Recombinant Pseudomonas putida Ribosomal RNA small subunit methyltransferase B (rsmB)

What are the Rsm family proteins in Pseudomonas putida and how are they classified?

Pseudomonas putida KT2440 harbors three known members of the CsrA/RsmA family of post-transcriptional regulators: RsmA, RsmE, and RsmI. These proteins function as global regulators that control various aspects of bacterial physiology including motility, biofilm formation, and environmental adaptation . While the search results don't specifically focus on RsmB, these Rsm proteins collectively belong to a family of RNA-binding proteins that post-transcriptionally regulate gene expression by affecting mRNA stability and translation efficiency.

The Rsm system also includes small regulatory RNAs (sRNAs) like rsmY and rsmZ that have been identified in P. putida KT2440. These sRNAs show 72% and 78% identity, respectively, to their counterparts in Pseudomonas protegens Pf-5. A potential rsmX has also been identified with lower sequence identity (55%) compared to the other two sRNAs .

What is the genomic context and evolutionary conservation of Rsm methyltransferases?

The Rsm methyltransferases in P. putida show significant conservation across Pseudomonas species. The genes encoding these proteins are located at different positions in the P. putida KT2440 genome: PP_4472 (rsmA), PP_3832 (rsmE), and PP_1746 (rsmI) . Comparative genomic analyses have revealed that these genes belong to the core genome of P. putida, comprising genes related to essential metabolic pathways and regulatory functions .

The pangenome of P. putida includes 3386 conserved genes belonging to the core genome, which includes genes involved in various metabolic pathways and regulatory functions . While P. putida shares approximately 85% of its coding regions with Pseudomonas aeruginosa, it lacks key virulence factors, exotoxins, and type III secretion systems found in the latter, making it a safer model organism for biotechnological applications .

What are the specific functional roles of different Rsm proteins in bacterial physiology?

Rsm proteins serve as post-transcriptional regulators that modulate various physiological processes in P. putida:

• Motility regulation: RsmE and RsmA play critical roles in swimming and swarming motility. The triple mutant (ΔIEA) and the ΔEA double mutant show reduced swimming motility compared to wild type .

• Biofilm development: All three Rsm proteins influence biofilm formation, with distinct patterns observed in different mutants. For example, the ΔI mutant initiates attachment similar to wild type but shows early detachment, while ΔE and ΔA strains exhibit reduced initial attachment .

• Exopolysaccharide production: Rsm proteins regulate the expression of genes involved in exopolysaccharide biosynthesis, affecting biofilm matrix composition .

• Stress response: There is evidence that Rsm proteins may be involved in stress responses, as indicated by their influence on various cellular processes related to environmental adaptation .

How do Rsm methyltransferases modify ribosomal RNA structure and function?

Rsm methyltransferases catalyze the addition of methyl groups to specific nucleotides in ribosomal RNA. For example, RsmD methylates G966 of the 16S rRNA, while RsmC modifies G1207 . These methylation events occur at functionally important and highly conserved sites on the ribosome, suggesting their significance in translation.

The modifications introduced by Rsm proteins can affect ribosome assembly, structure, and function. For example, RsmD acts late in the assembly process and can modify a completely assembled 30S subunit . It possesses superior binding properties toward the unmodified 30S subunit but is unable to bind a 30S subunit already modified at G966, indicating a precise recognition mechanism .

The crystal structure of RsmD has been determined at 2.05 Å resolution, revealing structural similarities to RsmC, whose target (m2G1207) is located very close in the spatial structure of the small ribosomal subunit . Comparison with RsmB, which methylates an adjacent residue with different base specificity (m5C967), provides insights into substrate specificity mechanisms .

What are the established protocols for generating null mutants of Rsm genes?

The generation of rsm null mutants involves a multi-step process as detailed in the literature. The following methodology has been successfully employed to create single, double, and triple mutants :

• PCR amplification: Regions flanking the target rsm gene are amplified using primers containing specific restriction sites.

• Cloning and verification: Amplified fragments are cloned into vectors like pGEM-T Easy or pCR2.1-TOPO, and the absence of missense mutations is confirmed by sequencing.

• Suicide vector construction: The null allele is subcloned into a suicide vector like pKNG101, which cannot replicate in Pseudomonas.

• Conjugation: The vector is introduced into P. putida KT2440 via conjugation using helper strains.

• Selection and counter-selection: Merodiploid exconjugants are selected, then subjected to counter-selection (e.g., with sucrose) to obtain clones where a second recombination event has removed the plasmid backbone.

• Verification: The presence of the null mutations is confirmed by PCR, sequencing, and Southern blotting.

The table below shows examples of primers used for rsm null mutant construction:

How can Rsm protein overexpression be achieved for functional studies?

