Recombinant Lactobacillus plantarum Ribosome-binding factor A (rbfA)

What is Ribosome-binding factor A (RbfA) and what is its role in bacterial cold adaptation?

RbfA is a cold-shock adaptation protein essential for efficient processing of 16S rRNA. It interacts with the 5'-terminal helix (helix I) of 16S rRNA and is critical for the formation of translation initiation-capable 30S ribosomal subunits at low temperatures. RbfA belongs to a large family of small proteins found in most bacterial organisms, making it an important target for structural proteomics. The structure of RbfAΔ25 (a 108-residue construct with 25 residues removed from the carboxyl terminus) has been determined by heteronuclear NMR methods, revealing an α+β fold containing three helices and three β-strands, arranged as α1-β1-β2-α2-α3-β3 . This structure has type-II KH-domain fold topology, related to conserved KH sequence family proteins, characterized by a helix-kink-helix motif with a conserved AxG sequence replacing the GxxG sequence found in other KH domains .

Why is Lactiplantibacillus plantarum a suitable host for recombinant protein expression?

L. plantarum represents an optimal expression system for recombinant proteins due to several key attributes:

• Safety profile: It has "Generally Recognized As Safe" (GRAS) status and a history of safe use in humans

• Genetic tractability: Its genome shows remarkable plasticity, facilitating genetic manipulation

• Inherent adjuvanticity: Self-adjuvant properties make it weakly immunogenic

• Ecological diversity: Found across various niches, demonstrating adaptability

• Probiotic capabilities: Confers health benefits through gut colonization

Research has demonstrated that L. plantarum can efficiently express heterologous proteins both intracellularly and on its cell surface. In comparative studies, recombinant L. plantarum has achieved protein expression levels ranging from moderate to strong, depending on the promoter and vector system employed . The development of specific expression vectors like pSIP-based systems has further enhanced its utility as a recombinant protein production platform .

What are the structural and functional similarities between RbfA and other bacterial cold-shock proteins?

The structure of RbfAΔ25 shows the greatest similarity to the KH domain of the E. coli Era GTPase, while its electrostatic field distribution most closely resembles the KH1 domain of the NusA protein from Thermotoga maritima, another cold-shock associated RNA-binding protein. Notably, both RbfA and NusA are regulated within the same E. coli operon. The structural and functional similarities between RbfA, NusA, and other bacterial type II KH domains suggest previously unsuspected evolutionary relationships between these cold-shock associated proteins .

Comparative analysis of their functional properties reveals:

What are the optimal promoter systems for expressing recombinant RbfA in L. plantarum?

For the efficient expression of recombinant RbfA in L. plantarum, several promoter systems have demonstrated notable efficacy. Based on comparative studies, the following promoter options present distinct advantages for different research objectives:

• Constitutive promoters:

  • The P11 promoter, derived from an rRNA promoter from L. plantarum WCSF1, shows strong activity comparable to inducible systems .

  • The Ptuf promoters from both L. plantarum (Ptuf33) and L. buchneri (Ptuf34) drive high-level constitutive expression .

  • The heterologous PtlpA promoter from Salmonella typhimurium has demonstrated five-fold higher expression levels than previously used strong promoters like P48 and P23 .

• Inducible systems:

  • The pSIP vectors encoding two-component signaling systems induced by autoinducer peptides offer dose-dependent expression control .

  • The newly identified phage-derived promoter/repressor system has shown nearly 9-fold higher expression than previously reported strongest promoters in L. plantarum WCFS1, with the repressor able to reduce expression nearly 500-fold .

For optimal RbfA expression, which may require fine-tuning due to its role in ribosome assembly, the following strategies are recommended:

• For constitutive expression: Use the PtlpA promoter with the high-copy pCDLbu-1ΔEc plasmid backbone for maximum yield.

• For controlled expression: Employ the phage-derived promoter/repressor system, which offers unprecedented control over expression levels in L. plantarum.

• For temperature-regulated expression: Consider naturally cold-inducible promoters that would complement RbfA's physiological role.

How can the spacer region between the Shine-Dalgarno sequence and start codon be optimized for RbfA expression?

