Recombinant Arcobacter butzleri Ribonuclease 3 (rnc)

What is Arcobacter butzleri and why is it significant for ribonuclease research?

Arcobacter butzleri is a Gram-negative foodborne pathogen belonging to the Arcobacteraceae family. This emerging pathogen is found in water, food, and various organisms including farm animals, clams, and fish. A. butzleri has been isolated from human stool samples and is associated with gastrointestinal symptoms such as diarrhea . The significance of studying ribonuclease 3 (rnc) in A. butzleri lies in understanding RNA processing mechanisms in this emerging pathogen. While limited research exists specifically on A. butzleri rnc, studies on related bacterial ribonucleases suggest important roles in RNA maturation, gene regulation, and potentially virulence. Drawing parallels from research on Rhodobacter capsulatus RNase III, which is involved in processing ribosomal RNA and removing extra stem-loop structures , A. butzleri rnc likely plays similar crucial roles in RNA metabolism that may influence pathogenicity and survival.

How does Ribonuclease III (RNase III) function in bacterial systems?

Ribonuclease III functions as an endoribonuclease that specifically cleaves double-stranded RNA (dsRNA) structures. In bacterial systems, RNase III plays essential roles in RNA processing and maturation pathways. From research on homologous systems like Rhodobacter capsulatus, we know that RNase III contains a double-stranded RNA-binding domain (dsRBD) that is critical for substrate recognition and binding . The enzyme is involved in processing ribosomal RNA precursors, where it removes stem-loop structures to generate functional rRNA molecules. In R. capsulatus, RNase III participates in the fragmentation of large subunit ribosomal RNA into pieces of 14S and 16S that together form the functional equivalent of intact 23S rRNA . Beyond rRNA processing, RNase III regulates gene expression by processing mRNAs and small RNAs, influencing their stability and translation efficiency. The cloned Rhodobacter enzyme has been shown to substitute RNase III activity in RNase III-deficient E. coli strains, demonstrating functional conservation across bacterial species .

What genomic characteristics of A. butzleri are relevant to rnc gene expression and function?

The genomic analysis of A. butzleri strains reveals several characteristics relevant to rnc gene expression and function. A. butzleri has a relatively small genome size of approximately 2.3 Mb with a notably low GC content of about 26.8% . This low GC content may influence codon usage and expression efficiency of the rnc gene. The genome typically contains around 2300-2400 predicted genes, including coding sequences (CDS), tRNAs, ncRNAs, and rRNAs . The genomic organization of rnc in A. butzleri has not been specifically characterized in the provided search results, but by drawing parallels with Rhodobacter, where rnc is located in an operon with the lep gene encoding leader peptidase , we might expect unique genomic arrangements in A. butzleri that could affect rnc regulation. Full genome characterization of A. butzleri, as shown in Table 1 below (adapted from search result #4), provides a foundation for understanding the genomic context in which rnc operates:

What are the optimal methods for isolating the rnc gene from A. butzleri clinical or environmental isolates?

Isolating the rnc gene from A. butzleri begins with proper sample collection and strain isolation. For environmental samples, especially from water sources, concentration techniques like centrifugation (at approximately 10,000 ×g for 20 minutes at 20°C) followed by selective enrichment in Bolton Broth with selective supplements is recommended . For clinical isolates, stool samples should be processed with appropriate selective media. After isolation and confirmation of A. butzleri strains, genomic DNA extraction can be performed using commercial kits designed for Gram-negative bacteria.

For targeted amplification of the rnc gene, design specific primers based on conserved regions identified through comparative genomics of available A. butzleri genomes. PCR conditions should be optimized considering the low GC content (26.8%) of A. butzleri . Alternatively, whole genome sequencing approaches combining single-molecule real-time (SMRT) and Illumina sequencing technologies can provide the complete genome sequence , from which the rnc gene can be identified through bioinformatic analysis. For confirmation, qPCR assays targeting species-specific genes like hsp60 can be used to verify the identity of A. butzleri isolates before proceeding with rnc isolation .

What expression systems are most effective for producing recombinant A. butzleri Ribonuclease III?

The selection of an appropriate expression system for recombinant A. butzleri Ribonuclease III requires careful consideration of several factors. E. coli-based expression systems, particularly those designed for toxic or membrane-associated proteins, often provide good yields for bacterial recombinant proteins. Given the functional similarity between bacterial RNase III enzymes, an RNase III-deficient E. coli strain would be an excellent host system, as demonstrated with the Rhodobacter RNase III, which successfully complemented RNase III activity in such strains .

When designing the expression construct, consider codon optimization to account for the low GC content (26.8%) of A. butzleri , which differs significantly from E. coli. The expression vector should include an appropriate promoter (e.g., T7 or tac), a suitable affinity tag (His-tag or GST) for purification, and potentially a solubility-enhancing fusion partner if protein solubility becomes an issue.

