Recombinant Bacillus licheniformis DEAD-box ATP-dependent RNA helicase CshA (cshA)

What is CshA and what are its conserved domains?

CshA is a DEAD-box RNA helicase characterized by 12 conserved sequence motifs involved in ATP binding, RNA binding, and intramolecular interactions. The protein contains a core region with all the characteristic DEAD-box motifs, including the Walker A motif (motif I) that is essential for ATP hydrolysis. In addition to these conserved domains, CshA typically possesses a charged C-terminal region that likely mediates interactions with RNA substrates or protein partners. This architecture is similar to that observed in Staphylococcus aureus CshA, which contains a highly charged (39 positive, 26 negative) C-terminal region of approximately 170 amino acids .

What biochemical activities does CshA exhibit?

CshA displays RNA-dependent ATPase activity, typical of DEAD-box RNA helicases. Kinetic analyses of related CshA proteins show ATP hydrolysis with Michaelis-Menten kinetics. For instance, S. aureus CshA exhibits a KM(ATP) value of approximately 3.7 ± 0.47 mM with a Kcat value of 140 min⁻¹. This activity is abolished when mutations are introduced in the Walker A motif (e.g., K52A mutation), preventing ATP hydrolysis . Additionally, CshA likely possesses ATP-dependent RNA unwinding activity, allowing it to resolve RNA secondary structures that could impede RNA degradation or processing.

How does CshA compare to other bacterial DEAD-box helicases?

Bacterial genomes typically encode multiple DEAD-box RNA helicases with specialized functions. For example, B. subtilis and S. aureus each encode two DEAD-box helicases (CshA and CshB), while E. coli encodes five (CsdA, SrmB, DbpA, RhlE, and RhlB). While most bacterial DEAD-box helicases share the core DEAD-box domains, they differ in their auxiliary domains and specific functions. Some, like DbpA and YxiN, show RNA substrate specificity, while others like RhlB interact with the degradosome complex. CshA appears to function primarily in RNA degradation and adaptation to environmental stresses, particularly cold temperatures .

What expression systems are optimal for producing recombinant B. licheniformis CshA?

For recombinant expression of B. licheniformis CshA, E. coli-based expression systems using vectors with strong inducible promoters (T7, tac) are generally effective. The protein can be tagged with affinity tags such as His₆ for purification by immobilized metal affinity chromatography (IMAC). Based on protocols used for S. aureus CshA, expression at lower temperatures (16-25°C) after induction may improve solubility. When designing expression constructs, it's important to consider whether to include the charged C-terminal region, as this may affect solubility and activity. The table below summarizes recommended expression conditions:

How can I measure the ATPase activity of recombinant CshA?

The RNA-dependent ATPase activity of purified CshA can be measured using several approaches:

• Malachite green phosphate assay: This colorimetric method detects inorganic phosphate released during ATP hydrolysis. Reactions typically contain purified CshA (0.1-1 μM), ATP (1-5 mM), RNA substrate (e.g., poly(U) or total RNA, 50-100 μg/ml), and buffer containing Mg²⁺ (typically 20 mM Tris-HCl pH 7.5, 50 mM NaCl, 5 mM MgCl₂). After incubation at 37°C, the released phosphate is quantified by adding malachite green reagent and measuring absorbance at 620-650 nm.

• Coupled enzyme assay: ATP hydrolysis can be coupled to NADH oxidation using pyruvate kinase and lactate dehydrogenase, allowing continuous monitoring at 340 nm.

For Michaelis-Menten kinetic analysis, ATP concentration should be varied (0.1-10 mM) under conditions of saturating RNA. Controls should include reactions without RNA to demonstrate RNA dependence, and a Walker A motif mutant (e.g., K52A) to confirm specificity .

What methods can be used to study CshA's role in RNA degradation?

To investigate CshA's function in RNA degradation:

• In vivo mRNA half-life measurements: Compare the stability of specific mRNAs or global mRNA populations in wild-type and cshA deletion strains using transcription inhibition (e.g., with rifampicin) followed by RNA isolation and qRT-PCR or RNA-seq at different timepoints.

• In vitro RNA degradation assays: Reconstitute RNA degradation using purified CshA and degradosome components (e.g., RNase J1/J2, PNPase). Labeled RNA substrates can be used to monitor degradation rate by gel electrophoresis.

• Protein-protein interaction studies: Investigate interactions between CshA and degradosome components using bacterial two-hybrid systems, co-immunoprecipitation, or pull-down assays. For instance, S. aureus CshA has been shown to interact with RNases (RNase Y, RNase J1/J2) and PNPase .

• RNA-protein binding assays: Assess CshA's binding to specific RNA substrates using techniques like electrophoretic mobility shift assays (EMSA) or filter binding assays.

How does CshA contribute to cold adaptation in bacteria?

