Life Science Journal 2014;11(1) http://www.lifesciencesite.com 353 Detection, Cloning, and Expression of catechol 2,3 dioxygenase Genes from Novel Polychlorinated Biphenyl (PCB) Degraders Hamdy A. Hassan Environmental Biotechnology Department, Genetic Engineering and Biotechnology Research Institute, Sadat City University, Sadat city, Egypt hamdy.hassan@gebri.usc.edu.eg Abstract: 27 bacterial isolates were isolated from PCBs contaminated site. A partial 16S rRNA gene sequence revealed that the isolates belonged to the genus Pseudomonas, Bacillus, Staphylococcus, Rhodococcus, Burkholderia, and Pandoraea, all the isolates have the capability to use monochlorinated biphenyl as the sole carbon source, ten of these isolates were belonged to genus Pseudomonas, Burkholderia, Bacillus and Rhodococcus had also the capability to use dichlorinated biphenyl as the sole carbon source and only two strains Burkholderia sp. HA- OP24 and Rhodococcus sp. HA-30 were capable of metabolizing the highly recalcitrant congener 2,4,5,2′,4′,5′- chlorobiphenyl. Two catechol 2,3 dioxygenase genes termed C23O 1 and C23O 2 were identified and characterized from Burkholderia sp. HA-OP24 and Pseudomonas sp. HA-OP22 respectively. These genes may act as functional genes in the metabolic pathway of the PCBs. These findings may benefit in the bioremediation of PCBs by selecting the vital C23O for the mineralization of PCBS compounds. [Hamdy A. Hassan. Detection, Cloning, and Expression of catechol 2,3 dioxygenase Genes from Novel Polychlorinated Biphenyl (PCB) Degraders. Life Sci J 2014;11(1):353-360] (ISSN:1097-8135). http://www.lifesciencesite.com. 54 Keywords: Polychlorinated Biphenyls, Catechol 2,3 dioxygenase / Extradiol dioxygenase. 1. Introduction PCBs are ubiquitous contaminants of public concern because of their persistence and bioaccumulation in the environment, as well as their potential toxicity to humans and ecotoxicology (Borlakoglu and Haegeles, 1991). One of the problems related to the persistence of PCBs can be explained by the inability of environmental microbial populations to degrade these compounds efficiently (Sylvestre, 2004). This problem could be solved by the introduction of specific PCBs degrading microorganisms into the environment. Cultivation of microorganisms isolated from PCBs contaminated sites or any variety of ecosystem samples by any chosen cultivation approach will inevitably favor the growth of some community members while others are inhibited or not cultured and allows only around 0.1 to 1% of the total viable bacterial cells present in a variety of ecosystems can be cultured (Amann et al., 1995). It is likely to generate more cultivation methods allow more detections of the PCBs degraders in the contaminated sites. Some aerobic bacteria capable of oxidizing PCBs have been reported (Pieper and Seeger 2008). Bacterial strains of Pseudomonas, Burkholderia, Comamonas, Cupriavidus, Sphingomonas, Acidovorax, Rhodococcus, Corneybacterium and Bacillus genera have been characterized (Furukawa and Fujihara, 2008; Seeger and Pieper, 2009) as well as some fungi, such as Phanerochaete chrysosporium (Beaudette et al., 1998). The degradation of PCBs by microorganisms isolated thus far is started with transforming PCBs into vicinal diols by initial dioxygenase enzymes belong to a large family of Rieske non-heme iron oxygenases (Iwasaki et al., 2007) by introducing two oxygen atoms into PCBs which dehydrogenated by a dehydrogenase to give chloro, 2,3' dihydroxybiphenyl which subjected to extradiol dioxygenase (Colbert, 2013), which can easily be identified due to their yellow pigmentation of the product (Pieper, 2005). In this study identification and characterization new PCBs degraders were performed based on culture-dependent bacterial community in PCBs contaminated site, and identify, characterize, clone, and expression of new catechol 2,3 dioxygenase genes type I from the new isolates for contributing in dissolve the problem of degradation of PCBs compounds. 2. Material and Methods Isolation, purification and preservation of bacterial isolates The samples were collected from the sediments of oxidation ponds which receives all the manufactories wastes of Sadat city – Egypt from the oxidation ponds 1 g of sediment was incubated in 1 L Erlenmeyer flasks containing 100 ml of mineral medium (MM) (Buffer:Na 2 HPO 4 x12H 2 O 14g, KH 2 PO 4 2g, H 2 O to 1L, pH 7.4;100x Salt-solution :Ca(NO3)2 x 4H 2 O 5g, MgSO 4 x 7H2O 20g, Fe III-ammonium-citrate 1g (28 % Fe),Trace elements solution 100 ml, H 2 O to 1L;