Recombinant Bacteroides thetaiotaomicron 50S ribosomal protein L21 (rplU)

What is Bacteroides thetaiotaomicron and why is it significant in microbial research?

Bacteroides thetaiotaomicron is a prominent human gut bacterium that has emerged as a promising candidate for therapeutic delivery in the gut microbiome. This anaerobic, gram-negative bacterium serves as an important model organism for studying host-microbe interactions within the gastrointestinal tract . B. thetaiotaomicron has been successfully engineered to produce specific enzymes, as demonstrated with strain BTX (a recombinant variant of strain 5482), which was engineered to produce high levels of xylanase, an enzyme crucial for hemicellulose degradation . Its genetic tractability makes it valuable for synthetic biology applications aimed at developing programmable bacterial therapeutics that can sense and respond to specific environmental conditions in the gut .

What is the 50S ribosomal protein L21 (rplU) and what is its function in prokaryotic ribosomes?

The 50S ribosomal protein L21, encoded by the rplU gene, is a component of the large subunit of bacterial ribosomes. It plays a structural role in the assembly and stability of the ribosome, contributing to protein synthesis functionality. Interestingly, L21 is one of the ribosomal proteins that can be absent in certain bacterial species, particularly in organisms with reduced genome sizes . Analysis of prokaryotic genomes has revealed that only about half of ribosomal proteins are universally conserved across bacteria and archaea, while others, including L21, exhibit patterns of lineage-specific gene loss . L21 is typically located on the periphery of the ribosome structure, which may explain why it is nonessential in some bacterial species such as Escherichia coli and Bacillus subtilis .

How is the essentiality of ribosomal proteins determined in prokaryotes?

The essentiality of ribosomal proteins in prokaryotes is determined through several experimental approaches:

• Genome-wide mutagenesis studies: Systematic gene disruption or deletion to identify which genes are required for cell viability under standard growth conditions.

• Gene expression suppression: Techniques using antisense RNA or other gene silencing methods to reduce expression of specific ribosomal protein genes and observe the resulting phenotypes .

• Comparative genomics: Analysis of ribosomal protein gene presence/absence across diverse bacterial genomes can indicate which proteins are dispensable. For example, comprehensive analysis of 1,309 prokaryote genomes in the Clusters of Orthologous Genes (COG) database has revealed patterns of ribosomal protein gene conservation and loss .

• Database resources: Findings from essentiality studies are compiled in databases such as the Database of Essential Genes (DEG) and the Online Gene Essentiality database (OGEE), providing researchers with valuable reference resources .

These approaches have revealed that many ribosomal proteins located on the periphery of the ribosome, including L21 in some species, are nonessential despite their role in ribosome structure and function.

What patterns of ribosomal protein gene loss have been observed in Bacteroides species?

Ribosomal protein gene loss in Bacteroides and other prokaryotes follows several noteworthy patterns:

• Size-dependent loss: Ribosomal protein genes are most commonly missing in organisms with small genomes (<1 Mb), particularly in host-associated bacteria and archaea that have undergone genome reduction during adaptation to their hosts .

• Peripheral location: Most ribosomal proteins that are frequently lost from bacterial genomes, potentially including L21, are located on the ribosome periphery rather than in core functional regions .

• Lineage-specific patterns: Six bacterial and nine archaeal-specific ribosomal proteins show clear patterns of lineage-specific gene loss, suggesting evolutionary processes have shaped ribosomal protein composition differently across prokaryotic lineages .

• Functional redundancy: The dispensability of certain ribosomal proteins suggests functional redundancy or compensatory mechanisms that maintain ribosome function despite the absence of specific components.

While specific information about L21 loss patterns in Bacteroides is limited in the provided search results, the general principles of ribosomal protein loss observed across prokaryotes likely apply to this genus as well.

What genetic engineering approaches are most effective for expressing recombinant ribosomal proteins in Bacteroides thetaiotaomicron?

Effective genetic engineering approaches for recombinant protein expression in B. thetaiotaomicron include:

• Genome integration: Stable expression can be achieved by integrating genes at specific attachment sites in the B. thetaiotaomicron genome, such as attBT1-1, attBT-1, and attBT2-1/2, as demonstrated in genetic circuit implementation studies .

• Promoter selection: Standard E. coli promoters function poorly in Bacteroides, necessitating the use of native promoters. Studies have utilized constitutive promoters like P PAM3 and P BT1311 for reliable expression .

• Reporter systems: Due to the anaerobic nature of Bacteroides, traditional reporters like GFP (which requires oxygen for chromophore maturation) may not function optimally unless highly expressed. Alternative reporters such as luciferase (nanoluc) have proven more effective for monitoring gene expression in B. thetaiotaomicron .

• Terminator selection: Strong terminators are crucial to prevent read-through to other genes. While terminators have not been extensively characterized in Bacteroides, studies have successfully adapted strong terminators from E. coli libraries for use in B. thetaiotaomicron .