Expression conditions require careful optimization. Since A. butzleri grows optimally at lower temperatures (30°C in microaerophilic conditions) , expressing the recombinant protein at lower temperatures (16-25°C) may improve proper folding and solubility. Additionally, consider using specialized E. coli strains that express rare tRNAs to accommodate the codon usage bias of A. butzleri. For functional verification, the recombinant enzyme can be tested by complementation assays in RNase III-deficient strains, similar to studies with Rhodobacter RNase III .

What purification strategy yields the highest activity of recombinant A. butzleri RNase III?

A multi-step purification strategy is recommended to obtain high-activity recombinant A. butzleri RNase III. After expression in an appropriate E. coli host system, begin with cell lysis under conditions that preserve enzyme activity. Since RNase III binds double-stranded RNA, it's crucial to include nuclease treatment steps to remove bound nucleic acids that might co-purify with the enzyme.

The initial purification step typically employs affinity chromatography based on the fusion tag incorporated in the expression construct. For His-tagged RNase III, immobilized metal affinity chromatography (IMAC) using Ni-NTA resin is effective, with elution performed using an imidazole gradient to minimize co-purification of contaminating proteins. This should be followed by ion-exchange chromatography to separate protein variants with different charge properties.

A final polishing step using size-exclusion chromatography helps remove aggregates and ensures a homogeneous preparation. Throughout the purification process, include reducing agents (DTT or β-mercaptoethanol) to protect critical cysteine residues within the enzyme. Activity assessments at each purification stage using standard RNase III substrates will help optimize the protocol. The final purified enzyme should be stored with glycerol (15-20%) at -80°C in small aliquots to minimize freeze-thaw cycles that could reduce activity.

How does the structure of A. butzleri RNase III compare to other bacterial ribonucleases?

Based on comparative analysis with known bacterial RNase III enzymes, particularly the Rhodobacter homolog, A. butzleri RNase III likely possesses the hallmark structural features of this enzyme family. RNase III enzymes typically consist of an N-terminal nuclease domain and a C-terminal double-stranded RNA-binding domain (dsRBD) . The nuclease domain contains the catalytic site responsible for RNA cleavage, while the dsRBD is essential for binding to double-stranded RNA substrates.

The Rhodobacter RNase III, which shares functional homology with other bacterial RNase III enzymes, has 226 amino acids with a molecular weight of 25.5 kDa . A. butzleri RNase III likely has a similar molecular weight and amino acid composition, though the exact sequence would need to be determined through gene cloning and sequencing. The functional conservation across bacterial species is demonstrated by the ability of Rhodobacter RNase III to substitute for E. coli RNase III activity , suggesting structural conservation of critical domains.

The dsRBD is particularly important, as demonstrated in the Rhodobacter Fm65 mutant, where a frameshift mutation resulting in loss of the dsRBD rendered the enzyme inactive . This highlights the essential nature of this domain for substrate recognition and enzyme function. Detailed structural analysis through techniques like X-ray crystallography or cryo-EM would be necessary to fully characterize the unique structural features of A. butzleri RNase III and compare them with other bacterial homologs.

What substrate specificity does A. butzleri RNase III exhibit in experimental settings?

A. butzleri RNase III, like other bacterial RNase III enzymes, likely exhibits specificity for double-stranded RNA structures. While specific experimental data on A. butzleri RNase III is not provided in the search results, insights can be drawn from studies on homologous enzymes. RNase III typically recognizes and cleaves RNA stem-loop structures, such as those found in ribosomal RNA precursors.

In experimental settings, the substrate specificity can be assessed using synthetic double-stranded RNA substrates with varying sequence and structural features. The enzyme would likely show preference for certain sequence contexts within double-stranded regions, similar to other bacterial RNase III enzymes. Additionally, the presence of specific structural elements, such as bulges or mismatches within the double-stranded region, might influence cleavage efficiency.

One relevant experimental approach would be to examine the role of A. butzleri RNase III in processing the 23S rRNA, similar to the function observed in Rhodobacter where RNase III removes an extra stem-loop structure from the 23S rRNA . This could involve in vitro cleavage assays using purified recombinant enzyme and transcribed RNA substrates, followed by analysis of cleavage products by gel electrophoresis or sequencing.

How do mutations in the rnc gene affect A. butzleri virulence and adaptive capabilities?

Mutations in the rnc gene likely have significant implications for A. butzleri virulence and adaptation, though specific experimental data linking rnc mutations to A. butzleri virulence is not presented in the search results. Drawing parallels from the Rhodobacter study, where a frameshift mutation resulting in loss of the dsRBD rendered the RNase III enzyme inactive , similar mutations in A. butzleri rnc would disrupt RNA processing functions.