Overexpression of Rsm proteins for functional studies involves cloning and expressing the genes using appropriate expression systems. Based on the methodologies described in the literature , the process typically includes:

• Gene amplification: The rsm gene of interest is amplified using PCR with primers containing appropriate restriction sites and tags (e.g., His-tag).

• Vector construction: The amplified gene is cloned into an expression vector such as pET15b for E. coli expression or a Pseudomonas-compatible vector.

• Expression: The recombinant protein is expressed in an appropriate host system under controlled conditions.

• Purification: The protein is purified using affinity chromatography or other suitable methods.

• Storage: Purified proteins are typically stored with glycerol (5-50%) at -20°C/-80°C to maintain stability.

For example, primers used for ectopic expression of Rsm proteins include:

What assays can be used to determine Rsm methyltransferase activity?

Several assays have been developed to measure the enzymatic activity of Rsm methyltransferases. These include:

• In vitro methylation assays: These typically involve incubating the purified enzyme with its substrate (rRNA or ribosomal subunits) and S-adenosyl-methionine (SAM) as the methyl donor. For RsmD, a standard assay uses:

  • 50 mM HepesK, pH 7.6

  • 2.5 mM Mg(OAc)2

  • 20 mM NH4Cl

  • 100 mM KCl

  • 8 mM β-mercaptoethanol

  • 0.2 mM AdoMet

  • 0.1 pmol of RsmD protein

  • 15 pmol of either rRNA or 30S subunits

  • Reaction volume: 50 μl

  • Incubation: 1 hour at 37°C

• Binding assays: These measure the interaction between the methyltransferase and its substrate, providing insights into affinity and specificity.

• Competition assays: These assess the ability of the enzyme to distinguish between modified and unmodified substrates, as demonstrated for RsmD, which binds preferentially to unmodified 30S subunits .

• Mutant complementation: This involves testing whether expression of wild-type or mutant methyltransferases can restore proper ribosome function in null mutants.

How can researchers measure the phenotypic effects of Rsm protein modifications?

The phenotypic effects of Rsm protein modifications can be assessed using various assays that measure bacterial behaviors and properties affected by these proteins:

• Motility assays: Swimming motility can be assessed using LB plates with 0.3% agar, while swarming motility can be tested on 0.5% agar plates .

• Biofilm assays: Biofilm formation can be measured by growing bacteria in polystyrene multiwell plates or glass tubes, followed by crystal violet staining of attached biomass. The dye can be solubilized with 30% acetic acid and quantified by measuring absorbance at 580 nm .

• Gene expression analysis: Quantitative reverse-transcription PCR (qRT-PCR) can be used to measure the expression of genes regulated by Rsm proteins, particularly those involved in exopolysaccharide biosynthesis and other relevant pathways .

• β-galactosidase assays: By creating translational fusions between rsm genes and lacZ, the expression patterns of Rsm proteins can be monitored by measuring β-galactosidase activity .

• Competitive fitness assays: These assess the relative fitness of wild-type and mutant strains when grown in mixed cultures, revealing the impact of Rsm proteins on bacterial competitiveness .

How do Rsm proteins influence biofilm formation at the molecular level?

Rsm proteins exert complex effects on biofilm formation through regulation of multiple factors involved in attachment, matrix production, and dispersal. Research has revealed several molecular mechanisms:

• Regulation of exopolysaccharide biosynthesis: Quantitative RT-PCR analysis has shown that the triple mutant (ΔIEA) exhibits significantly increased expression of genes involved in the biosynthesis of the exopolysaccharides Peb (2.5-fold) and Bcs (5-fold) compared to wild type after 48 hours of growth on solid medium . This suggests that Rsm proteins normally repress these pathways.

• Differential regulation by individual Rsm proteins: Analysis of single mutants revealed that for Peb expression, the lack of either RsmE or RsmA had an effect similar to that observed in the triple mutant, while RsmI did not appear to influence its expression. For Bcs, increased expression was observed in ΔE and ΔA mutants, but in neither case did it reach the levels observed in the triple mutant, suggesting a cumulative effect of both proteins .

• Biofilm attachment and detachment dynamics: Different rsm mutants show distinct biofilm development patterns. The ΔI mutant initiates attachment like the wild type but shows early detachment from the surface. The ΔE and ΔA strains present reduced attachment during the first hours but reach wild-type levels later. The double mutant ΔEA and the triple mutant ΔIEA show significantly higher attached biomass after 6 hours compared to wild type .

• Surface-dependent effects: Biofilms formed on glass surfaces by the triple mutant are more labile than those of the wild-type strain and are easily detached from the surface, a phenomenon not observed on plastic surfaces. This suggests that Rsm proteins influence the composition of the extracellular matrix in a way that affects adhesion to different surfaces .