Optimizing the spacer region between the Shine-Dalgarno sequence (SDS) and the start codon is critical for translation efficiency in L. plantarum. Experimental evidence has demonstrated that the optimal spacer length consists of 8 nucleotides, with both elongation and shortening of this sequence resulting in gradual down-regulation of gene expression .

For RbfA expression specifically, the following recommendations are supported by research data:

• Use an 8-nucleotide spacer between the SDS and start codon for optimal translation initiation.

• When designing the construct, consider using the RBS from the slpB gene from L. buchneri CD034, which shows better alignment with the SDS core sequence and corresponds to one of the most abundantly expressed genes in Lactobacillus species .

• Systematically test spacer lengths between 5-12 nucleotides if fine-tuning of expression levels is required, as this parameter can be used as a precise regulatory mechanism.

This optimization is particularly relevant for RbfA expression, as its physiological role involves ribosome binding and it may therefore be sensitive to translation initiation efficiency.

What vector systems are recommended for high-level expression of RbfA in L. plantarum?

For high-level expression of RbfA in L. plantarum, the choice of vector system is crucial. Research has identified several effective options:

• High-copy number vectors:

  • Vectors containing the origin of replication from L. buchneri CD034 plasmid pCD034-1 (e.g., pCDLbu-1ΔEc) have demonstrated approximately 200 copies per chromosome and consistently yield two-fold higher protein expression compared to low-copy alternatives .

• Low-copy number vectors:

  • Vectors based on the p256 origin of replication provide moderate expression levels that may be advantageous when expressing proteins that could cause cellular stress when overproduced .

• Specialized expression vectors:

  • The pLP expression vector series, which combines strong promoters with optimized RBS and terminator sequences, offers efficient expression of recombinant proteins .

  • pSIP-based vectors that allow inducible expression through peptide pheromone induction systems provide controlled expression that may be beneficial for proteins affecting cellular physiology .

For RbfA expression specifically, given its role in ribosome assembly, a balanced approach using a medium-copy vector with a strong but controllable promoter is recommended to prevent potential disruption of cellular translation machinery.

How can recombinant RbfA be used to improve cold adaptation in L. plantarum?

Recombinant expression of RbfA in L. plantarum presents a strategic approach to enhance cold adaptation, particularly relevant for applications in food fermentation and storage of probiotic formulations. Implementation strategies include:

• Controlled overexpression approach:

  • Design an expression system with temperature-responsive elements that increase RbfA production at low temperatures

  • Incorporate a moderately strong promoter to prevent excessive metabolic burden

  • Monitor growth kinetics at various temperatures (4-15°C) to quantify the improvement in cold adaptation

• Functional analysis methods:

  • Ribosome profile analysis using sucrose gradient ultracentrifugation to assess 30S subunit maturation at low temperatures

  • 16S rRNA processing assessment via northern blotting

  • Translation efficiency evaluation using reporter systems (e.g., luciferase) at various temperatures

  • Comparative proteomics at normal vs. reduced temperatures to identify global effects of RbfA overexpression

• Performance metrics:

  • Lag phase duration at low temperatures

  • Growth rate and final biomass yield under cold stress

  • Survival rates during freeze-thaw cycles

  • Maintenance of metabolic activities (e.g., acid production) at sub-optimal temperatures

By systematically optimizing RbfA expression levels in response to temperature reduction, researchers can develop L. plantarum strains with enhanced performance in cold environments, potentially improving their commercial applicability in refrigerated food products and extending shelf-life of probiotic preparations.

What methodologies are appropriate for analyzing RbfA-RNA interactions in recombinant L. plantarum?

