Recombinant Bdellovibrio bacteriovorus 30S ribosomal protein S21 (rpsU)

What is the functional role of ribosomal protein S21 in bacterial translation?

Ribosomal protein S21 (bS21) plays a crucial role in translation initiation. Although not absolutely required for in vitro translation systems, it is essential for bacterial viability in vivo, as demonstrated in Escherichia coli and other bacterial species . The protein is one of the last components to be incorporated during 30S ribosomal subunit assembly . Early studies have established its involvement in translation initiation efficiency, where it appears to help stabilize mRNA-ribosome interactions during the formation of the initiation complex . In bacteria like Francisella tularensis, bS21 can specifically enhance translation from mRNAs with certain leader sequences, demonstrating its regulatory capacity beyond mere structural functionality .

How does B. bacteriovorus S21 compare structurally to S21 proteins from other bacterial species?

While the search results don't provide specific structural information for B. bacteriovorus S21, comparative analysis with other bacterial S21 proteins reveals important evolutionary patterns. Unlike some bacteria such as F. tularensis that encode multiple homologs of bS21, most bacteria contain a single copy of the rpsU gene . The rpsU gene in Bacteroidia contains a conserved C-terminal region involved in anti-Shine-Dalgarno sequence (ASD) interactions, which is not conserved in Gammaproteobacteria . Functional domains of S21 typically include regions that interact with the 16S rRNA, particularly near the anti-Shine-Dalgarno sequence. In E. coli, the bS21 residue R17 contacts the 16S rRNA nucleotide C1539, which is part of the anti-Shine-Dalgarno sequence .

What genomic context surrounds the rpsU gene in B. bacteriovorus?

While specific genomic context information for B. bacteriovorus rpsU isn't provided in the search results, we can infer likely arrangements based on patterns in other bacterial species. In bacteria, the rpsU gene often exists in operons with other genes involved in translation or cell division. Researchers investigating the genomic context should examine whether B. bacteriovorus rpsU is part of a larger transcriptional unit with functionally related genes. This can be determined through:

• Bioinformatic analysis of intergenic distances and promoter predictions

• RT-PCR experiments to identify co-transcribed genes

• Comparative genomic analysis with related predatory bacteria

The genomic neighborhood may provide insights into co-regulated processes and evolutionary conservation of genetic arrangements around this essential ribosomal protein.

What are the optimal conditions for expression and purification of recombinant B. bacteriovorus S21 protein?

For optimal expression and purification of recombinant B. bacteriovorus S21 protein, researchers should consider:

• Expression system selection: E. coli BL21(DE3) is typically suitable for ribosomal protein expression. Alternative hosts such as C41(DE3) or C43(DE3) may be preferred if toxicity is observed.

• Vector design:

  • Include an N-terminal or C-terminal affinity tag (His6, GST, or MBP)

  • Consider a cleavable tag if native protein is required for functional studies

  • Optimize codon usage for the expression host

• Expression conditions:

  • Induce at OD600 of 0.6-0.8

  • Use lower temperatures (16-20°C) for overnight expression to enhance solubility

  • Test IPTG concentrations between 0.1-1.0 mM

• Purification protocol:

  • Extract using mild lysis conditions (avoid denaturation)

  • Include RNase treatment to remove bound RNA

  • Employ a two-step purification (affinity chromatography followed by size exclusion)

• Quality control:

  • Verify purity by SDS-PAGE

  • Confirm identity by mass spectrometry

  • Assess RNA contamination by measuring A260/A280 ratio

For functional studies, buffer optimization is critical as S21 interacts with RNA and requires proper folding for activity.

How can I establish a reliable assay to measure B. bacteriovorus S21 binding to ribosomes?