• CRISPR/Cas9-based systems: deactivated Cas9 (dCas9) with small guide RNAs (sgRNAs) has been successfully implemented in B. thetaiotaomicron without the toxicity issues observed in E. coli, providing a powerful tool for gene regulation and potentially for recombinant protein expression .

How does the absence of L21 ribosomal protein affect ribosome assembly and function in prokaryotes?

The absence of L21 ribosomal protein can influence ribosome assembly and function in several ways:

• Structural adaptations: Ribosomes lacking L21 likely undergo structural adaptations to maintain functionality, possibly through altered interactions between remaining ribosomal components or compensatory protein conformation changes.

• Species-specific effects: The impact of L21 absence varies between species. For example, Mitsuaria sp. strain 7 lacks genes for several ribosomal proteins including L21, which is described as "unique among genomes of this size," suggesting potential adaptation to this specific gene loss pattern .

• Peripheral location effect: As L21 is located on the ribosome periphery in many species, its absence may have less impact on core ribosomal functions compared to the loss of proteins in more central positions .

• Translation efficiency implications: While viable, organisms lacking nonessential ribosomal proteins like L21 may exhibit altered translation kinetics, accuracy, or efficiency under certain growth conditions or stresses.

• Evolutionary compensation: Long-term absence of L21 in certain lineages suggests evolutionary compensation through changes in other ribosomal components or translation factors.

What are the optimal conditions for expressing and purifying recombinant B. thetaiotaomicron ribosomal proteins?

When working with recombinant B. thetaiotaomicron ribosomal proteins, researchers must consider the anaerobic nature of this organism. Growth in pre-reduced media under strictly anaerobic conditions is essential, with careful attention to media composition to support optimal growth . For expression systems, genome integration at specific attachment sites has proven effective, with native Bacteroides promoters providing reliable expression levels . Purification strategies must be adapted to maintain protein stability and structure throughout the process.

How can CRISPR/Cas9 technology be used to study ribosomal protein function in B. thetaiotaomicron?

CRISPR/Cas9 technology offers powerful approaches for studying ribosomal protein function in B. thetaiotaomicron:

• Gene knockouts: CRISPR/Cas9 can be used to create precise deletions of ribosomal protein genes like rplU (encoding L21) to study the impact on ribosome assembly, stability, and function.

• CRISPRi for gene regulation: Unlike in E. coli where dCas9 expression can be toxic, B. thetaiotaomicron tolerates dCas9 expression well, making CRISPRi (CRISPR interference) an effective approach for controlled downregulation of ribosomal protein genes . This allows for studying phenotypes associated with reduced but not eliminated ribosomal protein expression.

• Promoter engineering: CRISPR systems can be used to modify native promoters of ribosomal protein genes, enabling studies of expression level effects on ribosome assembly and function.

• Tagged protein expression: CRISPR-mediated genome editing can facilitate the integration of affinity or fluorescent tags to ribosomal proteins, enabling purification and localization studies.

• Genetic circuit implementation: As demonstrated in recent research, CRISPR systems can be incorporated into genetic circuits in B. thetaiotaomicron that respond to environmental signals, potentially allowing for controlled expression of recombinant ribosomal proteins under specific conditions .

Implementation of this technology for ribosomal protein studies should consider the optimal design of sgRNAs, efficient delivery methods, and appropriate controls to account for any off-target effects.

What techniques are most effective for monitoring ribosome assembly and function in the absence of specific ribosomal proteins?

For effectively monitoring ribosome assembly and function in B. thetaiotaomicron strains lacking specific ribosomal proteins like L21, researchers should employ complementary approaches. Sucrose gradient centrifugation provides valuable information about ribosome assembly status, while ribosome profiling can reveal genome-wide translation effects resulting from ribosomal protein absence. For anaerobic organisms like B. thetaiotaomicron, adaptation of standard protocols may be necessary, particularly for approaches requiring cell lysis and purification steps.

How can survival studies be designed to assess the impact of ribosomal protein modifications on B. thetaiotaomicron fitness?

Designing effective survival studies for ribosomal protein-modified B. thetaiotaomicron requires careful consideration of experimental conditions:

• In vitro batch culture systems: Similar to those used for B. thetaiotaomicron strain BTX, consecutive batch culture (CBC) systems can be employed to assess competitive fitness under defined conditions . These systems should include appropriate growth media and transfer protocols that maintain stable bacterial communities.

• Environmental stress testing: Modified strains should be exposed to various stressors relevant to gut environments, including bile acid fluctuations, pH changes, nutrient limitation, and antimicrobial compounds to assess survival under challenging conditions.

