Recombinant Geobacter sulfurreducens Ribonuclease Y (rny), partial

What is the basic structure of RNase Y in G. sulfurreducens and how does it compare to other bacterial species?

RNase Y in G. sulfurreducens, like its counterparts in other bacteria, is likely to contain several conserved domains that are essential for its functionality. Based on comparative studies with other bacterial species, G. sulfurreducens RNase Y likely possesses:

• An N-terminal membrane-anchoring α-helix

• A disordered coiled-coil domain

• An RNA-binding KH domain

• A metal-chelating HD domain containing the catalytic active site

• A C-terminal region of unknown function

The enzyme belongs to a specific class of ribonucleases that constitute a subgroup within the superfamily of HD proteins, combining the metal-chelating HD domain with the RNA-binding KH domain . Mutational studies in related organisms have shown that single amino acid substitutions of the highly conserved HD doublet greatly reduce ribonucleolytic activity, indicating that the catalytic activity resides within this domain .

What expression systems are most effective for producing recombinant G. sulfurreducens RNase Y?

When expressing recombinant G. sulfurreducens RNase Y, researchers should consider the following approaches based on successful strategies with related enzymes:

Expression Systems Comparison:

When purifying the enzyme, it's crucial to maintain the integrity of the membrane domain if studying the full-length protein. Research with B. subtilis RNase Y has demonstrated that while the membrane anchor is not essential for enzymatic activity, deleting it can significantly alter the enzyme's behavior and cellular localization . For activity studies, consider expressing both the full-length protein and a truncated version lacking the membrane anchor for comparative analysis.

What are the optimal conditions for assessing RNase Y activity in vitro?

Based on studies with related bacterial RNases, the following conditions are recommended for assessing G. sulfurreducens RNase Y activity:

Buffer Components:

• 25-50 mM Tris-HCl (pH 7.5-8.0)

• 50-100 mM NaCl or KCl

• 5-10 mM MgCl₂ (essential cofactor for the HD domain)

• 1-5 mM DTT (reducing agent)

• 0.1-0.5% non-ionic detergent (for full-length membrane-anchored protein)

Key Experimental Parameters:

• Temperature: 30°C (optimal growth temperature for G. sulfurreducens)

• RNA substrates: 5'-monophosphorylated RNAs (preferred substrate in related RNases)

• Control reactions: Include EDTA-containing samples to chelate Mg²⁺ and confirm metal-dependent activity

• Detection methods: 5'-RACE for mapping cleavage sites, gel-based assays for quantifying activity

When designing RNA substrates, consider that RNase Y in S. aureus and S. pyogenes shows a strong preference for cleavage after a purine (particularly G) residue . This specificity might be conserved in G. sulfurreducens RNase Y.

How does RNase Y activity influence extracellular electron transfer in G. sulfurreducens?

G. sulfurreducens is known for its unique ability to transfer electrons to extracellular acceptors like metal oxides and electrodes. The role of RNase Y in this process likely involves:

• Regulation of transcripts encoding electron transfer components:

  • Type IV pili proteins

  • Outer membrane c-type cytochromes

  • Extracellular matrix components

• Processing of polycistronic mRNAs in electron transfer-related operons.

• Modulation of extracellular polysaccharide production:

  • Proper attachment and anchoring of external c-type cytochromes necessary for a conductive biofilm network requires extracellular polysaccharides

  • RNA processing likely influences the expression of genes involved in polysaccharide export

In metabolic studies, G. sulfurreducens mutants with defects in extracellular electron transfer components show altered growth phenotypes when cultivated with different electron donors/acceptors . Similar approaches could be used to evaluate the impact of RNase Y mutations on electron transfer capabilities.

What role might RNase Y play in G. sulfurreducens biofilm formation?

