Recombinant Photobacterium profundum Ribosomal RNA small subunit methyltransferase G (rsmG)

What is Photobacterium profundum and why is it significant for studying rRNA methyltransferases?

Photobacterium profundum is a deep-sea Gammaproteobacterium belonging to the family Vibrionaceae and genus Photobacterium. It's a marine organism containing two circular chromosomes with the ability to grow at temperatures from 0°C to 25°C and pressures from 0.1 MPa to 70 MPa, depending on the strain . The most studied strain, SS9, grows optimally at 15°C and 28 MPa, making it both a psychrophile and a piezophile .

P. profundum is particularly valuable for studying rRNA methyltransferases because it has evolved specific adaptations to function under extreme pressure conditions. Unlike mesophilic bacteria whose protein synthesis machinery (including ribosomes) is highly pressure-sensitive, P. profundum maintains functional ribosomes at high pressures . The genome of P. profundum contains 15 rRNAs, the largest reported in any bacterium, with significant variation within these rRNA operons that likely reflects its ability to rapidly respond to pressure changes .

This unique characteristic makes P. profundum an excellent model organism for studying how rRNA methyltransferases like rsmG contribute to ribosome stability and function under varying pressure conditions, providing insights into fundamental mechanisms of bacterial adaptation to extreme environments.

How do rRNA methyltransferases contribute to bacterial adaptation to environmental stressors?

RNA methyltransferases play critical roles in modifying ribosomal RNA, which directly affects ribosome assembly, stability, and function. In the context of environmental adaptation, these modifications can be crucial for maintaining protein synthesis under stressful conditions.

For P. profundum, which experiences extreme pressure in its natural deep-sea habitat, rRNA methylation likely contributes to ribosomal stability under high pressure. Research has shown that mesophilic ribosomes are among the most pressure-sensitive structures in bacterial cells due to the large volume change associated with ribosome assembly . P. profundum has evolved adaptations to counter this sensitivity, and rRNA methylation is likely one such adaptation.

Studies of other rRNA methyltransferases provide insights into potential mechanisms. For example, in E. coli, the absence of rRNA methyltransferases can lead to altered ribosome structure and function, affecting translation efficiency and accuracy. These modifications can also influence antibiotic susceptibility, as many antibiotics target the ribosome . In high-pressure environments, proper ribosome assembly and function become even more critical, suggesting that rRNA methyltransferases like rsmG may have evolved specialized functions in piezophilic bacteria.

The adaptability of P. profundum to different pressures may be partially attributed to its variable expression of ribosomal proteins and likely rRNA methyltransferases, allowing it to maintain functional ribosomes across a wide range of pressure conditions .

What methods are recommended for culturing P. profundum for recombinant protein expression?

Culturing P. profundum for recombinant protein expression requires careful consideration of its growth requirements. Based on established protocols, the following methodological approach is recommended:

Medium Composition:

• Use 75% strength 2216 Marine Agar for solid media cultures

• For liquid cultures, Marine Broth or a defined medium such as Vogel-Bonner medium with appropriate salt concentrations can be used

Growth Conditions:

• Temperature: Optimal growth occurs at 15°C for strain SS9 and 9-10°C for strains 3TCK and DSJ4

• Pressure: For strain SS9, optimal growth occurs at 28 MPa, while strain 3TCK grows best at 0.1 MPa and strain DSJ4 at 10 MPa

• For atmospheric pressure culturing (convenient for lab work), use strain 3TCK or culture SS9 at reduced temperature

Expression System:

• Broad-host-range plasmids like pGL10 have been successfully used in P. profundum

• For expression in E. coli, vectors such as pET28a, pCA24N, or pGEX4T-1 have been used for recombinant methyltransferases

Induction Conditions:

• For IPTG-inducible systems, 0.2 mM IPTG at 22°C for 12 hours has been effective for methyltransferase expression

• Reduced temperature during induction can improve solubility of recombinant proteins

When working with P. profundum, it's important to note that its ability to grow at atmospheric pressure allows for easier genetic manipulation compared to obligate piezophiles, making it an excellent model organism for studying high-pressure adaptation mechanisms .

