Date Published: July 28, 2013
Author(s): Yuka Sone, Yusuke Mochizuki, Keita Koizawa, Ryosuke Nakamura, Hidemitsu Pan-Hou, Tomoo Itoh, Masako Kiyono.
We report the complete nucleotide sequence of plasmid pMR68, isolated from Pseudomonas strain K-62, two plasmids contribute to broad-spectrum mercury resistance and that the mer operon from one of them (pMR26) has been previously characterized. The plasmid was 71,020 bp in length and contained 75 coding regions. Three mer gene clusters were identified. The first comprised merR-orf4-orf5-merT1-merP1-merF-merA-merB1, which confers bacterial resistance to mercuric ions and organomercury. The second and third clusters comprised merT2-merP2, which encodes a mercury transport system, and merB2, which encodes an organomercurial lyase, respectively. The deduced amino acid sequences for the proteins encoded by each of the mer genes identified in pMR68 bore greater similarity to sequences from Methylobacterium extorquens AM1 than to those from pMR26, a second mercury-resistance plasmid from Pseudomonas strain K-62. Escherichia coli cells carrying pMKY12 (containing merR-orf4-orf5-merT1-merP1-merF-merA-merB1 cloned from pMR68) and cells carrying pMRA114 (containing merR-merT-merP-merA-merG-merB1 cloned from plasmid pMR26) were more resistant to, and volatilized more, mercury from mercuric ions and phenylmercury than the control cells. The present results, together with our earlier findings, indicate that the high phenylmercury resistance noted for Pseudomonas strain K-62 seems to be achieved by multiple genes, particularly by the multiple merB encoding organomercurial lyase and one merG encoding cellular permeability to phenylmercury. The novel mer gene identified in pMR68 may help us to design new strategies aimed at the bioremediation of mercurials.
Pseudomonas strain K-62, a bacterial strain isolated from phenylmercury-polluted soil, is about 1,000 times more resistant to phenylmercury than sensitive strains of Escherichia coli (Tonomura et al. 1968). A study performed about 40 years ago showed that the biochemical mechanism underlying this mercurial resistance is based on the enzymatic degradation of organomercurials and the subsequent reduction of the resulting mercuric ions to the less toxic and more volatile metallic mercury (Tonomura et al. 1968). Two separate organomercurial lyases, designated S-1 and S-2, each showing somewhat different physical properties and substrate specificities, are thought to be responsible for the resistance of P. K-62 to phenylmercury (Tezuka and Tonomura 1976;Tezuka and Tonomura 1978). The organomercurial resistance of this soil strain is encoded by two plasmids, pMR26 and pMR68 (Kiyono et al. 1995b). In addition, pMR26 contains two mer operons that map about 1 kb apart (Kiyono et al. 1997). One comprises merR-o/p-merT-merP-merA-merG-merB1. The other is a defective mer operon comprising merR-o/p-merB2-merD (Kiyono and Pan-Hou 1999a).
The present study determined the complete nucleotide sequence of plasmid pMR68 (isolated from P. strain K-62). In addition, the mer genes in pMR68 and pMR26 were identified, sequenced, and cloned in E. coli. The pMR68 sequence contained 75 complete coding regions; however, we were not able to identify a predicted origin of replication (Additional file 2: Table S2 and Figure 1). Although most of the identified genes (44%) encoded mobile elements related to transfer functions, 12% encoded mercurial-resistance determinants, 16% encoded metabolism-related genes, and 28% encoded hypothetical proteins (HPs).
The authors declare that they have no competing interests.