To characterize RbfA-RNA interactions in recombinant L. plantarum, researchers should employ multiple complementary techniques:

• In vitro binding assays:

  • RNA Electrophoretic Mobility Shift Assay (EMSA) using purified recombinant RbfA and synthetic RNA oligonucleotides corresponding to the 5'-terminal helix (helix I) of 16S rRNA

  • Filter binding assays to determine binding constants (Kd)

  • Isothermal Titration Calorimetry (ITC) to measure thermodynamic parameters of binding

• Structural analysis approaches:

  • Nuclear Magnetic Resonance (NMR) spectroscopy to map the interaction interface between RbfA and RNA, similar to the approach used for E. coli RbfAΔ25

  • Chemical probing of RNA structure (SHAPE, DMS) in the presence and absence of RbfA

  • Cryo-electron microscopy of RbfA-30S ribosomal subunit complexes

• In vivo interaction studies:

  • RNA Immunoprecipitation (RIP) using epitope-tagged RbfA expressed in L. plantarum

  • Crosslinking and Immunoprecipitation (CLIP) to identify precise binding sites

  • Ribosome assembly analysis by sucrose gradient centrifugation to assess the impact of RbfA on 30S subunit maturation

• Functional validation:

  • Site-directed mutagenesis of key residues in the potential RNA-binding site (around the conserved polypeptide segment Ser76-Asp100)

  • Assessment of cold sensitivity phenotypes of L. plantarum expressing mutant RbfA proteins

  • Complementation studies in RbfA-deletion strains

These methodologies will provide comprehensive insights into the molecular basis of RbfA function in L. plantarum, particularly focusing on its RNA-binding properties and role in ribosome assembly during cold adaptation.

How can recombinant L. plantarum expressing RbfA be used as a delivery system for therapeutic applications?

Lactiplantibacillus plantarum expressing recombinant RbfA can be developed as a multifunctional therapeutic delivery system that combines the probiotic benefits of L. plantarum with the potential immunomodulatory effects of controlled protein expression. Implementation strategies include:

• Mucosal delivery system design:

  • Surface display of RbfA fused with immunogenic epitopes using cell wall anchoring domains

  • Secretion of RbfA or RbfA-fusion proteins using optimized signal peptides

  • Co-expression with immunomodulatory molecules to enhance therapeutic efficacy

• In vitro validation methods:

  • Dendritic cell maturation assays to evaluate immunostimulatory properties, similar to approaches used for other L. plantarum recombinant systems

  • Evaluation of expression stability without antibiotic selection using plasmid bioretention systems

  • Assessment of survival under gastrointestinal conditions

• In vivo assessment approaches:

  • Analysis of gut colonization dynamics using fluorescently labeled strains

  • Measurement of immune responses (serum IgG, IgG1, and fecal sIgA levels)

  • Evaluation of CD4+ T cell and IgA+ B cell populations in gut-associated lymphoid tissues

  • Metagenomic analysis to determine effects on gut microbiota composition using approaches like 16S rRNA sequencing and Shannon-Wiener diversity index analysis

• Safety and containment considerations:

  • Implementation of biocontainment strategies to prevent environmental release

  • Assessment of plasmid stability in the absence of selection

  • Monitoring of potential horizontal gene transfer

This approach leverages the demonstrated ability of L. plantarum to modulate gut microbial diversity and immune responses , potentially enhanced by the expression of RbfA, which could improve the strain's resilience during production, storage, and gastrointestinal transit by enhancing cold and stress tolerance.

How does heterologous expression of RbfA affect the native ribosome assembly pathway in L. plantarum?

The introduction of recombinant RbfA into L. plantarum could significantly impact endogenous ribosome assembly pathways, requiring careful characterization at multiple levels:

• Ribosome assembly dynamics assessment:

  • Quantitative analysis of ribosomal subunit profiles using sucrose gradient ultracentrifugation under varying temperatures

  • Pulse-chase labeling of rRNA to track maturation rates

  • Cryo-electron microscopy to visualize assembly intermediates

• Competition with native RbfA:

  • Quantification of native versus recombinant RbfA levels using targeted proteomics

  • Pull-down assays to determine if recombinant RbfA displaces native protein from ribosome binding sites

  • RNA immunoprecipitation to compare binding profiles

• Global physiological impacts:

  • Transcriptome analysis to identify compensatory responses

  • Ribosome profiling to assess translation efficiency across the genome

  • Growth kinetics analysis across temperature ranges

• Potential regulatory feedback mechanisms:

  • Analysis of native rbfA transcription in response to recombinant expression

  • Assessment of other cold-shock proteins' expression levels

  • Investigation of potential cross-talk with stress response pathways

Research has shown that ribosome assembly factors often function in concert, with potential compensatory mechanisms when specific factors are altered. The structured electrostatic field distribution of RbfA (bipolar with strongly negative and positive faces) suggests that non-specific interactions might occur when the protein is overexpressed, potentially sequestering rRNA or affecting other ribonucleoprotein complexes.

