterial physiology.

• Complementation studies: Reintroducing wild-type orn on plasmids to verify that phenotypes observed in the mutant are specifically due to orn deletion.

• Point mutations: Creating catalytically inactive variants through site-directed mutagenesis to distinguish between enzymatic and potential structural roles.

• Heterologous expression: Expressing orn orthologs from other bacterial species (e.g., E. coli) in P. aeruginosa Δorn mutants to assess functional conservation .

• Regulatable expression systems: Using inducible promoters to control orn expression levels and study dose-dependent effects.

How does orn affect c-di-GMP homeostasis in P. aeruginosa?

Orn plays a critical role in c-di-GMP homeostasis by degrading pGpG, the intermediate product of c-di-GMP degradation. In the c-di-GMP signaling pathway, diguanylate cyclases synthesize c-di-GMP, while EAL domain-containing phosphodiesterases (PDEs) hydrolyze it to produce pGpG. Orn then degrades pGpG to GMP, completing the degradation pathway .

When orn is deleted, pGpG accumulates to high levels (approximately 88 μM in Δorn cells compared to undetectable levels in wild-type). This accumulation inhibits EAL-dependent PDEs through product inhibition, leading to increased intracellular c-di-GMP levels (approximately 3.3 μM in Δorn cells compared to 1.7 μM in wild-type) . This inhibition can be alleviated by the addition of purified Orn. Thus, Orn provides homeostatic control of intracellular pGpG under native physiological conditions, which is fundamental to proper c-di-GMP signal transduction .

What is the molecular mechanism by which pGpG accumulation affects c-di-GMP signaling?

The molecular mechanism involves product inhibition of EAL-domain PDEs by pGpG. When pGpG accumulates in the absence of orn, it binds to the active site of EAL-domain PDEs, inhibiting their ability to degrade c-di-GMP. This creates a negative feedback loop: as PDEs degrade c-di-GMP to pGpG, the accumulating pGpG inhibits further PDE activity, leading to elevated c-di-GMP levels .

Experimental evidence shows that adding pGpG to cell lysates slows c-di-GMP degradation, and this inhibition can be relieved by adding purified orn. Similarly, pGpG inhibits the activity of purified EAL-domain PDEs (such as RocR, PA2133, and PvrR) from P. aeruginosa in vitro . This mechanism explains why deletion of orn leads to elevated c-di-GMP levels despite not directly affecting c-di-GMP synthesis or degradation enzymes.

How do intracellular nucleotide levels change in orn deletion mutants compared to wild-type P. aeruginosa?

The deletion of orn leads to dramatic changes in intracellular nucleotide concentrations, particularly for pGpG and related molecules. The estimated intracellular concentrations in wild-type versus Δorn mutant strains are summarized in the following table:

These data demonstrate that the absence of orn leads to massive accumulation of pGpG, a nearly 2-fold increase in c-di-GMP, and a substantial decrease in GMP levels . The decrease in GMP likely results from the failure to complete the c-di-GMP degradation pathway, while the elevated pGpG inhibits EAL-domain PDEs, leading to c-di-GMP accumulation.

How does orn deletion affect biofilm formation and extracellular polysaccharide production?

Deletion of orn in P. aeruginosa leads to significantly enhanced biofilm formation and overproduction of extracellular polysaccharides, particularly Pel and Psl . This phenotype is directly linked to elevated c-di-GMP levels in the Δorn mutant, as c-di-GMP promotes the production of these extracellular polysaccharides while repressing bacterial motility .

The overproduction of Pel polysaccharide in the Δorn mutant is particularly significant as it recruits extracellular DNA (eDNA) to the bacterial surface, forming a protective shield. This Pel-eDNA matrix can trap antimicrobials such as polymyxin B and provide extracellular protection to bacterial cells . Scanning electron microscopy reveals increased colony aggregation and enhanced biofilm architecture in Δorn mutants compared to wild-type strains .

What impact does orn deletion have on P. aeruginosa virulence and metabolism?