What analytical techniques are suitable for confirming the methyltransferase activity of recombinant rsmG?

Confirming the methyltransferase activity of recombinant rsmG from P. profundum requires targeted analytical approaches. Based on methodologies used for similar methyltransferases, the following techniques are recommended:

In vitro Methylation Assays:

• Using purified recombinant rsmG (typically at ~3 μM concentration) in a reaction with its substrate (16S rRNA fragments) and S-adenosyl-methionine (SAM) as the methyl donor

• The reaction is typically conducted in a buffer system that mimics physiological conditions, with incubation times of 30-60 minutes

HPLC Analysis:

• High-Performance Liquid Chromatography can be used to separate and quantify methylated and unmethylated rRNA fragments

• Methylated nucleosides show specific retention time shifts compared to unmethylated counterparts

• The relative abundance of methylated products can be quantified by comparing peak areas

Mass Spectrometry:

• LC-MS or LC-MS/MS analysis provides definitive identification of methylated nucleosides

• This approach can determine both the position and nature of the methylation

• Peptide-based LC-MS methods similar to those used in the P. profundum proteomic analysis can be adapted for analyzing rsmG activity

Comparative Analysis Under Different Conditions:

• To understand pressure effects, methylation assays can be conducted under different pressure conditions

• Pressurized reaction chambers capable of maintaining 0.1-70 MPa can be used to mimic natural conditions

• Activity comparisons between P. profundum rsmG and orthologues from non-piezophilic bacteria provide insights into pressure adaptation mechanisms

When designing experiments to analyze rsmG activity, it's important to consider control reactions without the enzyme or without SAM to confirm the specificity of the methylation reaction.

How do genomic characteristics of P. profundum inform approaches to recombinant rsmG expression?

The genomic architecture of P. profundum provides valuable insights for recombinant rsmG expression strategies. Understanding these characteristics can guide experimental design and optimize expression outcomes.

P. profundum possesses a genome ranging from 4.2 to 6.4 Mb in size, with P. profundum SS9 having one of the largest genomes in the genus, likely reflecting its environmental versatility . The genome is distributed across two circular chromosomes and an 80 kb plasmid , which has implications for gene expression regulation and protein production.

The GC content of P. profundum varies between 38.7% and 50.9%, clustering in two groups of approximately 40% and 50% . This GC variation affects codon usage, which should be considered when designing expression systems, particularly if expressing in heterologous hosts like E. coli. Codon optimization may be necessary for efficient translation in expression hosts with different GC content profiles.

P. profundum contains extensive mobile genetic elements and shows evidence of horizontal gene transfer , suggesting that some genes, potentially including those encoding rRNA modification enzymes, may have been acquired from other species. This genetic plasticity may impact regulatory elements and expression patterns of rsmG.

When designing expression constructs, consider the following approaches based on successful expression of other P. profundum genes:

• Use of native promoters for expression in P. profundum

• Selection of appropriate broad-host-range vectors like pGL10

• Inclusion of pressure-responsive regulatory elements if expression under different pressure conditions is desired

• Consideration of the extensive CRISPR-Cas systems present in some P. profundum strains that might interfere with plasmid stability

The expression of stress response genes (htpG, dnaK, dnaJ, and groEL) is upregulated in P. profundum in response to pressure changes , suggesting that co-expression with chaperones might improve the folding and solubility of recombinant rsmG, particularly when expressed under non-native conditions.

What methodological approaches are most effective for studying the impact of pressure on recombinant P. profundum rsmG activity?

