Identification of a chlorobenzene reductive dehalogenase in Dehalococcoides sp. strain CBDB1

Abstract

A chlorobenzene reductive dehalogenase of the anaerobic dehalorespiring bacterium Dehalococcoides sp. strain CBDB1 was identified. Due to poor biomass yields, standard protein isolation procedures were not applicable. Therefore, cell extracts from cultures grown on trichlorobenzenes were separated by native polyacrylamide gel electrophoresis and analyzed directly for chlorobenzene reductive dehalogenase activity within gel fragments. Activity was found in a single band, even though electrophoretic separation was performed under aerobic conditions. Matrix-assisted laser desorption ionization mass spectrometry (MALDI MS) and nano-liquid chromatography-MALDI MS analysis of silver-stained replicas of the active band on native polyacrylamide gels identified a protein product of the cbdbA84 gene, now called cbrA. The cbdbA84 gene is one of 32 reductive dehalogenase homologous genes present in the genome of strain CBDB1. The chlorobenzene reductive dehalogenase identified in our study represents a member of the family of corrinoid/iron-sulfur cluster-containing reductive dehalogenases. No orthologs of cbdbA84 were found in the completely sequenced genomes of Dehalococcoides sp. strains 195 and BAV1 nor among the genes amplified from Dehalococcoides sp. strain FL2 or mixed cultures containing Dehalococcoides. Another dehalogenase homologue (cbdbA80) was expressed in cultures that contained 1,2,4-trichlorobenzene, but its role is unclear. Other highly expressed proteins identified with our approach included the major subunit of a protein annotated as formate dehydrogenase, transporter subunits, and a putative S-layer protein.

FIG. 1.

Separation and detection scheme of chlorobenzene reductive dehalogenase using native PAGE. First, proteins were separated on a native polyacrylamide gel (a). The gel was then scaled and vertically cut into two parts (b). One part was silver stained and photographed. The mirror image and size-corrected picture was used as a template with which to cut the unstained part of the gel into fragments containing single bands (c). The fragments were analyzed for dechlorination activity in an in vitro activity assay. See text for details.

FIG. 2.

(a) Whole-cell proteins from strain CBDB1 separated by native PAGE. (b) Chlorobenzene dechlorinating protein (CbrA) analyzed by SDS-PAGE. Lanes: 1, molecular mass marker; 2, eluted protein from an active native gel fragment.

FIG. 3.

Protein sequence encoded by the cbdbA84 gene. Peptides identified by mass spectrometry are underlined. The twin-arginine motif and iron-sulfur cluster binding motifs are highlighted in gray; residues of the truncated cobalamin-binding consensus sequence are in bold. The arrow indicates the predicted leader peptide cleavage site.

FIG. 4.

Physical map of the cbr gene locus (cbdbA81-cbdbA86). The cbrAB operon is located downstream of a histidine kinase (cbrC) and a response regulator (cbrD). The cbrAB regions encoding the twin-arginine leader peptide and the iron-sulfur cluster binding motif are highlighted. The cbdbA81 and cbdbA86 genes encode amino acid sequences without similarity to characterized proteins.