Heat shock-induced plasmid conformational changes in Rhodococcus sp. strain DK17

Abstract

Rhodococcus sp. strain DK17 utilizes a wide variety of aromatic compounds including alkylbenzenes and phthalates as sole carbon and energy sources. DK17 possesses three linear megaplasmids (380-kb pDK1, 330-kb pDK2, and 750-kb pDK3). The genes encoding the initial steps in alkylbenzene metabolism are present on pDK2 while the phthalate operons are duplicated and are present on both pDK2 and pDK3. We previously noticed that pDK2 has tendency to be easily cured by UV radiation. DK 17 with a growth temperature optimum at 30oC showed no growth at 37oC. When transferred to 30 oC, however, the 37oC culture began to grow immediately. This indicates that the temperature of 37oC is not lethal but stressful to DK17. Thus, to investigate the environmental factors affecting the structural stability of its megaplsmids, cells of DK17 were exposed to a heat shock of 37℃, and then were plated with proper dilutions on minimal agar plates containing 20 mM glucose. Colonies were randomly picked for colony PCR using primers for akbC, ophC', and ophC. The former two genes and the latter gene are specific for pDK2 and pDK3, respectively. After screening 200 colonies a total of 29 strains were found to lose at least one of the three genes. Pulsed-field gel electrophoresis analysis revealed six different cases of plasmid conformational changes: 1) nine of pDK2 loss, 2) one of pDK3 loss, 3) ten of pDK2 loss with appearance of a new plasmid (~700 kb), 4) eight of pDK3 loss with appearance of a new plasmid (~400 kb), 5) one of pDK3 loss with appearance of a new plasmid (~600 kb), and 6) one of both pDK2 and pDK3 loss with appearance of two new plasmids (~400 and ~700 kb).