Recombinant Mannheimia succiniciproducens Ribosomal RNA small subunit methyltransferase G (rsmG)

What is Ribosomal RNA small subunit methyltransferase G (rsmG) and what is its role in bacterial cells?

Ribosomal RNA small subunit methyltransferase G (rsmG) is an enzyme that methylates the N7 position of nucleotide G535 in 16S rRNA of bacteria (corresponding to G527 in Escherichia coli) . This enzyme plays a critical role in ribosome biogenesis and function by catalyzing the formation of 7-methylguanosine (m7G) in 16S rRNA, which is the only naturally occurring m7G modification in bacterial 16S rRNA .

The enzyme utilizes S-adenosylmethionine (SAM) as a methyl donor for this reaction. Importantly, rsmG activity influences translational accuracy and has been linked to antibiotic sensitivity, particularly to streptomycin. Disruption or mutation of the rsmG gene has been shown to confer low-level streptomycin resistance in various bacteria including Bacillus subtilis, Escherichia coli, Mycobacterium tuberculosis, and Streptomyces coelicolor .

How do mutations in rsmG affect streptomycin resistance and what are the molecular mechanisms involved?

Mutations in the rsmG gene confer low-level streptomycin resistance (up to 100 μg/ml in LB medium for B. subtilis) . This resistance is causally related to the loss of methylation at position G535 in 16S rRNA. When rsmG is inactivated, the absence of the m7G modification alters ribosome structure in a way that reduces streptomycin binding affinity.

The molecular mechanism involves:

• Loss of the m7G modification in 16S rRNA due to rsmG mutation or deletion

• Altered ribosome structure that reduces streptomycin binding

• In some bacteria, such as B. subtilis, a detectable increase in translational accuracy is observed, though not as pronounced as in rpsL mutations

What's particularly significant is that rsmG mutants exhibit a 500- to 2,000-fold higher frequency of mutation to high-level streptomycin resistance compared to wild-type strains . This suggests that rsmG mutations create a genetic background that facilitates the acquisition of additional resistance mutations, particularly in rpsL.

What experimental methods are used to study rsmG methyltransferase activity and its effects?

Several methodological approaches are employed to study rsmG methyltransferase activity and its effects:

• Gene disruption and complementation studies:

  • Construction of rsmG knockout strains using targeted gene disruption

  • Complementation with functional rsmG genes to confirm phenotypes

  • Analysis of streptomycin resistance using minimum inhibitory concentration (MIC) determination

• RNA modification analysis:

  • Isolation of 16S rRNA from wild-type and mutant strains

  • Analysis of methylation status using HPLC or mass spectrometry

  • Identification of m7G modification at specific positions

• Translational accuracy assays:

  • Measurement of readthrough of nonsense codons as an indicator of translational accuracy

  • Comparison of readthrough frequencies between wild-type and rsmG mutant strains

• Growth and competition assays:

  • Comparison of growth rates between wild-type and rsmG mutant strains in various media

  • Competition assays to assess fitness costs of rsmG mutations

  • Long-term cultivation experiments to evaluate evolutionary stability

• Determination of mutation frequencies:

  • Analysis of spontaneous mutation rates to high-level streptomycin resistance

  • Sequencing of streptomycin resistance genes in mutants

How do rsmG mutations enhance enzyme production and secondary metabolism in bacteria?

rsmG mutations have been shown to dramatically enhance enzyme production and secondary metabolism in bacteria through several interrelated mechanisms:

• Enhanced expression of SAM synthetase gene (metK):

  • rsmG mutations lead to significantly increased expression of metK, which encodes S-adenosylmethionine synthetase

  • This results in higher intracellular SAM levels, which serve as a signal molecule for secondary metabolism

• Increased protein synthesis activity:

  • rsmG mutations, especially when combined with rpsL mutations, lead to 2.5-fold higher in vitro protein synthesis activity

  • Enhanced protein synthesis during transition and stationary phases contributes to increased enzyme production

• Cumulative effects with other mutations:

  • When combined with other mutations like rpsL (encoding ribosomal protein S12) and rpoB (encoding RNA polymerase β-subunit), rsmG mutations can lead to dramatic increases in enzyme production

  • For example, ribosome engineering with cumulative drug resistance mutations has shown >1,000-fold enhancement of enzyme production

A study with Paenibacillus agaridevorans demonstrated that introducing an rsmG mutation, combined with other mutations, led to substantially enhanced production of cycloisomaltooligosaccharide glucanotransferase (CITase) . This enzyme enhancement occurs due to the activation of otherwise silent or weakly expressed genes.