To establish a reliable assay for measuring B. bacteriovorus S21 binding to ribosomes, consider the following methodological approaches:

• Direct binding assays:

  • Microscale thermophoresis (MST) with fluorescently labeled S21

  • Bio-layer interferometry (BLI) with immobilized 30S subunits

  • Surface plasmon resonance (SPR) to measure binding kinetics

• Filter binding assays:

  • Incubate radiolabeled S21 with isolated 30S subunits

  • Wash away unbound protein through nitrocellulose filters

  • Quantify bound protein via scintillation counting

• Ribosome reconstitution experiments:

  • Prepare 30S subunits lacking S21 (through selective extraction)

  • Add recombinant S21 at varying concentrations

  • Measure reconstitution efficiency by sucrose gradient centrifugation

• Fluorescence-based approaches:

  • Label S21 with environment-sensitive fluorophores

  • Monitor fluorescence changes upon ribosome binding

  • Calculate binding constants from titration curves

When designing these experiments, it's essential to control for RNA contamination in S21 preparations and to verify that ribosomes maintain their integrity throughout the assay. Comparing binding parameters with S21 proteins from other species (like E. coli) can provide valuable benchmarks for interpreting results.

What techniques can be used to investigate the impact of S21 on translation initiation in B. bacteriovorus?

Several complementary techniques can be employed to investigate S21's impact on translation initiation in B. bacteriovorus:

• In vitro translation systems:

  • Reconstitute 30S subunits with and without S21

  • Measure translation efficiency using reporter mRNAs

  • Analyze initiation complex formation through toeprinting assays

• Translational reporter fusions:

  • Construct β-galactosidase or GFP translational reporters with various 5′ UTRs

  • Test reporters in wild-type and S21-depleted conditions

  • Compare translational efficiency with different leader sequences (as done with F. tularensis in the reference)

• Ribosome profiling:

  • Generate S21-depleted or mutant strains

  • Perform ribosome profiling to identify transcripts with altered translation efficiency

  • Analyze 5′ UTR features of affected mRNAs

• RNA structure probing:

  • Use SHAPE or DMS-MaPseq to analyze mRNA structure changes

  • Compare structural accessibility of Shine-Dalgarno sequences

  • Correlate structural changes with translation efficiency

• Cryo-EM structural analysis:

  • Capture initiation complexes with and without S21

  • Identify conformational changes in the 30S subunit

  • Visualize mRNA path differences at atomic resolution

These approaches would allow researchers to determine whether B. bacteriovorus S21 influences translation in a sequence-dependent manner, similar to what has been observed in F. tularensis where bS21-2 enhances translation of mRNAs with imperfect Shine-Dalgarno sequences .

How does S21 contribute to ribosome heterogeneity and specialized translation in B. bacteriovorus?

Ribosome heterogeneity represents an emerging field in bacterial gene regulation, with S21 playing a potentially significant role. In B. bacteriovorus, S21's contribution to ribosome heterogeneity may be investigated through:

• Quantitative ribosome composition analysis:

  • Mass spectrometry-based quantification of S21 occupancy in ribosomes

  • Single-molecule fluorescence to track S21-containing vs. S21-deficient ribosomes

  • Polysome profiling combined with proteomic analysis

• Environmental response patterns:

  • Monitor S21 incorporation under different growth conditions

  • Determine if predatory vs. saprophytic growth alters S21 ribosome occupancy

  • Investigate stress-induced changes in ribosome composition

• mRNA selectivity:

  • Identify transcripts preferentially translated by S21-containing ribosomes

  • Analyze common features in 5′ UTRs of these transcripts

  • Determine if S21 influences translation of predation-related genes

Current research in F. tularensis has shown that specific bS21 homologs regulate translation in a leader sequence-dependent manner . In F. tularensis, loss of the bS21-2 homolog impacts protein synthesis and virulence . The mechanism involves specific interactions with 5′ UTRs, particularly those with imperfect Shine-Dalgarno sequences . Whether B. bacteriovorus S21 functions similarly remains to be determined but seems plausible given the conservation of ribosomal protein functions.

What sequence determinants in mRNA leader regions influence S21-dependent translation in B. bacteriovorus?