G. sulfurreducens forms conductive biofilms that are essential for its interactions with metal oxides and electrodes. Based on findings from related bacteria and studies on G. sulfurreducens biofilm formation:

• RNase Y likely regulates transcripts encoding:

  • Extracellular matrix proteins

  • Polysaccharide synthesis and export systems

  • Cell-to-cell communication components

• The membrane localization of RNase Y positions it to potentially regulate transcripts at the cell envelope, where many biofilm formation processes occur .

Experimental approaches to investigate this relationship:

• Gene deletion or depletion studies targeting RNase Y

• Transcriptomic analysis comparing wild-type and RNase Y mutant biofilms

• Complementation studies with RNase Y variants (membrane-anchored vs. soluble)

• Microscopic visualization of biofilm architecture in mutant strains

How conserved is RNase Y function across different bacterial species compared to G. sulfurreducens?

RNase Y orthologs occur in approximately 40% of sequenced eubacterial species , allowing for comparative analysis:

Conservation Table of RNase Y Across Species:

RNase Y in B. subtilis has been shown to move rapidly along the membrane in the form of dynamic short-lived foci, which become more abundant and increase in size following transcription arrest . This behavior contrasts with RNase E in E. coli, where foci formation depends on RNA substrate presence, highlighting fundamental differences between RNase E- and RNase Y-based degradation machineries .

Organisms containing RNase Y also typically have either an RNase E/G-like enzyme or RNase J. Some bacteria have all three enzymes, as seen in certain Frankia and Salinispora species of Actinobacteria and 25-30% of Bacillales and Clostridia , raising interesting questions about functional redundancy and specialization.

What experimental approaches can be used to identify specific RNA targets of G. sulfurreducens RNase Y?

To identify RNA targets of G. sulfurreducens RNase Y, researchers can employ several complementary approaches:

• Global RNA end mapping:

  • Comparing RNA 5' ends between wild-type and RNase Y mutant strains

  • RNA ends present in wild-type but absent in mutants indicate potential RNase Y cleavage sites

• In vitro cleavage assays:

  • Testing synthetic or in vitro transcribed RNAs with purified recombinant RNase Y

  • Analyzing cleavage products by primer extension or high-resolution gel electrophoresis

• Transcriptome-wide approaches:

  • RNA-seq to identify differentially abundant transcripts in RNase Y mutants

  • CLIP-seq (Cross-linking immunoprecipitation followed by sequencing) to identify direct RNA-protein interactions

• Comparative genomics:

  • Identifying conserved RNA motifs in potential target RNAs across related Geobacter species

  • Analyzing sequence context around known cleavage sites in homologous transcripts

From studies in S. aureus and S. pyogenes, there is a strong preference for RNase Y cleavage immediately downstream of a guanosine (G) residue . Similar sequence preferences might exist in G. sulfurreducens RNase Y, which could guide the search for potential target sites.

How might RNase Y function in syntrophic relationships between G. sulfurreducens and other bacteria?

G. sulfurreducens forms important syntrophic relationships with other microorganisms, particularly in processes involving interspecies electron transfer. The role of RNase Y in these relationships may include:

• Regulation of transcripts involved in interspecies interactions:

  • Genes encoding electron transfer components

  • Metabolic pathways that enable syntrophic growth

• Response to environmental signals during syntrophic growth:

  • Processing of transcripts in response to metabolite exchange

  • Regulation of gene expression during aggregate formation

Studies have shown that G. sulfurreducens forms aggregates with denitrifying bacteria like Diaphorobacter, accelerating nitrate removal rates . In these aggregates, G. sulfurreducens accounts for approximately 17% of the microbial population, and the formation of these aggregates suggests a syntrophic interaction mechanism where G. sulfurreducens and denitrifiers combine their metabolism to create a fast and energy-efficient route to utilize acetate and reduce nitrate .

Research approaches to investigate RNase Y's role in syntrophy:

• Transcriptomic analysis of G. sulfurreducens during syntrophic growth versus pure culture

• Creation of RNase Y mutants and evaluation of their ability to form syntrophic relationships

• Identification of RNase Y-dependent changes in gene expression specific to syntrophic conditions

What are the methodological considerations for using RNase Y as a tool in RNA structure-function studies?

