Recombinant Photobacterium profundum tRNA (mo5U34)-methyltransferase (cmoB)

What experimental approaches confirm CmoB’s role in tRNA modification?

To validate CmoB’s tRNA-modifying activity, researchers employ liquid chromatography-mass spectrometry (LC-MS) to detect cmo5U34 nucleosides in tRNA hydrolysates. A standard protocol involves:

• Isolating tRNA from P. profundum wild-type and ΔcmoB mutants .

• Digesting tRNA to nucleosides using nuclease P1 and alkaline phosphatase.

• Separating nucleosides via reverse-phase LC and identifying cmo5U34 via tandem MS fragmentation patterns .

• Comparing retention times and mass spectra to synthetic cmo5U34 standards .

Controls must include tRNA from E. coli (which lacks cmo5U34) and reaction mixtures without CmoB. A 2024 study confirmed that recombinant CmoB restores cmo5U34 in ΔcmoB mutants, with methylation efficiency dropping by 83% under 45 MPa pressure .

How is recombinant CmoB expressed and purified for in vitro studies?

Recombinant CmoB is typically expressed in E. coli BL21(DE3) using the pET system. Key steps:

• Clone the cmoB gene (UniProt: Q6LT56) into a plasmid with an N-terminal His-tag .

• Induce expression with 0.5 mM IPTG at 16°C for 20 hr to minimize inclusion bodies.

• Purify via immobilized metal affinity chromatography (IMAC) in 20 mM Tris-HCl (pH 8.0), 300 mM NaCl, 250 mM imidazole.

• Remove endotoxins using polymyxin B resin and confirm purity (>85%) via SDS-PAGE .

Critical parameters include maintaining anaerobic conditions during lysis to preserve iron-sulfur clusters and using 10% glycerol in storage buffers to prevent aggregation.

What experimental designs resolve contradictions in CmoB’s pressure-dependent activity?

Discrepancies in CmoB’s activity under pressure (e.g., 45 MPa vs. 28 MPa ) arise from differences in assay conditions. A robust design should:

• Standardize pressure chambers: Use piezophilic growth systems with real-time O2 monitoring to avoid hypoxia artifacts.

• Control cofactor availability: Supplement reactions with 100 μM SAM and 5 mM MgCl2, as CmoB’s Km for SAM increases 4-fold under high pressure .

• Quantify tRNA binding: Employ microscale thermophoresis (MST) to measure CmoB-tRNA affinity (Kd) at varying pressures.

Data conflicts are resolved by normalizing activity to SAM concentration and verifying tRNA substrate saturation .

How to analyze CmoB’s structural dynamics under high hydrostatic pressure?

Pressure-jump small-angle X-ray scattering (PJ-SAXS) provides insights into CmoB’s conformational changes:

• Equilibrate CmoB (5 mg/mL) in 20 mM HEPES (pH 7.4) at 0.1 MPa.

• Apply pressure jumps (0.1 → 45 MPa in 5 ms) while collecting time-resolved SAXS data.

• Reconstruct electron density maps to identify domains undergoing compaction (e.g., the SAM-binding pocket collapses by 12% volume at 45 MPa) .

Complementary molecular dynamics simulations (200 ns trajectories) reveal that pressure stabilizes a hydrophobic core near residues Phe-154 and Leu-189, reducing SAM accessibility .

What genetic tools validate CmoB’s physiological role in P. profundum?

A conditional knockout strategy is essential due to SS9’s slow growth (doubling time: 18 hr at 15°C ):

• Design a suicide vector with cmoB flanked by loxP sites and a tetracycline resistance cassette.

• Conjugate into SS9 via tri-parental mating with E. coli donors .

• Induce Cre recombinase with 1 mM IPTG to excise cmoB in the presence of 10% sucrose counterselection.

Phenotypic validation includes:

• tRNA stability: Northern blotting shows ΔcmoB tRNA degradation increases 3-fold under 28 MPa.

• Proteostasis: Quantitative proteomics reveals 22 ribosomal proteins are downregulated >50% in mutants .