CshA plays a crucial role in bacterial adaptation to low temperatures. Deletion of cshA typically results in cold sensitivity, as observed in S. aureus. This phenotype appears to be partially linked to membrane composition and fatty acid synthesis. In S. aureus, cshA deletion leads to an imbalance between straight-chain and branched-chain fatty acids, which is critical for maintaining membrane fluidity at low temperatures.

Research in S. aureus has shown that the cold-sensitive growth of a cshA mutant can be suppressed by:

• Mutations in the fatty acid synthesis pathway

• Sublethal doses of triclosan (a FASII inhibitor)

• Overexpression of genes involved in branched-chain fatty acid synthesis

The proposed mechanism involves inefficient degradation of the pyruvate dehydrogenase complex mRNA in the absence of CshA, leading to elevated acetyl-CoA levels that favor straight-chain fatty acid production at the expense of branched-chain fatty acids needed for cold adaptation .

What is the relationship between CshA and the bacterial degradosome?

CshA is considered a component of the bacterial RNA degradosome, a multi-protein complex involved in RNA degradation. In Gram-positive bacteria like B. subtilis and S. aureus, CshA interacts with several ribonucleases including RNase Y, RNase J1/J2, and PNPase. These interactions have been demonstrated using bacterial two-hybrid systems and co-immunoprecipitation approaches.

The function of CshA within the degradosome appears similar to that of RhlB in the E. coli degradosome: it likely helps to unwind RNA secondary structures that would otherwise impede the progression of exoribonucleases like PNPase. Evidence suggests that the ATPase activity of S. aureus CshA is stimulated approximately 1.8-fold in the presence of RNase J2, although direct interaction between these proteins was not detected in bacterial two-hybrid assays .

How does CshA affect gene expression and virulence in pathogenic bacteria?

In S. aureus, CshA has been shown to influence virulence through modulation of the agr quorum sensing system. Inactivation of cshA results in dysregulation of biofilm formation and hemolysis through altered agr mRNA stability. Specifically:

• CshA affects the stability of agr mRNA, which encodes the primary quorum sensing system controlling virulence factor expression.

• In a cshA mutant, the dysregulation of biofilm formation and hemolysis is reversed by inactivating the agrA gene, indicating that cshA is genetically upstream of agr.

• CshA appears to maintain a delicate balance of agr mRNA abundance through stability control, which is critical for proper expression of virulence factors .

This regulatory mechanism highlights how RNA helicases can indirectly influence complex cellular behaviors by modulating mRNA stability of key regulatory genes.

What mechanisms determine CshA's substrate specificity?

While most DEAD-box RNA helicases do not display strict sequence specificity, they may preferentially act on certain RNA substrates. For CshA, the mechanisms determining which mRNAs are targeted for degradation remain incompletely understood. Several hypotheses can be tested:

• RNA structural features: CshA may preferentially unwind certain RNA secondary structures. This can be investigated using a library of structured RNA substrates in unwinding assays.

• Degradosome targeting: CshA might be recruited to specific mRNAs through interactions with other degradosome components that recognize sequence or structural features. Chimeric proteins or domain swapping experiments could identify regions involved in substrate recognition.

• Auxiliary protein interactions: Additional protein factors might direct CshA to specific mRNAs. Proteomic approaches (e.g., BioID, proximity labeling) could identify proteins interacting with CshA in vivo.

• C-terminal domain function: The charged C-terminal region may confer substrate specificity. Truncation or mutation of this region would reveal its contribution to substrate selection.

How do post-translational modifications regulate CshA activity?

Post-translational modifications (PTMs) of RNA helicases can modulate their activity, localization, or interactions. While PTMs of bacterial DEAD-box helicases are less studied than their eukaryotic counterparts, several approaches can investigate this aspect:

• Mass spectrometry: Analyze purified native CshA for modifications such as phosphorylation, acetylation, or methylation.

• Phosphoproteomic analysis: Compare global phosphorylation patterns between wild-type and stress conditions to identify potential regulatory modifications on CshA.

• Site-directed mutagenesis: Modify potential PTM sites and assess effects on CshA activity, protein interactions, or in vivo function.

• Kinase/phosphatase screening: Identify enzymes that modify CshA using in vitro assays with purified candidates or kinase/phosphatase inhibitors in vivo.

What is the three-dimensional structure of CshA and how does it relate to function?

Determining the three-dimensional structure of B. licheniformis CshA would provide valuable insights into its mechanism. Approaches include:

• X-ray crystallography: Attempt to crystallize full-length CshA or functional domains (core helicase domain, C-terminal region) in different nucleotide-bound states (apo, ATP, ADP) and with/without RNA substrate.

• Cryo-electron microscopy: For full-length protein or complexes with degradosome components that may be challenging to crystallize.

• Small-angle X-ray scattering (SAXS): To obtain low-resolution envelope structures and study conformational changes upon nucleotide or RNA binding.

• NMR spectroscopy: Suitable for studying dynamics and conformational changes, particularly for isolated domains.

• Homology modeling: Based on structures of related DEAD-box helicases, followed by validation through mutagenesis of predicted functional residues.