Based on findings in other bacterial species, specific sequence determinants in mRNA leader regions likely influence S21-dependent translation in B. bacteriovorus:

• Shine-Dalgarno sequence characteristics:
  In F. tularensis, genes positively affected by bS21-2 generally have weaker Shine-Dalgarno (SD) sequences, with only 39% having strong SD sequences (defined by four or more nucleotides complementary to the anti-SD), compared to 54% or 69% strong SDs in negatively affected or unaffected genes, respectively . This suggests that:

  SD Strength	% in bS21-2 Positively Affected Genes	% in bS21-2 Negatively Affected Genes	% in Unaffected Genes
  Strong	39%	54%	69%
  Weak/Imperfect	61%	46%	31%

• Specific sequence motifs:
  In the mraY 5′ UTR of F. tularensis, a six-nucleotide sequence (GACUCU) was identified as critical for bS21-2 responsiveness . This may represent a direct binding site for S21 that enhances translation initiation.

• Secondary structure considerations:
  Leader sequence folding may influence accessibility to ribosomes with or without S21. Researchers should examine:

  • Predicted secondary structures of leader sequences

  • Accessibility of start codons and SD sequences

  • Potential structural transitions upon S21 binding

To identify these determinants in B. bacteriovorus specifically, researchers should:

• Create a library of reporter constructs with varied leader sequences

• Perform systematic mutagenesis of candidate sequence motifs

• Compare translation efficiency in wild-type vs. S21-depleted conditions

• Apply computational approaches to identify enriched motifs in S21-responsive transcripts

What role does S21 play in regulating gene expression during the predatory lifecycle of B. bacteriovorus?

B. bacteriovorus has a unique biphasic lifecycle with distinct attack phase and growth phase transcriptional programs. The role of S21 in regulating gene expression during this predatory lifecycle represents an intriguing research question:

• Lifecycle-specific expression patterns:

  • Quantify S21 protein levels during different lifecycle stages

  • Determine if S21 is differentially incorporated into ribosomes during transition between free-living and intraperiplasmic growth

  • Analyze if S21 gene expression correlates with expression of predation-related genes

• Targeted gene regulation:

  • Identify if S21 preferentially enhances translation of predation-related transcripts

  • Examine if hydrolytic enzymes or structural components of the invasion machinery show S21-dependent translation

  • Determine if the transition between attack phase and growth phase involves changes in S21-mediated translation

• Experimental approaches:

  • Create conditional S21 depletion strains to examine effects on predatory efficiency

  • Perform ribosome profiling at different lifecycle stages with and without S21

  • Use reporter constructs containing promoters and 5′ UTRs from key predatory genes to measure S21 dependence

• Potential metabolic impact:

  • Investigate whether S21 influences translation of genes involved in nutrient acquisition

  • Determine if adaptation to different prey bacteria involves S21-mediated translational regulation

  • Examine energetic considerations during the transition between growth states

This research area represents a potentially groundbreaking intersection between ribosome heterogeneity and the specialized bacterial predation mechanism.

How has the function of S21 evolved in predatory bacteria compared to non-predatory relatives?

The evolutionary trajectory of S21 in predatory bacteria compared to non-predatory relatives represents an important research question:

• Phylogenetic analysis:

  • Construct phylogenetic trees of S21 sequences across the bacterial kingdom

  • Determine if predatory bacteria exhibit distinct S21 sequence clades

  • Identify specific amino acid changes potentially associated with predatory lifestyle

• Functional domain analysis:

  • Compare conserved domains across predatory and non-predatory species

  • Identify if RNA-binding regions show adaptive signatures

  • Examine conservation of specific amino acids like R17 (which in E. coli contacts the 16S rRNA nucleotide C1539)

• Horizontal gene transfer assessment:

  • Evaluate if predatory bacteria acquired specialized S21 variants

  • Analyze GC content and codon usage patterns for evidence of recent transfer

  • Compare to patterns seen in other horizontally transferred genes, like the β-lactam biosynthesis genes in Penicillium

• Experimental validation:

  • Perform complementation studies with S21 from different species

  • Test if predatory bacterial S21 can function in non-predatory hosts and vice versa

  • Examine functional consequences of key amino acid substitutions

Evolutionary insights could reveal whether S21's role in translation regulation has been specialized to support the unique lifecycle requirements of predatory bacteria.

