Organohalide-respiring Desulfoluna species isolated from marine environments

Abstract

The genus Desulfoluna comprises two anaerobic sulfate-reducing strains, D. spongiiphila AA1^T and D. butyratoxydans MSL71^T, of which only the former was shown to perform organohalide respiration (OHR). Here we isolated a third strain, designated D. spongiiphila strain DBB, from marine intertidal sediment using 1,4-dibromobenzene and sulfate as the electron acceptors and lactate as the electron donor. Each strain harbors three reductive dehalogenase gene clusters (rdhABC) and corrinoid biosynthesis genes in their genomes, and dehalogenated brominated but not chlorinated organohalogens. The Desulfoluna strains maintained OHR in the presence of 20 mM sulfate or 20 mM sulfide, which often negatively affect other organohalide-respiring bacteria. Strain DBB sustained OHR with 2% oxygen in the gas phase, in line with its genetic potential for reactive oxygen species detoxification. Reverse transcription-quantitative PCR revealed differential induction of rdhA genes in strain DBB in response to 1,4-dibromobenzene or 2,6-dibromophenol. Proteomic analysis confirmed expression of rdhA1 with 1,4-dibromobenzene, and revealed a partially shared electron transport chain from lactate to 1,4-dibromobenzene and sulfate, which may explain accelerated OHR during concurrent sulfate reduction. Versatility in using electron donors, de novo corrinoid biosynthesis, resistance to sulfate, sulfide and oxygen, and concurrent sulfate reduction and OHR may confer an advantage to marine Desulfoluna strains.

Fig. 1. Enrichment and isolation of D. spongiiphila DBB.

Intertidal sediment mainly composed of shore sediment used for isolation (a). Reductive debromination of 1,4-dibromobenzene (1,4-DBB) by: the original microcosms containing intertidal sediment (b), the sediment-free enrichment cultures (c), the most diluted culture (10⁷) in the dilution series (d). Phylogenetic analysis of bacterial communities in the microcosms from the shore sediment at time zero (left), the original 1,4-DBB debrominating enrichment culture after 104 days incubation (middle) and the 10⁷ dilution series culture (right) (e). Reductive debromination of 1,4-DBB to bromobenzene (BB) by the isolated pure culture (f). Sediment enrichment culture and sediment-free transfer cultures (b–d) were prepared in single bottles. Pure cultures (f) were prepared in duplicate bottles. Points and error bars represent the average and standard deviation of samples taken from the duplicate cultures. Phylogenetic data are shown at phylum level, except Deltaproteobacteria shown at class level and Desulfoluna at genus level. Taxa comprising less than 1% of the total bacterial community are categorized as ‘Others’.

Fig. 2. Comparison of the rdh gene clusters in D. spongiiphila DBB, D. spongiiphila AA1^T and D. butyratoxydans MSL71^T.

Numbers indicate the locus tags of the respective genes.

Fig. 3. Differential induction of rdhA genes during 1,4-DBB and 2,6-DBP debromination by D. spongiiphila DBB.

Debromination of 1,4-DBB (a) and 2,6-DBP (c) by strain DBB and RT-qPCR analysis of relative induction of its three rdhA genes during debromination of 1,4-DBB (b) and 2,6-DBP (d). Error bars in panels a and c indicate the standard deviation of two random cultures analyzed out of 10 replicates. The concentration of 1,4-DBB (>0.1 mM) could not be accurately measured due to large amount of undissolved compound and hence was not plotted. Error bars in panels b and d indicate standard deviation of triplicate RT-qPCRs performed on samples withdrawn from duplicate cultures at each time point (n = 2 × 3).

Fig. 4. Corrinoid biosynthesis and transporter gene clusters of Desulfoluna strains.

Numbers indicate the locus tags of the respective genes. The corresponding enzymes encoded by the genes and their functions in corrinoid biosynthesis are indicated in Table S4.

Fig. 5. Preliminary electron transport pathway scheme based on the genomic and proteomic analysis of D. spongiiphila DBB grown on lactate, sulfate and 1,4-DBB (LSD condition).

Corresponding gene locus tags are given for each protein. Proteins shown in dashed line square were not detected under the tested conditions. Probable electron flow path is shown in red arrows, and the dashed red arrows indicate reverse electron transport. The pmf is built up by ATPase using ATP generated by substrate-level phosphorylation via Por, Pta and Ack. Note that the distribution of electrons to the electron transport chains is not equal between sulfate respiration and OHR, but shifted heavily toward sulfate respiration due to excess sulfate (20 mM vs. 100 µM 1,4-DBB).