• Competition assays: Co-culturing modified strains with wild-type B. thetaiotaomicron or complex microbial communities (as in the CBC system with blended rumen contents) provides insights into relative fitness . Strain-specific markers are essential for tracking population dynamics over time.

• Growth substrate variation: Testing growth on different substrates, similar to how chondroitin sulfate supplementation affected BTX strain survival, can reveal condition-specific fitness effects of ribosomal protein modifications .

• Long-term stability assessment: As demonstrated with genetic circuits in B. thetaiotaomicron, stability should be monitored over extended periods (e.g., 12+ days) to detect any gradual loss of fitness or genetic instability .

• Cell adhesion models: For gut-relevant applications, epithelial cell monolayer models can assess both survival and functional performance of modified strains in host-associated conditions .

What approaches can be used to investigate the structural implications of L21 absence in the B. thetaiotaomicron ribosome?

Investigating structural implications of L21 absence requires multi-faceted approaches:

• Comparative cryo-EM analysis: High-resolution structures of wild-type and L21-deficient ribosomes can reveal structural adaptations and conformational changes that compensate for L21 absence.

• Crosslinking mass spectrometry: This technique can identify altered protein-protein or protein-RNA interactions in L21-deficient ribosomes compared to wild-type ribosomes.

• Molecular dynamics simulations: Computational modeling based on available ribosome structures can predict stability changes and conformational effects resulting from L21 absence.

• rRNA structure probing: Chemical and enzymatic probing methods can detect altered rRNA conformations in the absence of L21, revealing RNA structural adaptations.

• Functional domain mapping: Systematic analysis of translation functions (peptidyl transfer, translocation, etc.) can map specific functional defects to structural regions affected by L21 absence.

• Ribosome assembly kinetics: Pulse-chase experiments with labeled ribosomal components can reveal altered assembly pathways or kinetics in the absence of L21.

• Suppressor mutation analysis: Identification of compensatory mutations that restore optimal growth in L21-deficient strains can provide insights into structural adaptation mechanisms.

How might recombinant ribosomal proteins be utilized in synthetic biology applications in B. thetaiotaomicron?

Recombinant ribosomal proteins offer several promising applications in B. thetaiotaomicron synthetic biology:

• Specialized ribosomes: Engineering ribosomes with modified L21 or other ribosomal proteins could create specialized translation machinery with altered substrate specificity, codon preference, or environmental responsiveness.

• Environmental sensing: Coupling ribosomal protein expression to environmental signals using genetic circuits, as demonstrated with bile acid and aTc responsive systems, could create condition-specific translation regulation mechanisms .

• Therapeutic protein production: Modified ribosomes could potentially enhance production of therapeutic proteins in the gut environment, complementing the demonstrated ability to engineer B. thetaiotaomicron for controlled gene expression in gut-like conditions .

• Translation control switches: Ribosomal proteins could serve as components in genetic circuits that regulate translation in response to specific gut conditions, creating programmable bacterial therapeutics.

• Cross-kingdom regulation: Modified ribosomal proteins could potentially facilitate interactions between bacterial translation machinery and host regulatory factors, enabling novel therapeutic strategies.

• Stability enhancement: Engineered ribosomal proteins might improve stability and survival of B. thetaiotaomicron under challenging gut conditions, enhancing its utility as a therapeutic delivery platform.

The implementation of Cello circuit design automation for B. thetaiotaomicron provides a foundation for systematic design of genetic circuits that could incorporate ribosomal protein modifications for these applications .

What can comparative genomics reveal about the evolution of ribosomal protein L21 in Bacteroides and related genera?

Comparative genomic analysis of ribosomal protein L21 across Bacteroides and related genera can provide valuable evolutionary insights:

• Conservation patterns: Systematic analysis similar to the 1,309 prokaryote genome study can reveal whether L21 shows consistent presence/absence patterns within Bacteroidetes compared to other bacterial phyla .

• Sequence divergence: Analysis of L21 sequence conservation versus divergence can identify functional constraints and potentially adaptive regions of the protein.

• Genomic context: Examination of the genomic neighborhood of rplU across species can reveal conserved gene clusters and potential co-evolutionary relationships with other genes.

• Host association correlation: Correlation between L21 characteristics and host-association patterns may reveal adaptive significance of L21 variations in gut symbionts.

• Horizontal gene transfer: Analysis of phylogenetic incongruence could detect potential horizontal transfer events affecting L21 evolution in Bacteroides.

• Selection pressure analysis: Calculation of Ka/Ks ratios across L21 sequences can identify regions under purifying or positive selection in different lineages.

• Structural constraint mapping: Mapping sequence conservation onto ribosome structural data can reveal structure-function relationships specific to Bacteroides ribosomes.

This comparative approach could reveal whether the patterns of ribosomal protein gene loss observed more broadly in prokaryotes apply specifically to Bacteroides and its relatives, potentially identifying lineage-specific adaptations in ribosomal architecture.