What are the comparative differences between RbfA function in E. coli versus L. plantarum, and how might this impact recombinant expression strategies?

Understanding the evolutionary and functional differences between RbfA proteins from E. coli and L. plantarum is crucial for optimizing recombinant expression strategies. Key considerations include:

• Structural and sequence conservation analysis:

  • Sequence alignment reveals RbfA is widely conserved across bacterial species, but with notable variation in the C-terminal region

  • The E. coli RbfA contains the AxG sequence motif in place of the GxxG sequence found in typical KH domains , which should be compared with the L. plantarum sequence

  • Homology modeling of L. plantarum RbfA based on E. coli RbfAΔ25 structure to identify potential structural differences

• Comparative functional characterization:

  • Cold sensitivity phenotype comparison between E. coli and L. plantarum rbfA mutants

  • Cross-complementation studies to determine functional exchangeability

  • rRNA processing pattern analysis in both species

  • Binding affinity comparisons toward respective 16S rRNAs

• Expression optimization implications:

• Heterologous expression considerations:

  • When expressing E. coli RbfA in L. plantarum, codon optimization should account for the significant GC content difference between the species

  • For L. plantarum RbfA overexpression, the native regulatory elements might provide more physiologically relevant expression patterns

This comparative approach will help determine whether a heterologous or homologous RbfA expression strategy would be more effective for enhancing cold adaptation in L. plantarum.

How can extracellular electron transfer (EET) capabilities be integrated with RbfA expression in L. plantarum for enhanced stress tolerance?

Recent research has revealed that L. plantarum performs extracellular electron transfer (EET) through a blended metabolism combining features of respiration and fermentation . Integrating EET capabilities with RbfA expression presents an innovative approach to develop multifunctional stress-tolerant strains:

• Mechanistic basis for integration:

  • Both systems represent adaptation mechanisms: RbfA for cold stress and EET for redox balance

  • L. plantarum EET requires NADH dehydrogenase (ndh2) and flavin-binding membrane reductase (pplA)

  • Cold stress often creates redox imbalance, suggesting potential synergistic benefits

• Design strategies for co-optimization:

  • Coordinate expression using stress-responsive promoters that activate under both cold and oxidative stress

  • Engineer a polycistronic construct containing both RbfA and key EET components

  • Develop a dual-plasmid system with compatible origins of replication for separate optimization

• Experimental assessment approach:

  • Evaluate ferrihydrite reduction capacity at varying temperatures

  • Measure NAD+:NADH ratios during cold adaptation

  • Assess organic acid production profiles and environmental acidification rates

  • Quantify growth parameters under combined stresses (cold, oxidative)

• Potential synergistic mechanisms:

  • RbfA enhancement of translation machinery may support increased production of EET components

  • EET pathways may help maintain redox balance during cold adaptation

  • Combined expression may activate complementary stress response pathways

• Practical applications:

  • Enhanced survival in fermented food systems

  • Improved functionality in microbial fuel cells operating at variable temperatures

  • Development of robust biocatalysts for environmental remediation

This integrative approach leverages the finding that EET in L. plantarum results in shortened lag phase and increased fermentation flux , which could complement the effects of RbfA on cold adaptation, potentially creating strains with broad stress tolerance capabilities.

What are the common challenges in purifying recombinant RbfA from L. plantarum and how can they be addressed?