The impact of orn deletion on P. aeruginosa virulence and metabolism is multifaceted:

• Metabolism: Δorn mutants exhibit altered global metabolism and reduced intracellular energy metabolism . Proteomic analysis using TMT quantitative labeling reveals widespread changes in metabolic pathways.

• Quorum sensing: The quorum sensing system becomes dysregulated in Δorn mutants, affecting coordination of population-level behaviors .

• Motility: Bacterial motility is reduced in Δorn mutants, consistent with elevated c-di-GMP levels, which are known to repress motility .

• Virulence factors: Production of important virulence factors including pyocyanin and rhamnolipids is reduced . Additionally, the type three secretion system (T3SS) is affected by orn mutation .

• Pathogenicity: Despite the reduction in certain virulence factors, Δorn mutants show enhanced pathogenicity in both in vitro cell models and in vivo animal skin trauma models . This enhanced pathogenicity likely results from increased biofilm formation and resistance to host defenses.

What is the relationship between orn function and bacterial stress responses?

Orn plays a significant role in bacterial stress responses, particularly in relation to antibiotic exposure and oxidative stress:

• Oxidative stress: Orn affects the translation of the catalase gene katA, which is crucial for defense against oxidative stress. Mutation of orn reduces katA translation, increasing bacterial susceptibility to oxidative stresses .

• SOS response: Mutation of orn activates the SOS DNA damage response, which reduces PrtR protein levels. PrtR is a negative regulator of pyocin biosynthesis genes, so its reduction leads to overproduction of pyocins .

• Environmental adaptation: Orn is essential for P. aeruginosa to adapt to changing environmental conditions, with its deletion leading to dysregulation of stress response pathways .

• Biofilm-mediated protection: The enhanced biofilm formation in Δorn mutants provides protection against various environmental stressors, including antimicrobials and host immune defenses .

How does orn deletion affect P. aeruginosa susceptibility to polymyxin antibiotics?

Deletion of orn increases P. aeruginosa survival following polymyxin B treatment. In wild-type strain PA14, mutation of orn significantly enhanced bacterial survival when exposed to polymyxin B . This increased tolerance is attributed to the overproduction of Pel polysaccharide, which binds to extracellular DNA (eDNA) on the bacterial surface, trapping polymyxin B molecules and preventing them from reaching their targets in the bacterial membrane .

Importantly, while orn deletion increases survival during polymyxin B exposure, it does not affect the minimum inhibitory concentration (MIC) of polymyxin B. When treated with polymyxin B at the clinical breakpoint concentration (2 μg/mL), both wild-type and Δorn mutant strains show similar killing (>10⁶-fold reduction in viable cells) . This indicates that Pel-mediated protection is one of several mechanisms contributing to polymyxin resistance, providing approximately 20-fold increased survival under specific conditions .

What is the molecular mechanism behind the Pel-mediated polymyxin tolerance in orn mutants?

The molecular mechanism of Pel-mediated polymyxin tolerance in orn mutants involves a protective shield formed by Pel polysaccharide and extracellular DNA:

• The Δorn mutation leads to elevated c-di-GMP levels, which upregulate Pel polysaccharide production .

• Pel polysaccharide binds to extracellular DNA (eDNA) on the bacterial surface, forming a protective matrix .

• This Pel-eDNA matrix acts as a shield by trapping polymyxin B molecules, preventing them from reaching their targets in the bacterial membrane .

• Experiments using fluorescently labeled polymyxin B demonstrate increased surface-bound polymyxin B in Δorn mutants compared to wild-type strains .

• When Pel synthesis genes are deleted or cells are treated with a Pel hydrolase, the surface-bound polymyxin B is reduced, and bacterial survival decreases, confirming the protective role of Pel .

How does orn affect P. aeruginosa sensitivity to other classes of antibiotics?

Orn affects P. aeruginosa sensitivity to multiple antibiotic classes through distinct mechanisms:

• Fluoroquinolones: Mutation of orn increases susceptibility to fluoroquinolone antibiotics by activating the SOS response. This activation reduces PrtR protein levels, leading to overproduction of pyocins and increased cell lysis following fluoroquinolone treatment .