What genetic manipulation techniques are most effective for studying S21 function in B. bacteriovorus?

Genetic manipulation of B. bacteriovorus presents unique challenges due to its predatory lifestyle and fastidious growth requirements. For studying S21 function, researchers should consider:

• Gene deletion/replacement strategies:

  • As S21 may be essential, conditional knockdown systems are preferable

  • CRISPRi-based transcriptional repression to achieve tunable expression

  • Inducible antisense RNA to regulate S21 levels post-transcriptionally

  • Merodiploid approaches with complementation controlled by inducible promoters

• Reporter systems:

  • Translational fusions with mCherry or GFP to monitor S21 expression dynamics

  • Dual reporter systems to distinguish transcriptional vs. translational effects

  • Development of B. bacteriovorus-specific reporters optimized for its genetic background

• Protein tagging approaches:

  • Fluorescent protein fusions to track S21 localization during predatory lifecycle

  • Affinity tags for pulldown experiments to identify interacting partners

  • Split fluorescent protein systems to investigate incorporation into ribosomes in vivo

• Host-independent mutant utilization:

  • Compare S21 function in host-dependent vs. host-independent strains

  • Leverage the simplified cultivation of HI strains for high-throughput studies

  • Examine S21's role in the transition to host-independence

• Heterologous expression systems:

  • Express B. bacteriovorus S21 in E. coli to examine functional conservation

  • Create chimeric S21 proteins to map functional domains

  • Develop in vitro translation systems with purified B. bacteriovorus components

Each approach has specific advantages depending on the research question being addressed. Combining multiple genetic techniques will likely provide the most comprehensive understanding of S21 function.

How could understanding S21 function in B. bacteriovorus contribute to developing new antimicrobial strategies?

Understanding S21 function in B. bacteriovorus could contribute to antimicrobial strategies in several ways:

• Engineered predatory bacteria:

  • Modifying S21 to enhance translation of predation-related genes

  • Optimizing B. bacteriovorus predatory efficiency against specific pathogens

  • Creating synthetic regulatory circuits that respond to pathogen-specific signals

• Novel antibiotic targets:

  • Identifying S21-mRNA interactions that could be targeted by small molecules

  • Exploiting differences between pathogen and host S21 functions

  • Developing compounds that disrupt ribosome heterogeneity in pathogens

• Diagnostic applications:

  • Using knowledge of S21-specific translation to develop biosensors

  • Creating reporter systems based on S21-dependent translation

  • Applying insights to detect ribosome heterogeneity in clinical isolates

• Biotechnological applications:

  • Developing specialized expression systems with engineered S21 variants

  • Creating translation systems with altered mRNA preferences

  • Adapting B. bacteriovorus S21 for controlled gene expression in synthetic biology

Research on F. tularensis has shown that specific bS21 homologs regulate virulence gene expression , suggesting that S21-mediated translation regulation may be broadly important in bacterial pathogenesis and predation.

What are the challenges in studying ribosome heterogeneity in B. bacteriovorus compared to model organisms?

Studying ribosome heterogeneity in B. bacteriovorus presents several unique challenges compared to model organisms:

• Growth and cultivation challenges:

  • Limited biomass production due to predatory lifestyle

  • Requirement for prey bacteria complicates biochemical isolation

  • Host-independent variants may have altered ribosome composition

• Life cycle complexity:

  • Dynamic changes between attack and growth phases

  • Potential confounding factors from prey bacterial components

  • Synchronization difficulties for stage-specific analyses

• Technical limitations:

  • Limited genetic tools compared to model organisms

  • Challenges in isolating sufficient ribosomes for structural studies

  • Difficulties in establishing in vitro translation systems

• Analytical considerations:

  • Distinguishing predator vs. prey ribosomes in mixed samples

  • Detecting subtle changes in ribosome composition

  • Correlating ribosome heterogeneity with functional outcomes

• Evolutionary context:

  • Lack of closely related model organisms for comparative studies

  • Limited annotated genomic data for deltaproteobacteria

  • Few established functional assays for specialized predatory functions

These challenges necessitate innovative experimental approaches, potentially including:

• Single-cell analysis techniques to overcome biomass limitations

• Specialized ribosome isolation protocols to ensure purity

• Advanced mass spectrometry methods to detect low-abundance ribosomal variants

• Computational modeling to predict functional consequences of heterogeneity

How should researchers interpret discrepancies between in vitro and in vivo studies of S21 function?

Researchers frequently encounter discrepancies between in vitro and in vivo studies of ribosomal proteins. For B. bacteriovorus S21, consider these interpretation frameworks:

• Contextual factors:

  • In E. coli, S21 is not required for translation in vitro but is essential for viability in vivo

  • This suggests S21 functions may depend on cellular context absent in reconstituted systems

  • Consider if macromolecular crowding, ion concentrations, or co-factors present in vivo are missing in vitro

• Methodological considerations:

  • Evaluate if protein purification impacts native S21 structure or modifications

  • Consider if in vitro conditions recapitulate physiological parameters

  • Assess whether reporter systems may introduce artifacts

• Statistical approaches:

  • Implement appropriate statistical tests for different data types

  • Consider sample size requirements for both approaches

  • Use power analysis to determine required replication

• Reconciliation strategies:

  • Develop intermediate systems (e.g., cell extracts, semi-purified ribosomes)

  • Introduce systematic variations in experimental conditions

  • Use computational modeling to identify potential missing factors

• Data integration framework:

  Factor	In Vitro System	In Vivo System	Potential Reconciliation
  RNA modifications	Often absent	Present	Include modified rRNAs
  Macromolecular crowding	Dilute	Crowded	Add crowding agents
  Co-factors	Limited	Complete	Supplement with cellular extract
  Temporal dynamics	Static	Dynamic	Time-resolved measurements
  Interacting partners	Isolated	Network	Add predicted interactors

When interpreting contradictory results, consider that neither system represents the "ground truth" - rather, each provides different perspectives on S21 function.

What bioinformatic approaches can identify potential regulatory targets of S21-mediated translation in B. bacteriovorus?

Several bioinformatic approaches can help identify potential regulatory targets of S21-mediated translation in B. bacteriovorus:

• Sequence motif analysis:

  • Search for the six-nucleotide sequence (GACUCU) identified in F. tularensis as important for bS21-2 responsiveness

  • Perform de novo motif discovery in 5′ UTRs across the genome

  • Analyze positional preferences of sequence motifs relative to start codons

• Shine-Dalgarno strength assessment:

  • Calculate complementarity between each mRNA's SD region and the anti-SD in 16S rRNA

  • Categorize genes by SD strength (similar to the analysis in F. tularensis where genes positively affected by bS21-2 generally have weaker SD sequences)

  • Correlate SD strength with gene function and expression patterns

• RNA secondary structure prediction:

  • Predict folding of 5′ UTRs genome-wide

  • Analyze accessibility of start codons and Shine-Dalgarno sequences

  • Identify potential structural switches that might be influenced by S21 binding

• Comparative genomics:

  • Analyze conservation of 5′ UTR features across related species

  • Identify co-evolution patterns between S21 sequences and potential target mRNAs

  • Compare with patterns observed in other predatory bacteria

• Machine learning approaches:

  • Train models on known S21-responsive transcripts from other species

  • Apply transfer learning to predict B. bacteriovorus targets

  • Validate predictions experimentally with reporter constructs

• Integration with transcriptomic/proteomic data:

  • Correlate mRNA features with translation efficiency data

  • Identify transcripts with discordant mRNA/protein ratios

  • Look for patterns in lifecycle-specific expression datasets

These computational predictions should be followed by experimental validation, as the exact sequence determinants for S21-responsiveness may differ between bacterial species.