Brezna Barbara, Khan Ashraf A, Cerniglia Carl E
Division of Microbiology, National Center for Toxicological Research, U.S. Food and Drug Administration, Jefferson, AR 72079, USA.
FEMS Microbiol Lett. 2003 Jun 27;223(2):177-83. doi: 10.1016/S0378-1097(03)00328-8.
Polycyclic aromatic hydrocarbon (PAH)-degrading genes nidA and nidB that encode the alpha and beta subunits of the aromatic ring-hydroxylating dioxygenase have been cloned and sequenced from Mycobacterium vanbaalenii PYR-1 [Khan et al., Appl. Environ Microbiol. 67 (2001) 3577-3585]. In this study, the presence of nidA and nidB in 12 other Mycobacterium or Rhodococcus strains was investigated. Initially, all strains were screened for their ability to degrade PAHs by a spray plate method, and for the presence of the dioxygenase Rieske center region by polymerase chain reaction (PCR). Only Mycobacterium sp. PAH 2.135 (RJGII-135), M. flavescens PYR-GCK (ATCC 700033), M. gilvum BB1 (DSM 9487) and M. frederiksbergense FAn9T (DSM 44346), all previously known PAH degraders, were positive in both tests. From the three positive strains, complete open reading frames of the nidA and nidB genes were amplified by PCR, using primers designed according to the known nidA and nidB sequences from PYR-1, cloned in the pBAD/Thio-TOPO vector and sequenced. The sequences showed >98% identity with the M. vanbaalenii PYR-1 nidA and nidB genes. Southern DNA-DNA hybridization using nidA and nidB probes from PYR-1 revealed that there is more than one copy of nidA and nidB genes in the strains PYR-1, BB1, PYR-GCK and FAn9T. However, only one copy of each gene was observed in PAH2.135.
编码芳香环羟基化双加氧酶α和β亚基的多环芳烃（PAH）降解基因nidA和nidB已从范氏分枝杆菌PYR-1中克隆并测序[Khan等人，《应用与环境微生物学》67（2001）3577 - 3585]。在本研究中，调查了其他12株分枝杆菌或红球菌菌株中nidA和nidB的存在情况。最初，通过喷雾平板法筛选所有菌株降解PAHs的能力，并通过聚合酶链反应（PCR）检测双加氧酶里氏中心区域的存在情况。只有分枝杆菌属的PAH 2.135（RJGII - 135）、微黄分枝杆菌PYR - GCK（ATCC 700033）、吉尔文分枝杆菌BB1（DSM 9487）和弗雷德里克斯堡分枝杆菌FAn9T（DSM 44346），这几种先前已知的PAH降解菌，在两项测试中均呈阳性。从这三株阳性菌株中，使用根据PYR - 1已知的nidA和nidB序列设计的引物，通过PCR扩增nidA和nidB基因的完整开放阅读框，克隆到pBAD/Thio - TOPO载体中并进行测序。序列显示与范氏分枝杆菌PYR - 1的nidA和nidB基因有>98%的同一性。使用来自PYR - 1的nidA和nidB探针进行的Southern DNA - DNA杂交显示，在菌株PYR - 1、BB1、PYR - GCK和FAn9T中存在多个nidA和nidB基因拷贝。然而，在PAH2.135中每个基因仅观察到一个拷贝。