Since RNase III plays crucial roles in RNA maturation and gene regulation, mutations affecting its function could impact several virulence-associated processes. A. butzleri possesses various virulence genes, as evidenced by transcriptome analysis showing upregulation of putative virulence genes during infection of human intestinal cell models . Impaired RNase III function could alter the expression or processing of these virulence factors.

Additionally, RNase III is involved in processing rRNA, which is essential for protein synthesis and bacterial growth. Mutations in rnc might lead to altered ribosome biogenesis, potentially affecting growth rates and stress responses. This could impact the ability of A. butzleri to adapt to various environmental conditions or host niches.

The relationship between rnc mutations and antibiotic resistance is another important consideration. A. butzleri exhibits variable resistance to antibiotics such as ampicillin, ciprofloxacin, erythromycin, and tetracycline . If rnc mutations affect the expression of antibiotic resistance genes or stress response pathways, they could potentially modulate antibiotic susceptibility patterns, further influencing the adaptive capabilities of A. butzleri in clinical settings.

How can recombinant A. butzleri RNase III be utilized for studying RNA processing mechanisms in emerging pathogens?

Recombinant A. butzleri RNase III serves as a valuable tool for investigating RNA processing mechanisms in emerging pathogens. By producing and characterizing this enzyme, researchers can conduct comparative studies of RNA processing across various bacterial species, including other emerging foodborne pathogens. This approach allows for the identification of conserved and divergent aspects of RNA metabolism that may contribute to pathogenicity.

In vitro RNA processing assays using purified recombinant A. butzleri RNase III can reveal specific cleavage patterns and substrate preferences. These findings can be compared with transcriptomic data from A. butzleri infection models, such as the mucus-producing human intestinal in vitro model (Caco-2/HT29-MTX-E12) described in search result #1, to correlate RNA processing events with gene expression patterns during infection.

Additionally, recombinant A. butzleri RNase III can be used to identify novel RNA substrates through techniques like CLIP-seq (crosslinking immunoprecipitation followed by sequencing), providing insights into the role of RNA processing in regulating virulence gene expression. The enzyme can also serve as a target for screening potential inhibitors that might disrupt essential RNA processing events, potentially leading to novel antimicrobial strategies against A. butzleri and related emerging pathogens that are increasingly showing resistance to conventional antibiotics .

What role does A. butzleri RNase III play in regulating virulence gene expression during host infection?

Although the search results don't directly address the role of A. butzleri RNase III in virulence gene regulation, we can hypothesize its involvement based on general RNase III functions and available data on A. butzleri virulence. Transcriptome analysis of A. butzleri during infection of human intestinal cell models revealed upregulation of several putative virulence genes . RNase III likely participates in processing and regulating the expression of these virulence factors through various mechanisms.

RNase III may process polycistronic mRNAs encoding virulence factors, influencing their stability and translation efficiency. Additionally, it might regulate the expression of virulence genes indirectly by processing small regulatory RNAs that control virulence gene expression post-transcriptionally. The TonB complex genes (tonB, exbB, and exbD), which were upregulated in A. butzleri strain LMG 11119 with the greatest colonization ability , represent potential targets for RNase III-mediated regulation.

Iron acquisition is critical for bacterial pathogenesis, and genes related to iron transport were differentially expressed during cell model colonization by A. butzleri . RNase III may regulate iron acquisition systems at the post-transcriptional level, thereby influencing the bacterium's ability to obtain essential iron during infection. Furthermore, stress responses are crucial for bacterial adaptation to the host environment, and RNase III might regulate stress response pathways that contribute to A. butzleri survival during infection.

What are the implications of A. butzleri RNase III research for developing novel antimicrobial strategies?

Research on A. butzleri RNase III opens several avenues for developing novel antimicrobial strategies against this emerging pathogen. As an essential enzyme involved in RNA processing and gene regulation, RNase III represents a potential target for antimicrobial development. The unique structural features of A. butzleri RNase III, particularly in the dsRNA-binding domain and catalytic site, might allow for the design of specific inhibitors that disrupt its function without affecting host enzymes.

The growing concern about antibiotic resistance in A. butzleri, as evidenced by studies identifying resistance to multiple antibiotics including ampicillin, ciprofloxacin, erythromycin, and tetracycline , necessitates the exploration of alternative antimicrobial approaches. Targeting essential RNA processing pathways through RNase III inhibition represents one such strategy. Additionally, understanding the role of RNase III in regulating virulence gene expression could lead to anti-virulence strategies that disarm the pathogen without imposing strong selective pressure for resistance development.

Structural analysis of A. butzleri RNase III in complex with its RNA substrates could provide templates for structure-based drug design, potentially leading to the development of small molecule inhibitors. Furthermore, the identification of RNase III-dependent regulatory RNA elements might reveal novel RNA-based therapeutic targets. As molecular cut-off values for A. butzleri susceptibility testing continue to be refined , incorporating RNase III inhibition assays into antimicrobial susceptibility testing protocols could provide valuable information for developing combination therapies against this emerging pathogen.

