Megaplasmids pDK2 and pRHL2 as well as pDK3 and pRHL1 Share Almost Identical Catabolic Gene Clusters

Rhodococcus sp. DK17 was isolated by Kim et al. (2002) by enrichment on o-xylene as the sole source of carbon and energy, but it is also able to grow on ethylbenzene, isopropylbenzene, toluene, benzene, phthalate, and terephthalate. Three megaplasmids pDK1, pDK2, and pDK3 were identified in this strain (Table 1). Genes for alkylbenzene dioxygenases (AkbA) cz's-dihydrodiol dehydrogenase (AkbB), meta-cleavage enzyme (AkbC), and downstream catabolic enzymes (AkbDEF) are located on the 330-kb plasmid pDK2 (Kim et al. 2004). Gene organization as well as nucleotide sequences of the akb regions are identical to corresponding regions of pRHL2 of Rhodococ-

Fig. 5 Organization of genes involved in biphenyl and alkylbenzene degradation. pDK2, akb genes located on the linear plasmid pDK2 of Rhodococcus sp. DK17 (Kim et al. 2004; AY502075); pRHL2, bph genes on pRHL2 of Rhodococcus sp. RHA1 (NC_008270); pRHLl, bph genes on pRHLl of strain RHA1 (NC_008296); pBD2, ipb genes of linear plasmid pBD2 of R. erythropolis BD2 (Stecker et al. 2003; NC_005073.1); pTA421, bphC3 locus on pTA421 of R. erythropolis TA421 (Arai et al. 1998; accession numbers AB014348, D88015, and D88020). The akbA/etbA/bphA/ipbA genes code for the oxygenase (A1A2/AaAb), ferredoxin (A3/Ac), and ferredoxin reductase (A4/Ad) components of ring-hydroxylating dioxygenases; akbB/bphB/ipbB, akbC/etbC/bphC, and akbD/bphD/ipbD encode cis-dihydrodiol dehydrogenases, meta-cleavage dioxygenase, and C-C hydrolases, respectively; akbE/bphE and akbF/bphF encode the downstream hydratase and aldolase. Genes akbST/bphST/ipbST code for putative sensor kinase and response regulator of a two-component signal transduction system. ORFs 12 and 13 of pDK2 and their homologues on pRHL2, pRHL1, and pBD2 may encode an enoyl-CoA hydratase and an acyl-CoA ligase (alkK), respectively. The gene products of ro10124 and ro10142 and their homologues on pDK2 are conserved hypothetical proteins; ro10140 of pRHL2 and ORF21 of pDK2 may code for an oxidoreductase (www.rhodococcus.ca; Kim et al. 2004). The arrows indicate the size, location, and direction of transcription of the genes and ORFs. Homologous ORFs feature the same pattern. Truncated regions are marked with the symbol )

cus sp. strain RHA1 (Fig. 5) (Kim et al. 2004). In fact, pDK2 shares nearly complete nucleotide sequence identity with pRHL2 of Rhodococcus sp. RHA1, except for a 0.9-kb insertion in pRHL2, indicating recent horizontal transfer (Gon^alves et al. 2006).

Plasmid pDK2 and the 750-kb plasmid pDK3 each contain a copy of oph and tph operons coding for the transformation of phthalate and terephtha-late, respectively, to protocatechuate. These operons are separated by a region containing a putative phthalate ester hydrolase gene and putative trans-

porter genes, which is organized like the patEBCAD cluster of strain RHA1 (Choi et al. 2005). Plasmids pDK3 of Rhodococcus sp. DK17 and pRHL1 from Rhodococcus sp. RHA1 actually share two regions of 99% sequence identity, namely, a 73-kb region that includes the genes encoding phtha-late and terephthalate metabolism, and another 22-kb segment (Patrauchan et al. 2005).

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