Cell Disruption and RNA Extraction from Rhodococcus and Gordonia Bacteria Grown in the Presence of Hydrophobic Organic Compounds and Heavy Metal Salts

Abstract

Non-pathogenic actinomycetes play a key role in the degradation of xenobiotics and the biotransformation of complex organic compounds. Studying gene expression in these bacteria can be challenging due to the rigid cell wall that hinders RNA extraction. Here we present the first comprehensive study of total RNA isolation from Rhodococcus and Gordonia genera, encompassing 21 strains grown under 34 conditions, including n-alkanes C3–C16, terpenoids, non-steroidal anti-inflammatory drugs, used frying oil, and mercury chloride. We compared no lysis strategy and three cell lysis approaches (freezing-thawing, bead beating, and lysozyme treatment) combined with the TRIzol or silica column-based RNA extraction kits. Bead beating, despite aggressive mechanical action, failed to reliably yield intact RNA. We show for the first time that 40 mg/mL of lysozyme with prolonged exposure (1 h) reliably lyses Rhodococcus and Gordonia cells (96–100% successful isolations), yielding pure integral RNA (118.5–485.8 ng/µL) with the RNA Solo Kit, without requiring detergents. We also developed the first protocol for RNA extraction from Rhodococcus cells grown on used frying oil, requiring pre-washing with 0.1 vol.% Tween 80 to disintegrate hard-to-harvest aggregates. These findings establish a robust strategy for RNA isolation from hydrophobic Rhodococcus and Gordonia bacteria.