Purification of recombinant RbfA from L. plantarum presents several technical challenges due to its RNA-binding properties and potential for aggregation. These challenges and their solutions include:

• Protein solubility issues:

  • Challenge: RbfA may form inclusion bodies when overexpressed

  • Solutions:

    • Lower induction temperature to 20-25°C when using inducible systems

    • Express as a fusion with solubility-enhancing tags (e.g., MBP, SUMO)

    • Co-express with molecular chaperones

    • Optimize induction parameters (concentration, duration) using response surface methodology

• RNA contamination:

  • Challenge: RbfA's strong RNA-binding properties can result in co-purification of RNA

  • Solutions:

    • Include high salt (0.5-1M NaCl) in lysis and wash buffers

    • Add RNase treatment steps during purification

    • Include competitive RNA-binding molecules in wash buffers

    • Perform on-column nuclease digestion

• Cell lysis efficiency:

  • Challenge: L. plantarum has a thick peptidoglycan layer that can be difficult to disrupt

  • Solutions:

    • Use combined enzymatic (lysozyme) and mechanical (sonication/homogenization) methods

    • Include cell wall hydrolases specific to Lactobacillus species

    • Optimize growth phase for harvest (early stationary phase often yields easier lysis)

• Optimal purification strategy:

• Yield optimization:

  • Systematic testing of cell disruption methods and buffer conditions

  • Scale-up considerations using design of experiments (DoE) approach

  • Stability assessment during storage (glycerol addition, optimal temperature)

By addressing these challenges systematically, researchers can obtain pure, functional RbfA protein for structural and biochemical studies, enabling deeper understanding of its role in ribosome assembly and cold adaptation.

How can researchers assess the impact of recombinant RbfA expression on L. plantarum physiology under various stress conditions?

To comprehensively evaluate how recombinant RbfA expression affects L. plantarum physiology under stress conditions, researchers should employ a multi-faceted approach:

• Growth and viability assessment:

  • Growth curve analysis across temperature ranges (4-45°C)

  • Colony-forming unit (CFU) determination after exposure to multiple stresses

  • BioLector™ cultivation for real-time monitoring of growth parameters

  • Flow cytometry with viability dyes to distinguish live/dead populations

• Stress response characterization:

  • Transcriptomics analysis comparing wild-type and RbfA-expressing strains under cold, oxidative, and acid stress

  • Quantitative proteomics focusing on stress response pathways

  • Metabolomic profiling to identify shifts in central metabolism

  • Differential expression analysis of key stress genes (e.g., cold shock proteins, chaperones)

• Ribosome functionality metrics:

  • Polysome profiling to assess translation efficiency

  • In vivo translation rate measurement using puromycin incorporation

  • 16S rRNA processing analysis via northern blotting

  • Ribosome assembly kinetics at varying temperatures

• Comprehensive stress testing protocol:

• Long-term adaptation assessment:

  • Evolution experiments under cold stress with and without RbfA expression

  • Genetic stability of expression constructs through repeated subculturing

  • Competitive fitness compared to wild-type in mixed cultures

This methodical approach will provide a comprehensive understanding of how RbfA expression modulates stress responses in L. plantarum, potentially revealing unexpected cross-protection mechanisms and informing the development of robust probiotic and expression systems.

What are the best practices for optimizing codon usage in recombinant RbfA genes for expression in L. plantarum?

Optimizing codon usage for recombinant RbfA expression in L. plantarum requires careful consideration of several factors to ensure efficient translation and maximum protein yield:

• Codon adaptation approach:

  • Analyze the codon usage bias in highly expressed L. plantarum genes, particularly those encoding ribosomal proteins and translation factors

  • Adjust the RbfA coding sequence to preferentially use the most frequent codons in the L. plantarum genome

  • Consider using the Codon Adaptation Index (CAI) as a metric, targeting values >0.8 for optimal expression

• Critical codon optimization parameters:

  • GC content adjustment: L. plantarum has a lower GC content (~44-45%) compared to E. coli (~50-51%)

  • Avoid rare codons, particularly those recognized by low-abundance tRNAs

  • Eliminate potential ribosome stalling sites with consecutive rare codons

  • Remove sequences that could form stable mRNA secondary structures, especially near the translation initiation region

• Beyond simple codon replacement:

  • Harmonize codon usage rather than maximizing it, mimicking the natural codon usage pattern of L. plantarum