Investigating the effects of pressure on recombinant P. profundum rsmG activity requires specialized methodologies that can simulate deep-sea conditions while allowing for precise enzymatic analysis. Based on established research with piezophilic organisms, the following approaches are recommended:

High-Pressure Enzymatic Assays:

• Use pressure chambers capable of maintaining stable hydrostatic pressure during enzymatic reactions

• For real-time activity monitoring, specialized pressure vessels with optical windows or fiber optic probes can be employed

• Pressure ranges should span 0.1 MPa (atmospheric) to at least 70 MPa to cover P. profundum's natural range

Comparative Kinetic Analysis:

• Determine enzyme kinetic parameters (Km, Vmax, kcat) at various pressures to generate pressure-dependent activity profiles

• Compare these profiles between recombinant rsmG from different P. profundum strains (e.g., piezophilic SS9 vs. less pressure-adapted 3TCK) to isolate pressure-specific adaptations

• Perform parallel analyses with rsmG orthologues from non-piezophilic bacteria as controls

Structural Stability Assessment:

• Employ circular dichroism (CD) spectroscopy under pressure to monitor pressure-induced conformational changes

• Fluorescence spectroscopy with pressure cells can track changes in tertiary structure

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS) can identify regions of the protein with altered stability under pressure

In vitro Translation Systems:

• Develop reconstituted translation systems using P. profundum ribosomes with and without rsmG-mediated methylation

• Monitor translation efficiency and accuracy under varying pressure conditions

• Use fluorescent reporters or radiolabeled amino acids to quantify translation output

Advanced Microscopic Techniques:

• Adapt high-pressure microscopic chambers similar to those used for swimming velocity measurements of P. profundum for single-molecule enzymatic studies

• This allows direct visualization of enzyme-substrate interactions under pressure

When designing these experiments, it's crucial to consider that pressure effects might be modulated by other environmental factors. For example, the study of P. profundum swimming motility demonstrated that the effects of pressure were more pronounced under conditions of high viscosity , suggesting that buffer composition and reaction conditions should be carefully controlled and varied systematically.

How does c-di-GMP signaling potentially regulate rsmG activity in P. profundum, and what methods can be used to investigate this relationship?

The relationship between c-di-GMP signaling and rRNA methyltransferase activity presents an intriguing area for investigation in P. profundum, especially given recent findings in other bacterial systems. Based on research with related methyltransferases, the following methodological approach would be appropriate for investigating this potential regulatory mechanism:

c-di-GMP Binding Assays:

• Differential radial capillary action of ligand assay (DRaCALA) can detect direct binding between purified recombinant rsmG and radiolabeled c-di-GMP

• Isothermal titration calorimetry (ITC) provides quantitative binding parameters (Kd, ΔH, ΔS)

• Surface plasmon resonance (SPR) offers real-time binding kinetics

• Protein microarray approaches have successfully identified c-di-GMP-binding methyltransferases in E. coli

Functional Impact Analysis:

• In vitro methylation assays comparing rsmG activity in the presence and absence of c-di-GMP at various concentrations

• HPLC or mass spectrometry to quantify methylation products under different c-di-GMP concentrations, similar to methods used for RlmI and RlmE

• Structure-based mutational analysis of potential c-di-GMP binding sites in rsmG

Research has shown that c-di-GMP binds to and inhibits the activity of 23S rRNA methyltransferases like RlmI and RlmE in a dose-dependent manner. For example, 5 μM c-di-GMP inhibited RlmI activity by 49% and RlmE activity by 31% . Similar inhibitory effects might occur with rsmG, potentially as part of a broader regulatory mechanism affecting ribosome assembly under different environmental conditions.

In vivo Correlation Studies:

• Create P. profundum strains with modified c-di-GMP levels by manipulating diguanylate cyclases (like DgcZ) and phosphodiesterases

• Compare rsmG expression and 16S rRNA methylation patterns in these strains using RT-PCR and mass spectrometry

• Analyze ribosome assembly and function under different pressure conditions in strains with altered c-di-GMP levels

Pressure-Dependent Regulation Analysis:

• Investigate whether pressure affects the interaction between c-di-GMP and rsmG

• Combine high-pressure enzymatic assays with c-di-GMP titrations

• Examine changes in c-di-GMP levels in P. profundum under different pressure conditions using LC-MS/MS

This research direction is particularly relevant given that c-di-GMP has been shown to promote antibiotic tolerance by regulating rRNA methyltransferase activity in some bacteria , which could have implications for understanding P. profundum's natural resistance mechanisms.