How can rsmG be utilized in metabolic engineering strategies for enhanced succinic acid production?

Based on the research data, rsmG could be utilized in several strategic approaches for enhancing succinic acid production in M. succiniciproducens:

• Ribosome engineering approach:

  • Introduction of rsmG mutations could enhance the expression of key enzymes in the succinic acid production pathway

  • Combined mutations in rsmG, rpsL, and rpoB could potentially lead to dramatic increases in enzyme production, as demonstrated in other bacteria

• Enhancement of specific pathway enzymes:

  • The succinic acid production pathway in M. succiniciproducens involves several key enzymes:

    • PEP carboxykinase (most important CO2-fixing enzyme)

    • Malate dehydrogenase (MDH)

    • Fumarase

    • Fumarate reductase

  • rsmG mutations could enhance the expression of these enzymes, potentially increasing succinic acid production

• Elimination of competing pathways:

  • Successful metabolic engineering of M. succiniciproducens has involved disrupting genes for by-product formation (ldhA, pflB, pta, and ackA)

  • The LPK7 strain produced 13.4 g/liter of succinic acid with a yield of 0.97 mol succinic acid per mol glucose

  • rsmG mutations could potentially enhance the expression of the remaining pathway enzymes in these engineered strains

• Integration with enzyme optimization:

  • Research has shown that replacing native M. succiniciproducens MDH (MsMDH) with Corynebacterium glutamicum MDH (CgMDH) significantly enhanced succinic acid production

  • CgMDH showed higher specific activity and less substrate inhibition (ki of 588.9 μM compared to 67.4 μM for MsMDH)

  • rsmG mutations could potentially enhance the expression of such optimized enzymes

What methods are used to express and purify recombinant rsmG from Mannheimia succiniciproducens?

Based on standard protocols for similar recombinant proteins, the following methods would be applicable for expression and purification of recombinant M. succiniciproducens rsmG:

• Gene cloning and expression system selection:

  • PCR amplification of the rsmG gene from M. succiniciproducens genomic DNA

  • Cloning into an appropriate expression vector (pET, pBAD, etc.)

  • Selection of an appropriate host (E. coli, yeast) based on protein characteristics

  • For rsmG from related proteins, yeast has been used as an expression system

• Expression conditions optimization:

  • Temperature optimization (typically 16-37°C)

  • Induction conditions (IPTG concentration, time)

  • Media composition and cultivation parameters

• Purification protocol:

  • Cell lysis using methods such as sonication or French press

  • Initial capture using affinity chromatography (His-tag, GST-tag, etc.)

  • Further purification using ion exchange or size exclusion chromatography

  • Purity assessment using SDS-PAGE (target >85% purity)

• Protein characterization:

  • Mass spectrometry for molecular weight confirmation

  • Activity assays to verify methyltransferase function

  • Stability studies at different temperatures and buffer conditions

• Storage recommendations:

  • Addition of 5-50% glycerol and aliquoting for long-term storage at -20°C/-80°C

  • For liquid form, shelf life is typically 6 months at -20°C/-80°C

  • For lyophilized form, shelf life is typically 12 months at -20°C/-80°C

  • Avoiding repeated freeze-thaw cycles

How does rsmG methyltransferase activity differ among bacterial species, and what implications does this have for research?

rsmG methyltransferase shows both conservation and variation across bacterial species:

• Conservation of target site:

  • The rsmG methylation target (G535 in B. subtilis, corresponding to G527 in E. coli) is highly conserved across bacteria

  • This conservation suggests the fundamental importance of this modification for ribosome function

• Species-specific variations:

  • Different bacterial species show variations in rsmG activity and the phenotypic consequences of rsmG mutations

  • In B. subtilis, rsmG mutations lead to increased translational accuracy, while in E. coli, they do not

  • The frequency of spontaneous mutation to high-level streptomycin resistance varies among species

• Implications for antibiotic resistance research:

  • In M. tuberculosis, mutations in rsmG are an important cause of clinical streptomycin resistance

  • Understanding species-specific differences is crucial for predicting the emergence of antibiotic resistance

• Taxonomic considerations:

  • M. succiniciproducens is taxonomically related to other Pasteurellaceae

  • Comparative studies between M. succiniciproducens and its close relative Actinobacillus succinogenes show that they share many metabolic traits despite significant differences in genome structure

What is the effect of combining rsmG mutations with other genetic modifications for enhanced enzyme production?

