Open AccessDetection by PCR of reductive dehalogenase motifs in a sulfidogenic 2‐bromophenol‐degrading consortium enriched from estuarine sediment
Author(s) -
Rhee SungKeun,
Fennell Donna E,
Häggblom Max M,
Kerkhof Lee J
Publication year - 2003
Publication title -
fems microbiology ecology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.377
H-Index - 155
eISSN - 1574-6941
pISSN - 0168-6496
DOI - 10.1111/j.1574-6941.2003.tb01072.x
Subject(s) - biology , gene , dehalogenase , genetics , inverse polymerase chain reaction , conserved sequence , polymerase chain reaction , biochemistry , microbiology and biotechnology , peptide sequence , multiplex polymerase chain reaction
Polymerase chain reaction (PCR) primers were developed based on three known reductive dehalogenase (RDH) genes ( pceA from Dehalospirillum multivorans , cprA from Desulfitobacterium dehalogenans , tceA from Dehalococcoides ethenogenes ) and used to amplify bands of the appropriate size from a microbial consortium inoculated with contaminated estuarine sediment and enriched under sulfidogenic conditions using 2‐bromophenol (2‐BP) as the sole carbon source. These PCR fragments were found to contain many of the conserved amino acids and motifs present in the known RDH genes. The three cloned PCR products (2bprdh61, 2bprdh63, 2bprdh81 – designated 2‐bromophenol RDH) shared 21 of the 31 conserved amino acids present in the C‐terminus of the RDHs in GenBank. (The N‐terminus of the RDH protein shares very little homology for the known RDH genes.) All 2‐BP PCR products and the known RDH genes were found to contain two iron–sulfur cluster binding domains as well as conserved PCR priming sites. Southern hybridization of genomic DNA revealed multiple bands, implying additional RDH‐like motifs in the sulfidogenic 2‐BP‐degrading consortium. In order to gain information on upstream regions of the RDH‐like motifs, DNA fragments containing 2bprdh61, 2bprdh63, and 2bprdh81 were cloned and sequenced via an inverse PCR approach. The results indicated the presence of transposase gene homologues upstream of 2‐bprdh61A and 2‐bprdh81A . Therefore, some RDH‐like PCR products in our consortium are possibly pseudogenes and some RDH genetic diversity may be generated by transposition.