• Timing of matrix synthesis: The altered biofilm phenotypes in rsm mutants are likely due to changes in the timing of synthesis of matrix components, as indicated by gene expression patterns .

What are the molecular interactions between Rsm proteins and their RNA targets?

The molecular interactions between Rsm proteins and their RNA targets involve specific recognition of RNA sequence and structural motifs:

• Target recognition: Rsm proteins typically bind to specific sequences in the 5' untranslated regions of target mRNAs, often at or near ribosome binding sites. This binding can either block ribosome access, inhibiting translation, or protect the mRNA from degradation, enhancing translation .

• Competition with sRNAs: Small regulatory RNAs like rsmY and rsmZ can sequester Rsm proteins by mimicking their binding sites on target mRNAs, thereby relieving repression of target genes .

• Cooperative binding: Multiple Rsm proteins can bind to the same target, enhancing regulatory effects through cooperative interactions.

• Structural basis: Crystal structures of related Rsm proteins reveal how they interact with RNA. For example, the structure of RsmD has been determined at 2.05 Å resolution, showing similarities to other RNA methyltransferases like RsmC . These structures provide insights into the molecular basis of substrate specificity and catalytic mechanism.

• Global identification of targets: Genome-wide analysis of targets for post-transcriptional regulation by RsmA, RsmE, and RsmI in P. putida KT2440 has been performed to identify RNA sequences bound in vivo, revealing the extensive regulatory network controlled by these proteins .

How do mutations in Rsm proteins affect bacterial motility and what are the underlying mechanisms?

Mutations in Rsm proteins have significant effects on bacterial motility, with different proteins playing distinct roles:

• Swimming motility: The triple mutant (ΔIEA) and the ΔEA double mutant show reduced swimming motility compared to wild type and other mutants in LB plates with 0.3% agar . This suggests that RsmE and RsmA normally enhance swimming motility, possibly by regulating flagellar genes.

• Swarming motility: When tested on 0.5% agar plates, only the ΔI mutant showed movement after 24 hours, although it was unable to completely cover the plate surface like the wild type. All other mutants, including the double and triple mutants, showed no swarming at this time point .

• Link to pyoverdine production: Since swarming motility in P. putida KT2440 requires pyoverdine-mediated iron acquisition, the researchers measured pyoverdine levels in culture supernatants. All mutant strains showed reduced pyoverdine production compared to wild type, which could partially explain the swarming defects .

• Complex regulation: The difference in motility between ΔI and the remaining mutants cannot be explained simply by differences in pyoverdine production, which were not significantly different among the mutants. This suggests additional regulatory mechanisms involving Rsm proteins .

• Connection to GacS-GacA system: Previous research has shown that mutants in the GacS-GacA regulatory system display hypermotility, and a regulatory cascade involving RsmE and RsmI has been proposed. This suggests that Rsm proteins are part of a larger regulatory network controlling motility in response to environmental signals .

What role do Rsm proteins play in bacterial stress responses and adaptation?

While the search results don't directly address the role of RsmB in stress responses, they do provide insights into how related Rsm proteins might be involved in stress adaptation:

• sRNA-mediated regulation: Research on a novel small RNA in P. putida KT2440 revealed that it is stress-inducible and affects the expression of genes involved in amino acid metabolism and stress responses . The expression of this sRNA was altered by plasmid carriage, suggesting a role in adaptation to plasmid-imposed stress.

• Metabolic regulation: Rsm proteins regulate genes involved in various metabolic pathways, potentially allowing the bacterium to adapt to changing nutrient conditions. The triple rsm mutant showed altered expression of genes involved in exopolysaccharide biosynthesis, suggesting broader metabolic effects .

• Environmental resilience: P. putida is known for its robustness upon challenges with oxidative stress and toxic chemicals. The regulatory networks involving Rsm proteins may contribute to this resilience by modulating gene expression in response to environmental signals .

• Biofilm regulation: The ability to form biofilms is an important stress response that provides protection against various environmental threats. By regulating biofilm formation, Rsm proteins indirectly influence stress resistance .

• Cross-talk with other regulatory systems: Rsm proteins interact with the GacS-GacA two-component system, which is known to respond to environmental signals and stress conditions. This connection places Rsm proteins within broader stress-response networks .

What are the latest genomic engineering tools for studying Rsm proteins?