Combining rsmG mutations with other genetic modifications can lead to synergistic effects on enzyme production:

• rsmG and rpsL double mutations:

  • Double mutants containing both rsmG and rpsL mutations show greater ability to produce antibiotics and enzymes than single mutants

  • The rpsL mutation (in ribosomal protein S12) increases translational accuracy, which complements the effects of rsmG mutations

• Triple mutations including rpoB:

  • Addition of rpoB mutations (in RNA polymerase β-subunit) to rsmG and rpsL mutations can further enhance enzyme production

  • The rpoB mutation may mimic the ppGpp-bound form of RNA polymerase, activating expression of biosynthetic gene clusters

• Cumulative effects of multiple mutations:

  • Studies have shown that cumulative drug resistance mutations (up to eight mutations) can dramatically enhance production (>1,000-fold)

  • This approach does not require detailed genetic information and can be applied to various bacteria

• Application to M. succiniciproducens:

  • In M. succiniciproducens, combining rsmG mutations with targeted genetic modifications of metabolic pathways could significantly enhance succinic acid production

  • For example, combining rsmG mutations with the deletion of by-product forming pathways (ldhA, pflB, pta, ackA) and optimization of key enzymes like MDH could potentially lead to superior production strains

How can response surface methodology (RSM) be applied to optimize recombinant rsmG expression and activity?

Response Surface Methodology (RSM) can be effectively applied to optimize recombinant rsmG expression and activity:

• Experimental design approach:

  • RSM employs statistical designs such as rotatable central composite design (RCCD) to study interactions among multiple variables

  • This approach minimizes the number of experiments needed while maximizing information gained

• Parameter selection for optimization:

  • Key parameters for rsmG expression optimization might include:

    • Temperature

    • pH

    • Inducer concentration

    • Media composition

    • Oxygen levels

  • For M. succiniciproducens, which is capnophilic, CO2 levels would be particularly important

• Mathematical modeling:

  • RSM generates second-order polynomial equations relating the response (enzyme activity) to the variables

  • The general form is: Y = β₀ + ∑βᵢXᵢ + ∑βᵢᵢXᵢ² + ∑βᵢⱼXᵢXⱼ

  • Where Y is the predicted response, X represents variables, and β represents coefficients

• Optimization and validation:

  • The model identifies optimal parameter combinations for maximum enzyme activity

  • These predictions are validated experimentally

  • For example, when optimizing enzyme production using RSM, maximum residual enzyme activity of 38.7% was achieved at specific temperature and air flow conditions

• Application to M. succiniciproducens:

  • RSM could be particularly valuable for optimizing rsmG expression in M. succiniciproducens, given its sensitivity to cultivation conditions

  • Dissolved CO2 concentration significantly affects M. succiniciproducens growth and metabolism, with concentrations below 8.74 mM severely suppressing growth

  • Optimizing CO2 levels alongside other parameters could maximize both cell growth and enzyme production

What are the functional differences between rsmG and other ribosomal RNA methyltransferases in Mannheimia succiniciproducens?

Ribosomal RNA methyltransferases in bacteria serve various functions in ribosome biogenesis and function. While specific comparative data for M. succiniciproducens is limited in the search results, we can infer differences based on related research:

• Target site specificity:

  • rsmG specifically methylates G535 (B. subtilis numbering) at the N7 position in 16S rRNA

  • rsmJ, another methyltransferase found in M. succiniciproducens, is described as a "16S rRNA m2G1516 methyltransferase" or "rRNA (guanine-N(2))-methyltransferase"

  • These enzymes have distinct target sites and potentially different effects on ribosome function

• Protein structure comparison:

  • The full sequence of rsmJ from M. succiniciproducens consists of 252 amino acids

  • Based on related methyltransferases, rsmG and rsmJ likely have distinct structural domains despite both being S-adenosylmethionine-dependent methyltransferases

• Functional consequences of mutation:

  • rsmG mutations confer low-level streptomycin resistance and enhance secondary metabolism

  • The effects of rsmJ mutations have not been as extensively characterized in the provided search results

• Evolutionary conservation:

  • Both rsmG and rsmJ are conserved across bacterial species, suggesting essential roles in ribosome function

  • The specific conservation patterns may differ, reflecting their distinct functional roles

How does the presence of rsmG affect the growth characteristics of Mannheimia succiniciproducens under different cultivation conditions?