RNase Y's specific cleavage preferences make it potentially valuable as a tool for RNA structure-function studies:

• Mapping accessible regions in RNA structures:

  • Single-stranded regions accessible to RNase Y cleavage

  • Identification of structural motifs that protect from or promote cleavage

• Generating defined RNA fragments:

  • Site-specific cleavage to produce RNA fragments for functional studies

  • Creation of truncated RNAs to map functional domains

• Methodological considerations:

  • Use a truncated, soluble version of RNase Y without the membrane anchor for in vitro applications

  • Optimize reaction conditions to achieve controlled, limited cleavage

  • Include proper controls to account for non-specific degradation

Based on studies with RNase Y from S. aureus, researchers should design experiments considering that the nucleotide sequence surrounding a cleavage site is sufficient for recognition and can convert a non-substrate RNA into a substrate . Additionally, systematic mutational analyses can determine which nucleotides in the vicinity of the cleavage site are required for efficient cleavage and which determine the exact cleavage position .

How can manipulation of RNase Y expression be used to enhance G. sulfurreducens performance in bioremediation applications?

G. sulfurreducens is valued for its ability to reduce toxic metals and remediate contaminated environments. Manipulating RNase Y expression could potentially enhance these capabilities:

• Optimizing expression of metal reduction pathways:

  • Fine-tuning mRNA stability of key cytochromes and electron transfer components

  • Enhancing expression of genes involved in metal binding and reduction

• Improving biofilm formation on contaminated surfaces:

  • Modulating degradation of transcripts encoding extracellular matrix components

  • Enhancing cell-to-surface attachment mechanisms

• Adaptation to variable environmental conditions:

  • Regulating stress response pathways during exposure to toxic metals

  • Modifying gene expression patterns to optimize growth in contaminated environments

Studies have shown that G. sulfurreducens can be used for uranium bioremediation, with its interactions with sulfate-reducing bacteria playing an important role in this process . The dynamics of these interactions could potentially be optimized through targeted manipulation of RNA processing and stability via RNase Y.

What methodological approaches can be used to study the impact of RNase Y on riboswitch function in G. sulfurreducens?

G. sulfurreducens utilizes riboswitch sensors, such as those for the signaling molecule cyclic AMP-GMP (cAG), to regulate gene expression . RNase Y may play a role in riboswitch turnover, as seen in other bacteria . To investigate this:

• In vitro analysis of riboswitch processing:

  • Synthesize riboswitch RNAs with different ligand-bound states

  • Assess cleavage patterns with purified recombinant RNase Y

  • Determine how ligand binding affects RNase Y accessibility

• In vivo riboswitch function analysis:

  • Create reporter constructs with riboswitch elements

  • Compare reporter expression in wild-type and RNase Y mutant strains

  • Analyze riboswitch RNA stability and processing in vivo

• Structure-function relationships:

  • Map RNase Y cleavage sites within riboswitch structures

  • Determine how structural changes upon ligand binding affect RNase Y activity

  • Investigate the kinetics of riboswitch degradation relative to regulatory function

Research has shown that G. sulfurreducens uses GEMM-I riboswitch sensors for the signaling molecule cAG to regulate extracellular metal-reducing activity . Understanding how RNase Y affects the turnover of these regulatory elements could provide insights into the control of this important metabolic process.

What are the major challenges in expressing and purifying functional recombinant G. sulfurreducens RNase Y?