Recent advances in genomic engineering have expanded the toolkit available for studying Rsm proteins and their functions:

• CRISPR/Cas9 technologies: These have been applied to P. putida for various purposes, including:

  • Efficient curing of helper plasmids

  • Counterselection of infrequent mutations created through recombineering

  • Metabolic engineering

  • CRISPR interference-mediated gene regulation

• Recombineering methods: Novel approaches include:

  • RecET-based markerless recombineering for deletion and integration of large-sized genes and clusters

  • Efficient single-stranded recombineering using thermoinducible systems

  • Improved methods for marker removal and genome streamlining

• Modular vector systems: The Standard European Vector Architecture (SEVA) platform provides a valuable resource for constructing recombinant P. putida strains, offering modular vectors that can be used to express and study Rsm proteins .

• Transposon-based tools: Synthetic derivatives of Tn5- and Tn7-based transposon vectors allow for random or site-specific insertion of genetic constructs into the P. putida genome .

• Self-curing plasmids: Recent innovations include synthetic control of helper plasmid replication, enabling self-curing of the plasmid in a mere overnight cultivation, which significantly speeds up genetic modification procedures .

How can systems biology approaches enhance our understanding of Rsm protein functions?

Systems biology approaches can provide comprehensive insights into the functions and regulatory networks of Rsm proteins:

• Genome-scale metabolic models: Several genome-scale metabolic models (GSMM) are available for P. putida, such as iJN1462, which is comparable to high-quality E. coli models in size and level of detail. These models can help predict the effects of rsm mutations on metabolic fluxes and cellular phenotypes .

• Transcriptome analysis: Global transcriptome profiling using RNA-seq can reveal the complete set of genes regulated by Rsm proteins under different conditions, providing a systems-level view of their regulatory functions .

• Proteomics: Mass spectrometry-based proteomics can identify changes in protein abundance and post-translational modifications resulting from Rsm protein activity or absence.

• Interactomics: Techniques like co-immunoprecipitation followed by mass spectrometry can identify proteins that interact with Rsm proteins, revealing their integration in broader regulatory networks.

• Multi-omics integration: Combining data from genomics, transcriptomics, proteomics, and metabolomics can provide a holistic view of how Rsm proteins influence cellular physiology at multiple levels.

• Data fusion: Advanced statistical methods like those developed by RSMB can enable the integration of diverse datasets to gain deeper insights into complex biological systems and regulatory networks .

What are the potential biotechnological applications of engineered Rsm proteins?

While the search results don't directly address biotechnological applications of RsmB, we can infer potential applications based on the functions of related Rsm proteins:

• Enhanced biofilm formation: Engineered Rsm proteins could be used to modulate biofilm formation for beneficial applications such as bioremediation or industrial bioprocessing. The triple rsm mutant shows increased biofilm formation under certain conditions, suggesting that manipulating these proteins could enhance biofilm-based biotechnological processes .

• Metabolic engineering: Rsm proteins regulate various metabolic pathways, including exopolysaccharide biosynthesis. Engineering these regulatory proteins could potentially enhance the production of valuable biopolymers or other metabolites .

• Stress resistance: By modulating stress response pathways, engineered Rsm proteins might improve the robustness of P. putida in industrial settings or environmental applications .

• Controlled gene expression: The post-transcriptional regulatory function of Rsm proteins could be harnessed to develop novel gene expression control systems for biotechnological applications.

• Bioremediation: P. putida is already known for its potential in bioremediation of polluted sites. Engineered Rsm proteins might enhance this capability by optimizing relevant metabolic pathways and stress responses .

How do Rsm proteins interact with other regulatory networks in bacterial cells?

Rsm proteins are integrated into complex regulatory networks that control various aspects of bacterial physiology:

What are the major knowledge gaps and future research directions in the field of Rsm methyltransferases?

Despite significant advances in understanding Rsm methyltransferases, several important knowledge gaps remain:

• Complete target identification: While some targets of Rsm proteins have been identified, a comprehensive catalog of all mRNAs and other RNA molecules regulated by each Rsm protein in P. putida is still lacking.

• Structural insights: Detailed structural information about P. putida Rsm proteins in complex with their RNA targets would provide valuable insights into the molecular basis of their specificity and function.

• Environmental regulation: The environmental signals and conditions that regulate the expression and activity of Rsm proteins in P. putida need further investigation.

• Functional redundancy: The extent of functional overlap among different Rsm proteins and the evolutionary pressures maintaining multiple family members in the same organism require further study.

• Integration with global regulatory networks: A more comprehensive understanding of how Rsm proteins interact with other regulatory systems, including two-component systems and global regulators, would provide a more complete picture of bacterial gene regulation.

Future research directions might include:

• Application of advanced structural biology techniques to elucidate Rsm-RNA interactions

• Development of high-throughput methods to identify all targets of Rsm proteins

• Investigation of the roles of Rsm proteins in stress responses and adaptation to different environments

• Exploration of the potential of Rsm proteins as targets for antimicrobial development

• Engineering of Rsm proteins for biotechnological applications