  • Consider the translation elongation rate profile of the gene to maintain proper protein folding

  • Avoid introducing sequences that resemble Shine-Dalgarno motifs within the coding region

  • Eliminate potential cryptic splice sites or premature termination signals

• Experimental validation approach:

  • Test multiple codon optimization strategies in parallel

  • Compare expression levels using quantitative methods (Western blotting, reporter fusions)

  • Verify protein solubility and activity to ensure proper folding

  • Perform ribosome profiling to identify any remaining translation bottlenecks

• Specialized considerations for RbfA:

  • Pay particular attention to optimizing the region encoding the RNA-binding domain (Ser76-Asp100)

  • Consider the impact of translation rate on co-translational folding of structural elements like the helix-kink-helix motif

  • If expressing E. coli RbfA, compare native sequence performance versus optimized versions

By implementing these codon optimization strategies, researchers can enhance the expression efficiency of recombinant RbfA in L. plantarum, facilitating downstream applications in both basic research and biotechnological applications.

How can CRISPR-Cas technology be applied to optimize RbfA expression and function in L. plantarum?

CRISPR-Cas systems offer powerful tools for precise genetic manipulation of L. plantarum to optimize RbfA expression and function. Strategic applications include:

• Genome editing approaches:

  • Precise modification of the native rbfA promoter to alter expression patterns

  • Introduction of specific mutations in the rbfA coding sequence to enhance cold adaptation

  • Multiplex editing to simultaneously modify rbfA and related cold-shock genes

  • Knock-in of heterologous rbfA variants from extremophiles

• CRISPR interference (CRISPRi) applications:

  • Tunable repression of competing ribosome assembly factors

  • Temporal control of rbfA expression using inducible dCas9 systems

  • Creation of synthetic regulatory circuits responding to temperature shifts

  • Screening for genes that synergize with rbfA in enhancing cold tolerance

• CRISPR activation (CRISPRa) strategies:

  • Upregulation of native rbfA during cold shock

  • Coordinated activation of cold-shock response genes

  • Enhancement of complementary stress response pathways

• Implementation considerations for L. plantarum:

  • Selection of appropriate Cas variants with demonstrated activity in lactic acid bacteria

  • Optimization of guide RNA design for the AT-rich regions common in L. plantarum

  • Development of delivery methods suitable for industrial L. plantarum strains

  • Establishment of marker-free genome editing protocols

• Emerging CRISPR applications:

  • Base editing technologies for precise nucleotide substitutions

  • Prime editing for targeted insertions and deletions

  • RNA-targeting Cas systems for post-transcriptional regulation

  • CRISPR-based biosensors to monitor cold stress responses

This technology presents significant advantages over traditional genetic engineering approaches by offering increased precision, multiplexing capabilities, and the potential for marker-free modifications, ultimately accelerating the development of industrially relevant L. plantarum strains with enhanced stress tolerance.

What is the potential for developing biosensors based on RbfA-regulated systems in L. plantarum?

The temperature-responsive nature of RbfA regulation presents an intriguing foundation for developing biosensor systems in L. plantarum with various applications:

• Temperature-responsive biosensor designs:

  • RbfA promoter-driven reporter systems (fluorescent proteins, luciferase) for real-time temperature monitoring

  • Synthetic circuits incorporating rbfA regulatory elements controlling expression of detectable outputs

  • FRET-based systems using RbfA conformational changes upon RNA binding

  • Surface display of RbfA-responsive elements for whole-cell biosensing

• Detection mechanisms and outputs:

  • Colorimetric changes for visual detection

  • Bioluminescence for non-invasive monitoring

  • Electrochemical outputs for integration with microfluidic systems

  • Enzyme-based cascades amplifying detection sensitivity

• Potential applications:

  • Food quality monitoring during cold chain logistics

  • Environmental temperature logging with cellular memory

  • In vivo tracking of temperature gradients in research models

  • Process monitoring in industrial fermentations

• Advanced design strategies:

  • Multi-input logic gates combining temperature sensing with other parameters

  • Tunable threshold responses using synthetic biology principles

  • Integration with extracellular electron transfer pathways for electronic outputs

  • Development of graded responses across temperature ranges

• Validation methodologies:

  • Calibration against standard temperature measurement techniques

  • Assessment of response time and recovery kinetics

  • Evaluation of signal-to-noise ratio and detection limits

  • Testing for cross-reactivity with other stress conditions

The development of such biosensors would leverage the natural cold-sensing machinery of RbfA while potentially providing industrially relevant tools for monitoring environmental conditions in applications ranging from food safety to bioprocess control.

