Sequencing and transcriptional analysis of the chlorite dismutase gene of Dechloromonas agitata and its use as a metabolic probe

Abstract

The dismutation of chlorite into chloride and O(2) represents a central step in the reductive pathway of perchlorate that is common to all dissimilatory perchlorate-reducing bacteria and is mediated by a single enzyme, chlorite dismutase. The chlorite dismutase gene cld was isolated and sequenced from the perchlorate-reducing bacterium Dechloromonas agitata strain CKB. Sequence analysis identified an open reading frame of 834 bp that would encode a mature protein with an N-terminal sequence identical to that of the previously purified D. agitata chlorite dismutase enzyme. The predicted translation product of the D. agitata cld gene is a protein of 277 amino acids (aa), including a leader peptide of 26 aa. Primer extension analysis identified a single transcription start site directly downstream of an AT-rich region that could represent the -10 promoter region of the D. agitata cld gene. Northern blot analysis indicated that the cld gene was transcriptionally up-regulated when D. agitata cells were grown in perchlorate-reducing versus aerobic conditions. Slot blot hybridizations with a D. agitata cld probe demonstrated the conservation of the cld gene among perchlorate-reducing bacteria. This study represents the first description of a functional gene associated with microbial perchlorate reduction.

FIG. 1.

Nucleotide and predicted amino acid sequence of the upstream region of the D. agitata CD gene. The asterisk indicates the transcription start site, and the putative −10 promoter region immediately upstream of the start is double-underlined. The RBS is underlined. The leader peptide is indicated; the hydrophobic region and cleavage site of the leader are shaded.

FIG. 2.

Amino acid sequence alignment of the mature D. agitata and putative I. dechloratans and M. magnetotacticum CD proteins. Sequence identities are shaded.

FIG. 3.

Primer extension using RNA from a perchlorate-grown culture of D. agitata. The sequencing ladder was generated using the same primer as the primer extension reaction. The relevant sequence is 5′-GAAATTTGTTGAGTCGCCAA-3′, with the underlined nucleotide denoting the start site of transcription.

FIG. 4.

Northern blot of D. agitata RNA hybridized with the D. agitata probe to the 3′ end of the CD gene. Lanes: 1, aerobic growth; 2, anaerobic growth with perchlorate as the electron acceptor.

FIG. 5.

(A) Slot blot hybridization of gDNAs from DPRB and non-perchlorate-reducing close relatives using the D. agitata probe to the 3′ end of the CD gene. Organisms capable of chlorate or perchlorate reduction are underlined. Row A (left to right): lane 1, D. agitata; lane 2, Rhodocyclus tenuis; lane 3, Dechloromonas aromatica; lane 4, Dechloromonas sp. strain JJ. Row B: lane 1, Dechlorospirillum anomalous strain WD; lane 2, Magnetospirillum magnetotacticum; lane 3, Pseudomonas sp. strain PK; lane 4, Pseudomonas stutzeri. Row C: lane 1, Dechloromarinus sp. strain NSS; lane 2, Dechlorosoma suillum; lane 3, Azoarcus sp. strain LT-1; lane 4, I. dechloratans. (B) Phylogenetic tree of the organisms used in the slot blot hybridization based on 16S rDNA sequences (1, 9).