While direct studies on rsmG effects on M. succiniciproducens growth are not explicitly detailed in the search results, we can synthesize related information:

What analytical methods are most effective for assessing rsmG methyltransferase activity in vitro?

Several analytical methods can be employed to assess rsmG methyltransferase activity in vitro:

• Radioisotope-based assays:

  • Using [³H-methyl]-S-adenosylmethionine as methyl donor

  • Measuring transfer of radioactive methyl groups to 16S rRNA substrate

  • Quantifying incorporation using liquid scintillation counting

• HPLC-based methods:

  • Analysis of nucleosides after enzymatic digestion of methylated RNA

  • Detection of 7-methylguanosine using UV absorbance or mass spectrometry

  • Comparison with standards for quantification

• Mass spectrometry approaches:

  • LC-MS/MS analysis of nucleosides from digested RNA

  • Direct detection of methylated oligonucleotides

  • Structural confirmation of methylation position

• Coupled enzyme assays:

  • Monitoring S-adenosylhomocysteine (SAH) production using SAH nucleosidase and adenine deaminase

  • Spectrophotometric detection of the resulting hypoxanthine

• Fluorescence-based assays:

  • Using methyltransferase-coupled fluorescence assays

  • Detection of byproducts of the methylation reaction through fluorescence changes

For optimal activity assessment, these methods should be performed under conditions that mimic physiological parameters of M. succiniciproducens, including:

• Appropriate pH (near neutral)

• Physiological temperature

• Presence of necessary cofactors

• Proper substrate (16S rRNA or appropriate oligonucleotides)

How does rsmG expression correlate with the different growth phases of Mannheimia succiniciproducens?

While specific data on rsmG expression patterns in M. succiniciproducens is not detailed in the search results, we can extrapolate based on related findings:

• Expression patterns in related bacteria:

  • In Streptomyces, rsmG expression is regulated in a growth phase-dependent manner

  • Expression of rsmG-related pathways is often enhanced during transition and stationary phases

• Relation to metabolic phases in M. succiniciproducens:

  • M. succiniciproducens exhibits distinct metabolic phases during batch culture

  • The transition from exponential growth to stationary phase is accompanied by changes in carbon flux distribution

  • rsmG expression may correlate with these metabolic shifts

• Impact on succinic acid production:

  • Succinic acid production in M. succiniciproducens is influenced by growth phase

  • Maximum productivity is typically observed in late exponential and early stationary phases

  • rsmG may play a role in regulating the expression of key enzymes involved in succinic acid production during these phases

• Potential regulatory mechanisms:

  • rsmG expression may be regulated by global stress response systems

  • Environmental factors such as CO2 availability, which significantly affects M. succiniciproducens growth , may indirectly influence rsmG expression

What strategies can be employed to enhance the stability and activity of recombinant rsmG?

Several strategies can be employed to enhance the stability and activity of recombinant rsmG:

• Protein engineering approaches:

  • Site-directed mutagenesis to improve thermostability

  • Fusion with stability-enhancing tags or domains

  • Rational design based on structural information from related methyltransferases

• Formulation optimization:

  • Addition of stabilizing agents such as glycerol (5-50%)

  • Optimization of buffer composition and pH

  • Addition of reducing agents to prevent oxidation of cysteine residues

• Storage conditions:

  • For liquid formulations, storage at -20°C/-80°C provides a shelf life of approximately 6 months

  • Lyophilized formulations can be stored at -20°C/-80°C for approximately 12 months

  • Avoiding repeated freeze-thaw cycles to maintain activity

• Expression system selection:

  • Choice of appropriate expression host (E. coli, yeast, etc.)

  • Codon optimization for the chosen host

  • Use of solubility-enhancing fusion partners

• Post-translational considerations:

  • Ensuring proper folding through chaperone co-expression

  • Maintaining appropriate redox environment

  • Removal of destabilizing elements in the protein sequence

These strategies should be evaluated systematically, potentially using statistical approaches like Response Surface Methodology (RSM) to identify optimal conditions for maximum stability and activity.