• Aminoglycosides and β-lactams: Orn contributes to tolerance against these antibiotics by controlling translation of the catalase gene katA. Mutation of orn reduces katA translation, increasing susceptibility to oxidative stress generated during antibiotic treatment .

• Polymyxins: As detailed above, orn deletion increases survival following polymyxin B treatment through a Pel-eDNA shield mechanism .

These contrasting effects (increased susceptibility to some antibiotics but increased tolerance to others) highlight orn's complex role in bacterial physiology and stress responses.

What experimental approaches can be used to study the interplay between orn, pGpG, and EAL-domain PDEs?

Researchers investigating the complex interactions between orn, pGpG, and EAL-domain PDEs can employ several sophisticated experimental approaches:

• In vitro enzyme kinetics: Using purified recombinant orn and EAL-domain PDEs to measure enzyme kinetics with varying concentrations of pGpG. This allows determination of inhibition constants (Ki) and mechanisms (competitive, non-competitive, or uncompetitive) .

• Isothermal titration calorimetry (ITC): To measure binding affinities between pGpG and EAL-domain PDEs directly, providing thermodynamic parameters of the interaction.

• Structural biology approaches: X-ray crystallography or cryo-electron microscopy of EAL domains co-crystallized with pGpG to elucidate the molecular basis of inhibition.

• Genetic approaches: Creating point mutations in EAL domains that maintain catalytic activity but alter pGpG binding affinity, potentially creating PDEs resistant to product inhibition.

• Systems biology approaches: Global transcriptomic and proteomic analyses comparing wild-type, Δorn, and PDE mutant strains to understand downstream effects of pGpG accumulation on gene expression networks.

How can orn be targeted for potential antimicrobial development?

Orn represents a promising target for antimicrobial development due to its essential role in c-di-GMP signaling and its effects on antibiotic susceptibility:

• Direct inhibition: Small molecule inhibitors of orn enzymatic activity could mimic the Δorn phenotype, potentially increasing susceptibility to fluoroquinolones, aminoglycosides, and β-lactams while disrupting bacterial adaptation mechanisms .

• Combination therapy: Orn inhibitors could be used in combination with existing antibiotics to enhance their efficacy. For example, an orn inhibitor might increase fluoroquinolone effectiveness by promoting pyocin-mediated cell lysis .

• Anti-biofilm strategies: Targeting orn could disrupt the c-di-GMP signaling network, potentially interfering with biofilm formation, which is a major contributor to chronic infections and antibiotic tolerance .

• Virulence attenuation: By modulating c-di-GMP levels, orn inhibition could alter bacterial virulence factor expression, potentially attenuating infection without directly killing bacteria .

• Rational drug design: Structural information about orn's active site could guide the development of specific inhibitors with limited off-target effects.

What remaining questions exist about orn's role in bacterial physiology and pathogenesis?

Despite significant advances in understanding orn function, several important questions remain:

• Regulatory mechanisms: How is orn expression and activity regulated in response to environmental signals? Are there post-translational modifications that affect its function?

• Species-specific differences: How does orn function differ among various bacterial pathogens, and can these differences be exploited for species-specific targeting?

• Host interactions: Does orn activity affect host-pathogen interactions beyond biofilm formation and antibiotic tolerance? Are there effects on immune evasion or persistence in chronic infections?

• Alternative substrates: Besides pGpG and small RNAs, does orn have other physiologically relevant substrates that contribute to its effects on bacterial physiology?

• Compensatory mechanisms: Are there alternative pathways for pGpG degradation that can compensate for orn loss under specific conditions?

• Clinical relevance: Do naturally occurring variations in orn expression or activity contribute to P. aeruginosa virulence or antibiotic resistance in clinical isolates from chronic infections?

Addressing these questions will provide deeper insights into the fundamental biology of P. aeruginosa and potentially reveal new strategies for combating this important opportunistic pathogen.