Recently, Shen W et al. identified five genes (pnpACC1C2R) in another gram-negative PNP-degrading bacterium, Pseudomonas putida DLL-E4, but the XL184 in vitro rest of the genes (pnpBDE) in this gene cluster were not

identified [12]. To date, all the studies have focused on identifying the upper stream genes in the HQ pathway, while the knowledge of the lower stream pathway genes, especially that of the 4-HS dehydrogenase [13], remains limited. In this study, a gram-negative bacterium Pseudomonas sp. 1-7, with the ability to degrade both MP and PNP, was isolated from MP-polluted activated sludge. Microbial degradation studies showed that the intermediate JQEZ5 purchase products were HQ and 4-NC, which indicated that both the HQ pathway and BT pathway were utilized in Pseudomonas sp. 1-7. Additionally, a 10.6 Kb gene cluster (pdcEDGFCBA) was identified from a genomic library. Genes: pdcDE, pdcF and pdcG were selleck chemicals llc chosen to be expressed in Escherichia coli for characterization. Methods Strains, plasmids, and chemicals The plasmids and bacterial strains used in this study are listed in Table 1. Pseudomonas sp. 1-7 was grown at 30°C in Luria Bertani (LB) medium and Burk mineral medium [14] with 1 mM MP or 0.5 mM PNP as the sole carbon and nitrogen source, respectively. E. coli strains were grown in LB medium at 37°C and were transformed as described [15]. The primer sequences used for PCR are listed in Additional file 1: Table S1. All Janus kinase (JAK) reagents

used in this study were purchased from Sigma Chemical (St. Louis, MO, 113 USA) and Amresco Chemical (Solon, OH 44139 USA). Table 1 Bacterial strains and plasmids used in this study Strains and plasmids Relevant genotype or characteristic(s) Reference or source Pseudomonas sp     Strain 1-7 methyl parathion and p-nitrophenol utilizer, wild type This study E.coli     Trans10 F-Φ80(lacZ) M15 lacX74hsdR(rK -mK +) recA1398endA1tonA TransGen BL21(DE3) F- ompT hsdS (rB- mB-) gal dcm lacY1(DE3) Novagen Plasmids     pET30a Kmr, Expression vector Novagen pET22b Ampr, Expression vector Novagen pET2230 Ampr, Expression vector This

study pEASY-T3 Ampr, Cloning vector TransGen pET30- pdcF BamHI-HindIII fragment containing pdcF inserted into pET30a This study pET30- pdcG BamHI-XhoI fragment containing pdcG inserted into pET30a This study pET30- pdcD BamHI-XhoI fragment containing pdcD inserted into pET30a This study pET2230- pdcE BamHI-XhoI fragment containing pdcE inserted into pET2230 This study Isolation of Pseudomonas degrading MP and PNP Activated sludge (0.5 g) collected from a pesticide factory (Tianjin, China) was cultured overnight at 30°C in 100 ml liquid Burk medium, before being diluted and spread on solid Burk medium containing 0.1% (v/v) MP pesticide and incubated at 30°C. The positive strain able to degrade MP produced a visible hydrolysis halo around the colonies on the plate. Positive colonies were inoculated in liquid Burk medium containing 0.1% (v/v) MP pesticide and cultured overnight at 30°C.