Reductive dehalogenation of brominated phenolic compounds by microorganisms associated with the marine sponge Aplysina aerophoba

Abstract

Marine sponges are natural sources of brominated organic compounds, including bromoindoles, bromophenols, and bromopyrroles, that may comprise up to 12% of the sponge dry weight. Aplysina aerophoba sponges harbor large numbers of bacteria that can amount to 40% of the biomass of the animal. We postulated that there might be mechanisms for microbially mediated degradation of these halogenated chemicals within the sponges. The capability of anaerobic microorganisms associated with the marine sponge to transform haloaromatic compounds was tested under different electron-accepting conditions (i.e., denitrifying, sulfidogenic, and methanogenic). We observed dehalogenation activity of sponge-associated microorganisms with various haloaromatics. 2-Bromo-, 3-bromo-, 4-bromo-, 2,6-dibromo-, and 2,4,6-tribromophenol, and 3,5-dibromo-4-hydroxybenzoate were reductively debrominated under methanogenic and sulfidogenic conditions with no activity observed in the presence of nitrate. Monochlorinated phenols were not transformed over a period of 1 year. Debromination of 2,4,6-tribromophenol, and 2,6-dibromophenol to 2-bromophenol was more rapid than the debromination of the monobrominated phenols. Ampicillin and chloramphenicol inhibited activity, suggesting that dehalogenation was mediated by bacteria. Characterization of the debrominating methanogenic consortia by using terminal restriction fragment length polymorphism (TRFLP) and denaturing gradient gel electrophoresis analysis indicated that different 16S ribosomal DNA (rDNA) phylotypes were enriched on the different halogenated substrates. Sponge-associated microorganisms enriched on organobromine compounds had distinct 16S rDNA TRFLP patterns and were most closely related to the delta subgroup of the proteobacteria. The presence of homologous reductive dehalogenase gene motifs in the sponge-associated microorganisms suggested that reductive dehalogenation might be coupled to dehalorespiration.

FIG. 1.

Dehalogenation of brominated compounds in sponge enrichment cultures under methanogenic conditions. The results are means of four replicate cultures: 2-, 3-, and 4-BP (A); 2,6-DBP (B); and 2,4,6-TBP (C).

FIG. 2.

TRFLP profiles of MnlI-digested 16S rDNAs amplified from different anaerobic cultures enriched on brominated phenolic compounds. (A) Native sponge-associated microorganisms; (B) control with only 200 μM lactate; (C) a mixture of 2-, 3- and 4-BP (100 μM each); (D) 100 μM 2-BP; (E) 100 μM 3-BP; (F) 100 μM 4-BP; (G) 100 μM 3,5-DB-4-HB; (H) 100 μM 2,6-DBP; and (I) 100 μM 2,4,6-TBP.

FIG. 3.

DGGE profiles of 16S rDNA genes from different enrichment cultures on brominated phenolic compounds. The arrow highlights a band with the same relative migration distance found in most of the cultures. Lanes: 1, native sponge-associated microorganisms; 2, control with only 200 μM lactate; 3, mixture of 2-, 3-, and 4-BP (100 μM each); 4, 100 μM 2-BP; 5, 100 μM 3-BP; 6, 100 μM 4-BP; 7, 100 μM 2,6-DBP; and 8, 100 μM 3,5-DB-4-HB. Numbers indicate sequenced 16S rDNA fragments.

FIG. 4.

Phylogenetic tree of DGGE band sequences of 16S rDNA. Bootstrap values at nodes are the percentages of 100 iterations. Values less than 50% are not included. The reference bar indicates 10 nucleotide exchanges per 100 nucleotides. The sequences are numbered as in Fig. 3.

FIG. 5.

C-terminal amino acid sequence alignment of reductive dehalogenase from the sponge-associated microorganisms. Conserved sequence motifs (FeS clusters) are indicated by boxes. Group I, N1, N2, N5, N7, N9, E13, E32, E36, and E411; group II, N4, E29, E34, E37, E310, E41, E42, and E45 (E, DNA from enrichment cultures; N, DNA from native sponge without enrichment); PceA Y51, PceA dehalogenase from Desulfitobacterium sp. strain Y51 (GenBank accession no. AB070709); CprA Dd, ortho-chlorophenol reductase from D. dehalogenans (GenBank accession no. AF115542); TceA De, TCE dehalogenase from D. ethenogenes strain 195 (GenBank accession no. AF228507); Pce1 Ds, Pce1 dehalogenase from Desulfitobacterium sp. strain PCE-1 (GenBank accession no. AY013361); and PceA Dm, PCE dehalogenase from D. multivorans (GenBank accession no. AF022812).

FIG. 6.

Phylogenetic tree of deduced partial amino acid sequences of reductive dehalogenases. Multiple alignment and construction of the phylogenetic tree with the neighbor-joining method with 1,000 bootstrap resamplings were performed by using the Clustal X and TreeView programs. The reference bar indicates 10 amino acid exchanges per 100 amino acids. For names of sequences, see Fig. 5.