What approaches can be used to investigate the role of recombinant P. profundum rsmG in antibiotic resistance mechanisms?

Investigating the role of P. profundum rsmG in antibiotic resistance requires a multifaceted approach that combines genetic, biochemical, and microbiological methods. Recent research has highlighted connections between rRNA methylation and antibiotic resistance, making this an important area for investigation.

Gene Deletion and Complementation Studies:

• Generate rsmG deletion mutants in P. profundum using techniques similar to those used for recD functional studies

• Complement these mutants with wild-type rsmG or site-directed mutants

• Assess growth under various pressure conditions and in the presence of different antibiotics

• Compare results with a methodology similar to the pressure-sensitive growth phenotype characterization of recD mutants

Minimum Inhibitory Concentration (MIC) Determination:

• Measure antibiotic susceptibility profiles of wild-type, ΔrsmG, and complemented strains

• Test across a range of pressures from 0.1 MPa to the strain's optimal pressure

• Focus on aminoglycosides and other ribosome-targeting antibiotics that might be affected by 16S rRNA methylation

Ribosome Binding Studies:

• Compare antibiotic binding to ribosomes isolated from wild-type and ΔrsmG strains

• Use techniques such as filter binding assays or fluorescence polarization

• Determine if methylation by rsmG affects the affinity of antibiotics for their ribosomal targets

Heterologous Expression Analysis:

• Express P. profundum rsmG in antibiotic-sensitive E. coli strains

• Determine if expression confers resistance to specific antibiotics

• Compare with effects observed when expressing rsmG orthologues from non-piezophilic bacteria

Pressure-Dependent Resistance Profiles:

• Establish growth curves under antibiotic treatment at different pressures

• Use methodology similar to that described for the Vogel-Bonner medium growth curve experiments

• Compare growth at 0, 1.5, 3, 6, and 9 μg/mL antibiotic concentrations under different pressure conditions

Recent research has demonstrated that rRNA methylation serves as a significant mechanism for bacterial resistance against ribosome-targeting antibiotics . In clinical contexts, 16S rRNA methyltransferases can confer resistance by modifying conserved rRNA residues in functional sites targeted by antibiotics. While rsmG's specific role in P. profundum hasn't been fully characterized, its function as a 16S rRNA methyltransferase suggests it could contribute to intrinsic resistance mechanisms, particularly under the organism's natural high-pressure conditions.

How can structural biology techniques be optimized to determine the pressure-adapted features of P. profundum rsmG?

Determining the structural adaptations that enable P. profundum rsmG to function under high pressure requires specialized approaches that can capture structural information under native-like conditions. The following methodological framework addresses the unique challenges of studying pressure-adapted proteins:

X-ray Crystallography with Pressure Considerations:

• Crystallize recombinant rsmG using conditions that mimic aspects of the high-pressure environment (e.g., osmolytes, kosmotropic salts)

• Perform diffraction studies at cryogenic temperatures to "trap" pressure-adapted conformations

• Compare structures of rsmG from P. profundum SS9 (piezophilic) and 3TCK (less pressure-adapted) to identify structural differences

High-Pressure NMR Spectroscopy:

• Use specialized high-pressure NMR cells capable of maintaining stable pressure during data collection

• Acquire 2D and 3D NMR spectra at various pressures from 0.1 to 50 MPa

• Monitor chemical shift perturbations to identify pressure-sensitive regions of the protein

• Compare backbone dynamics at different pressures using relaxation measurements

Molecular Dynamics Simulations:

• Perform simulations of rsmG under various pressure conditions

• Analyze conformational ensembles, cavity volumes, and hydration patterns

• Identify pressure-sensitive regions that may be involved in catalysis or substrate binding