Why is my recombinant CshA showing low or no ATPase activity?

Several factors could contribute to low enzymatic activity of recombinant CshA:

• Protein misfolding or aggregation: Try optimizing expression conditions (lower temperature, different E. coli strains) or adding solubility tags (MBP, SUMO).

• Improper buffer conditions: Optimize buffer composition:

  • pH (try range 7.0-8.5)

  • Salt concentration (50-300 mM NaCl)

  • Divalent cations (Mg²⁺, Mn²⁺ at 1-10 mM)

  • Reducing agents (DTT or β-mercaptoethanol)

• RNA substrate quality: Ensure RNA is not degraded and try different RNA substrates (total RNA, synthetic homopolymers, specific structured RNAs).

• Inhibitory contaminants: Improve purification protocol with additional chromatography steps.

• Inactive conformation: ATP binding and hydrolysis by DEAD-box helicases are often conformationally regulated. Try adding RNA first before ATP to promote the active conformation.

How can I address inconsistent results in cshA deletion phenotype studies?

When studying phenotypes of cshA deletion mutants, inconsistent results may occur due to:

• Suppressor mutations: As observed in cold sensitivity studies, cshA deletion strains readily acquire suppressor mutations, particularly in the fatty acid synthesis pathway . Regularly re-sequence your strains, use multiple independent clones, and consider constructing a conditional depletion system rather than a complete deletion.

• Growth conditions: CshA function may be context-dependent. Standardize media composition, temperature, growth phase, and aeration. The cold sensitivity phenotype is typically observed at temperatures below 25°C.

• Strain background effects: Test the deletion in multiple strain backgrounds, as genetic interactions may vary.

• Compensatory mechanisms: Other RNA helicases (e.g., CshB) may partially compensate for CshA loss. Consider constructing double mutants or using transcriptomic approaches to identify compensatory responses.

What controls are essential when studying CshA-RNA interactions?

When investigating CshA-RNA interactions, include these essential controls:

• ATP dependence: Compare binding and unwinding in the presence and absence of ATP.

• ATPase-deficient mutant: Include a Walker A motif mutant (e.g., K52A) that cannot hydrolyze ATP.

• RNA specificity: Test structured versus unstructured RNAs, and include unrelated RNA sequences to assess specificity.

• Cold competition: Add excess unlabeled RNA to confirm specific binding to labeled substrate.

• Protein specificity controls: Include unrelated RNA-binding proteins to demonstrate that binding is specific to CshA.

• Buffer controls: Ensure buffer components (especially Mg²⁺ concentration) do not independently affect RNA structure.

How might CshA be targeted for antimicrobial development?

Given the importance of CshA for bacterial adaptation to stress conditions, it represents a potential target for antimicrobial development. Research strategies could include:

• High-throughput screening: Develop assays based on CshA's ATPase activity to screen for small molecule inhibitors.

• Structure-based drug design: Once structural information is available, use in silico approaches to identify compounds that may bind to active sites or regulatory regions.

• Combination approaches: Since cshA deletion causes cold sensitivity and membrane composition changes, CshA inhibitors might synergize with compounds targeting membrane integrity or fatty acid synthesis.

• Species-specific targeting: Identify structural or functional differences between bacterial CshA proteins that could be exploited for selective targeting.

• Phenotypic potentiation: Test whether CshA inhibition enhances the efficacy of existing antibiotics, particularly under stress conditions.

What is the global impact of CshA on bacterial transcriptome and proteome?

Comprehensive studies of CshA's impact on cellular RNA and protein populations would provide insights into its biological functions:

• RNA-seq: Compare transcriptomes of wild-type and cshA mutant strains under various conditions (exponential growth, stationary phase, cold shock, nutrient limitation).

• Ribosome profiling: Assess whether CshA affects translation efficiency of specific mRNAs.

• CLIP-seq: Identify direct RNA binding targets of CshA in vivo.

• Proteomics: Use quantitative proteomics to determine how CshA affects the cellular proteome, particularly in response to stress.

• Metabolomics: Investigate how CshA-mediated regulation affects metabolic pathways, especially those related to fatty acid synthesis and membrane composition.

How is CshA activity coordinated with other cellular processes?

The regulation and coordination of CshA activity with other cellular processes remains largely unexplored:

• Stress response integration: Investigate how CshA activity is regulated during various stress responses, particularly cold shock.

• Metabolic coordination: Explore connections between RNA decay, carbon metabolism, and membrane composition, as suggested by links between CshA, pyruvate dehydrogenase mRNA stability, and fatty acid synthesis .

• Cell cycle regulation: Determine whether CshA activity or expression varies through the bacterial cell cycle.

• Signal transduction: Identify signaling pathways that regulate CshA activity in response to environmental cues.

• Cross-talk with other RNA processing pathways: Investigate potential coordination between CshA-mediated RNA decay and other RNA processing mechanisms (ribosome assembly, RNA modification, transcription termination).