What are the key databases and resources for researching RbfA structure-function relationships across bacterial species?

For comprehensive investigation of RbfA structure-function relationships, researchers should utilize the following specialized databases and bioinformatic resources:

• Structural databases:

  • Protein Data Bank (PDB): Contains the solved structure of E. coli RbfAΔ25 and T. maritima RbfA

  • CATH/SCOP: For classification of RbfA within the KH domain structural family

  • Electron Microscopy Data Bank (EMDB): For cryo-EM structures of RbfA-ribosome complexes

  • AlphaFold Database: For predicted structures of RbfA from various organisms including L. plantarum

• Sequence analysis resources:

  • Pfam (PF02033): RbfA family domain database entry

  • InterPro (IPR000238): Integrated resource for protein families and domains

  • PROSITE (PS00827): Database of protein domains, families and functional sites

  • ConSurf Server: For mapping conservation patterns onto protein structures

• Specialized ribosome assembly databases:

  • RAIN (RNA-protein Association and Interaction Networks): For RNA-binding protein interactions

  • Ribosomal Protein Gene Database: For contextual information on ribosomal assembly factors

  • STRING database: For protein-protein interaction networks involving RbfA

• Comparative genomics resources:

  • KEGG Orthology (K07559): RbfA ortholog mapping across species

  • EggNOG database: Evolutionary genealogy of genes and non-supervised orthologous groups

  • Microbes Online: Comparative genomics platform for bacterial genes

• Literature mining tools:

  • PubTator: For finding RbfA-related publications with annotated biological entities

  • BRENDA Enzyme Database: For functional annotations of RbfA across species

  • RegulonDB: For regulatory network information in model organisms

These resources collectively provide a comprehensive foundation for investigating RbfA structure-function relationships, enabling researchers to develop informed hypotheses about its role in L. plantarum and design optimal recombinant expression strategies.

What methodological standards should be followed when reporting recombinant RbfA expression in L. plantarum?

To ensure reproducibility and facilitate comparative analysis across studies, researchers should adhere to the following methodological standards when reporting recombinant RbfA expression in L. plantarum:

• Strain and vector documentation:

  • Complete taxonomic identification of the L. plantarum strain (e.g., WCFS1, CD033)

  • Full vector sequence including all genetic elements (promoters, terminators, RBS)

  • Detailed description of any modifications to the RbfA coding sequence

  • Accession numbers for all genetic components

• Expression conditions reporting:

  • Media composition with exact concentrations of all components

  • Growth parameters (temperature, pH, agitation, aeration)

  • Induction conditions if using inducible systems

  • Cell density at induction and harvest (OD600)

  • Growth curve data including specific growth rates

• Protein production quantification:

  • Absolute quantification methods (μg protein/mL culture or % of total cellular protein)

  • Western blot analysis with appropriate controls

  • Activity assays if applicable

  • Standardized reporting units to enable cross-study comparisons

• Quality control metrics:

  • Plasmid stability assessment (% cells retaining expression construct)

  • Protein solubility analysis (soluble vs. insoluble fraction)

  • Protein purity determination by SDS-PAGE and/or other methods

  • Mass spectrometry confirmation of protein identity

• Reproducibility considerations:

  • Statistical analysis of biological and technical replicates

  • Sample size and power calculations

  • Potential batch effects and their control measures

  • Raw data availability in public repositories

• Functional validation:

  • Cold adaptation phenotype assessment methodology

  • Ribosome assembly analysis if relevant

  • RNA binding characterization if performed

  • Comparison with native RbfA function