Working with recombinant G. sulfurreducens RNase Y presents several methodological challenges:

• Membrane protein expression issues:

  • Potential toxicity due to membrane disruption in heterologous hosts

  • Protein aggregation and inclusion body formation

  • Improper folding in non-native membrane environments

• Purification considerations:

  • Requirement for detergents or membrane mimetics to maintain native conformation

  • Potential loss of activity during solubilization and purification steps

  • Need for metal cofactors to maintain enzymatic activity

• Stability concerns:

  • Membrane proteins often have reduced stability when removed from their native environment

  • Potential for proteolytic degradation during purification

  • Activity may be sensitive to buffer conditions and storage

Recommended approaches:

• Consider expressing the catalytic domain separately from the membrane anchor

• Use mild detergents (DDM, LMNG) for membrane protein extraction

• Include protease inhibitors throughout the purification process

• Test activity immediately after purification

Studies with RNase Y from B. subtilis have shown that the membrane anchor, while not essential for enzymatic activity in vitro, is important for proper cellular function . This suggests that both full-length and truncated versions of G. sulfurreducens RNase Y should be characterized to understand its complete functional properties.

How can researchers address discrepancies between in vitro and in vivo activities of RNase Y in G. sulfurreducens?

Researchers often encounter differences between the behavior of RNase Y in purified in vitro systems versus its activity in the cellular context:

• Factors contributing to discrepancies:

  • Absence of protein partners that modify activity in vivo

  • Different RNA structural landscapes in vitro versus in cells

  • Loss of membrane context that may influence substrate recognition

  • Simplified buffer conditions that don't reflect cellular complexity

• Methodological solutions:

  • Use membrane vesicles or proteoliposomes to better mimic native environment

  • Reconstitute with known interacting proteins identified through proteomics

  • Compare results from both in vitro and in vivo experimental approaches

  • Develop cell extract systems that maintain native protein interactions

In B. subtilis, the Y-complex (composed of YaaT, YlbF, and YmcA) influences the specificity of RNase Y activity in vivo and shifts the assembly status of RNase Y toward fewer and smaller complexes, thereby increasing cleavage efficiency . Similar specificity factors might exist in G. sulfurreducens and should be considered when interpreting in vitro results.

How might CRISPR-Cas technologies be applied to study RNase Y function in G. sulfurreducens?

CRISPR-Cas technologies offer powerful approaches for investigating RNase Y function in G. sulfurreducens:

• Gene editing applications:

  • Creating precise mutations in the rny gene to study domain-specific functions

  • Introducing tagged versions of RNase Y for localization and interaction studies

  • Generating conditional expression systems for temporal control of RNase Y levels

• CRISPR interference (CRISPRi) approaches:

  • Partial knockdown of RNase Y expression to avoid lethal effects

  • Tunable repression to study dose-dependent effects

  • Targeting specific transcripts to study their processing by RNase Y

• CRISPR activation (CRISPRa) strategies:

  • Upregulation of RNase Y to study the effects of increased RNA processing

  • Enhancing expression of potential RNase Y interacting partners

  • Activating compensatory pathways in RNase Y-depleted cells

A gene disruption approach has been successfully applied in G. sulfurreducens using methods like recombinant PCR and single-step recombination . These established genetic manipulation techniques could be combined with newer CRISPR-based approaches for more precise genetic analysis.

What are the implications of RNase Y-mediated regulation for metabolic engineering of G. sulfurreducens?

Understanding RNase Y function has significant implications for metabolic engineering efforts in G. sulfurreducens:

• Optimizing RNA stability for enhanced metabolic flux:

  • Modifying RNase Y recognition sites in key metabolic transcripts

  • Engineering mRNA structures to control degradation rates

  • Balancing expression levels in multi-enzyme pathways

• Enhancing extracellular electron transfer capabilities:

  • Stabilizing transcripts encoding electron transfer components

  • Optimizing expression of biofilm formation genes

  • Fine-tuning metabolic pathways that support electron transfer

• Adaptation to different growth conditions:

  • Engineering RNA processing patterns for optimal growth with different electron donors/acceptors

  • Modifying post-transcriptional regulation for adaptation to different carbon sources

  • Enhancing stress tolerance through RNA stability modulation

Model-based analysis of the G. sulfurreducens metabolic network has identified several redundant pathways , and understanding how RNase Y regulates the expression of enzymes in these pathways could help optimize metabolic flux for specific applications.