How can CRISPR-Cas9 technology be applied for studying rnc gene function in A. butzleri?

CRISPR-Cas9 technology offers powerful approaches for studying rnc gene function in A. butzleri through precise genetic manipulation. To implement this methodology, researchers should first design guide RNAs (gRNAs) targeting specific regions of the rnc gene, taking into account the low GC content (26.8%) of the A. butzleri genome which may require optimization of gRNA design parameters.

For gene knockout studies, the CRISPR-Cas9 system can be delivered via plasmid vectors optimized for A. butzleri, potentially using shuttle vectors that can replicate in both E. coli and A. butzleri. The system should include a selectable marker appropriate for A. butzleri. For homology-directed repair and precise gene editing, homology arms of sufficient length (typically 500-1000 bp) flanking the target site should be included.

Verification of successful editing can be performed using a combination of PCR, sequencing, and functional assays. For functional characterization, phenotypic changes in rnc mutants can be assessed by examining RNA processing patterns, particularly of ribosomal RNAs, which are known substrates of RNase III . Additionally, transcriptome analysis of wild-type versus rnc mutants can reveal the broader impact of RNase III on gene expression profiles.

CRISPR interference (CRISPRi) using catalytically inactive Cas9 (dCas9) offers an alternative approach for studying rnc gene function through transcriptional repression rather than DNA cleavage. This method may be particularly useful for studying essential genes where complete knockout might be lethal. The impact of rnc repression on virulence can be assessed using in vitro infection models such as the mucus-producing human intestinal model described in search result #1.

What high-throughput screening methods can identify novel substrates for A. butzleri RNase III?

Several high-throughput screening approaches can effectively identify novel substrates for A. butzleri RNase III. CLIP-seq (UV crosslinking and immunoprecipitation followed by high-throughput sequencing) represents a powerful method for this purpose. In this approach, purified recombinant A. butzleri RNase III with an appropriate tag (e.g., His-tag or FLAG-tag) is expressed in A. butzleri or a heterologous host. UV crosslinking creates covalent bonds between the enzyme and its RNA substrates, which are then immunoprecipitated using antibodies against the tag. The captured RNA-protein complexes are treated with proteases, and the RNA fragments are sequenced to identify binding sites and potential cleavage targets.

An alternative approach involves RNA-seq analysis comparing wild-type A. butzleri with rnc mutants or knockdowns. This method can reveal RNA species that accumulate or diminish in the absence of functional RNase III, providing insights into its substrate repertoire. For in vitro substrate screening, a library of structured RNAs can be incubated with purified recombinant A. butzleri RNase III, followed by high-throughput sequencing to identify cleaved species.

Structure-based screening methods can also be employed, where computational approaches are used to predict RNA secondary structures across the A. butzleri transcriptome, identifying potential stem-loop structures that resemble known RNase III substrates. These predicted substrates can then be validated experimentally. Additionally, ribosome profiling comparing wild-type and rnc mutants can reveal translational impacts of RNase III deficiency, potentially identifying mRNAs subject to RNase III-mediated regulation.

How can multi-omics approaches integrate rnc gene function with A. butzleri virulence mechanisms?

Multi-omics approaches offer comprehensive strategies for connecting rnc gene function with A. butzleri virulence mechanisms. A coordinated genomics, transcriptomics, and proteomics approach can reveal the cascading effects of rnc gene activity across multiple levels of cellular function. Genome sequencing of multiple A. butzleri strains with varying virulence potentials, similar to the three strains with variable virulence described in search result #1, can identify rnc gene variants and their genomic context, providing the foundation for comparative studies.

Transcriptome analysis using RNA-seq comparing wild-type and rnc mutants during infection of cell models can identify genes and pathways affected by RNase III deficiency, with particular attention to known virulence factors such as those related to the TonB complex and iron transport . This should be complemented with ribosome profiling to assess translational changes, as RNase III-mediated RNA processing can influence translation efficiency.

Proteomics analysis using LC-MS/MS can detect changes in protein abundance resulting from altered RNA processing, particularly focusing on virulence factors. Post-translational modification profiling can reveal downstream effects on signaling pathways involved in virulence regulation. Additionally, metabolomics analysis can identify changes in metabolic pathways that might contribute to virulence or adaptation to host environments.

Integration of these multi-omics datasets requires sophisticated bioinformatics approaches, including network analysis to identify regulatory networks connecting rnc to virulence mechanisms. Time-course experiments during infection can capture the dynamic nature of these regulatory processes. Finally, validation of key findings through targeted experiments, such as deletion or complementation of specific genes identified in the multi-omics analysis, can confirm the mechanistic links between rnc and virulence.