• Use approaches similar to those that revealed c-di-GMP binding mechanisms in RlmI, where structural simulation showed that binding induced closure of the catalytic pocket

Comparative Structural Analysis:

• Identify key residues that differ between piezophilic and non-piezophilic rsmG orthologues

• Focus on regions involved in substrate binding, catalysis, and structural stability

• Create chimeric proteins to test the contribution of specific regions to pressure adaptation

Small-Angle X-ray Scattering (SAXS) Under Pressure:

When analyzing structural data, particular attention should be paid to specific features associated with pressure adaptation in other proteins, including:

• Reduced internal cavities and improved packing

• Increased use of salt bridges and hydrogen bonds

• Modifications in surface charge distribution and hydration

• Altered flexibility in key functional regions

Understanding these structural adaptations would provide insights not only into how P. profundum rsmG functions under pressure but also into general principles of protein adaptation to extreme environments, potentially informing the engineering of pressure-stable enzymes for biotechnological applications.

What genomic and transcriptomic approaches can reveal the regulatory network controlling rsmG expression in P. profundum under varying pressure conditions?

Understanding the regulatory mechanisms controlling rsmG expression in P. profundum under different pressure conditions requires integrated genomic and transcriptomic approaches that can capture the complexity of pressure-responsive gene regulation. The following methodological framework provides a comprehensive strategy:

Comparative Transcriptomics:

• Perform RNA-seq analysis of P. profundum cultures grown at different pressures (0.1, 15, 28, and 40 MPa)

• Identify co-regulated gene clusters that include rsmG

• Analyze upstream regulatory regions for shared motifs

• Compare expression profiles across different P. profundum strains with varying pressure optima

Chromatin Immunoprecipitation Sequencing (ChIP-seq):

• Identify transcription factors binding to the rsmG promoter region under different pressure conditions

• Focus on pressure-responsive regulators such as those in the MarR family, which has been found to be differentially expressed between high and atmospheric pressure (7.8-fold decrease at high pressure)

• Compare binding patterns at different pressures to identify pressure-dependent regulatory mechanisms

Promoter Analysis and Reporter Systems:

• Clone the rsmG promoter region into reporter plasmids (e.g., using GFP or luciferase)

• Introduce systematic mutations to identify key regulatory elements

• Measure reporter activity under varying pressure conditions using high-pressure cultivation chambers

• Create a series of promoter truncations to define minimal pressure-responsive elements

Global Regulatory Network Analysis:

• Identify transcription factors differentially expressed under varying pressure conditions

• Create knockout strains for candidate regulators and assess impact on rsmG expression

• Perform epistasis analysis to establish hierarchy in the regulatory network

• Apply network analysis to RNA-seq data to identify regulatory hubs controlling pressure-responsive genes

Integration with Proteomics:

• Correlate transcriptomic data with proteomic profiles obtained under similar conditions

• Use label-free quantitation and mass spectrometry approaches similar to those used in previous P. profundum studies

• Identify post-transcriptional regulatory mechanisms by comparing mRNA and protein abundance changes

Secondary Messenger Signaling Analysis:

• Measure levels of signaling molecules like c-di-GMP under different pressure conditions

• Investigate whether these secondary messengers affect rsmG expression in addition to potentially regulating enzyme activity

• Create strains with altered c-di-GMP levels by manipulating diguanylate cyclases (dgcZ) and phosphodiesterases, using approaches similar to those described for E. coli

When analyzing data from these experiments, it's important to consider that P. profundum has evolved to respond to multiple environmental variables simultaneously. The extreme variations in pressure, temperature, and nutrient availability in deep-sea environments likely result in complex regulatory networks with multiple inputs and redundant control mechanisms.

Understanding these regulatory pathways has implications beyond basic science, as insights into how P. profundum maintains ribosome function under pressure could inform strategies for engineering pressure-tolerant microorganisms for biotechnological applications.