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. Author manuscript; available in PMC: 2025 Sep 12.
Published in final edited form as: Environ Microbiol Rep. 2017 Jul 21;9(5):537–549. doi: 10.1111/1758-2229.12556

Novel reductive dehalogenases from the marine sponge associated bacterium Desulfoluna spongiiphila

Jie Liu 1, Nora Lopez 1,2, Youngbeom Ahn 1,†, Tatyana Goldberg 3, Yana Bromberg 1, Lee J Kerkhof 2, Max M Häggblom 1,*
PMCID: PMC12423780  NIHMSID: NIHMS2106454  PMID: 28618195

Summary

Desulfoluna spongiiphila strain AA1 is an organohalide respiring bacterium, isolated from the marine sponge Aplysina aerophoba, that can use brominated and iodinated phenols, in addition to sulfate and thiosulfate as terminal electron acceptors. The genome of Desulfoluna spongiiphila strain AA1 is approximately 6.5 Mb. Three putative reductive dehalogenase (rdhA) genes involved in respiratory metabolism of organohalides were identified within the sequence. Conserved motifs found in respiratory reductive dehalogenases (a twin arginine translocation signal sequence and two iron-sulfur clusters) were present in all three putative AA1 rdhA genes. Transcription of one of the three rdhA genes was significantly upregulated during respiration of 2,6-dibromophenol and sponge extracts. Strain AA1 appears to have the ability to synthesize cobalamin, the key cofactor of most characterized reductive dehalogenase enzymes. The genome contains genes involved in cobalamin synthesis and uptake and can grow without cobalamin supplementation. Identification of this target gene associated with debromination lays the foundation for understanding how dehalogenating bacteria control the fate of organohalide compounds in sponges and their role in a symbiotic organobromine cycle. In the sponge environment, D. spongiiphila strain AA1 may thus take advantage of both brominated compounds and sulfate as electron acceptors for respiration.

Introduction

The marine environment is a major source of naturally occurring organohalides produced by algae, jellyfish, acorn worms and sponges (Gribble, 1998). These compounds include bromopyrroles, bromoindoles, bromophenols and brominated diphenyl ethers and are hypothesized to function as chemical defense to protect sponges from predators and biofouling (Teeyapant and Proksch, 1993; Ebel et al., 1997). These natural sources of brominated compounds also appear to select for dehalogenating bacteria living within the host animal (Ahn et al., 2003). Considering the extraordinary pumping capacity and abundant microbial communities of sponges (Hentschel et al., 2012), an understanding of the microbial processes that control the fate of organohalide compounds in sponges is needed in order to understand the role that these dehalogenating bacteria play within the animal and a marine organobromine cycle.

Reductive dehalogenation is a key process in the degradation of both natural and anthropogenic halogenated compounds (for reviews see Häggblom and Bossert, 2003; Adrian and Löffler, 2016). Cleavage of the carbon-halogen bond can be mediated by anaerobic bacteria that utilize the organohalide as terminal electron acceptor for respiration. These organohalide respiring bacteria are distributed among diverse phyla (Maphosa et al., 2010) and can be grouped into either obligate or non-obligate organohalide respirers based on their metabolic versatility (Hug et al., 2013). The Deltaproteobacterium, Desulfoluna spongiiphila strain AA1, isolated from the marine sponge Aplysina aerophoba is of interest because of its ability to reductively dehalogenate various brominated and iodinated phenolic compounds (Ahn et al., 2009). D. spongiiphila is a non-obligate organohalide respiring bacterium; in addition to organohalide respiration it has the ability to also grow with sulfate and thiosulfate as the terminal electron acceptor.

Organohalide respiration is mediated by reductive dehalogenases (RDases), encoded by homologous rdhA genes. Available genomes reveal that organohalide-respiring bacteria (ORB) can possess over 30 putative rdhA genes (Kube et al., 2005; Wang et al., 2014). The rdh operon contains rdhA, the gene coding for the active enzyme, and rdhB, the gene coding for a putative membrane-anchoring protein (Jugder et al., 2015). Putative regulatory genes are often found to be associated with rdhAB, that is, a two-component regulatory system (RdhCD) or MarR-type regulators (RdhR) in Dehalococcoides spp., and CRP/FNR type regulators (CprK) in Desulfitobacterium spp. (Gábor et al., 2006; Joyce et al., 2006; Kemp et al., 2013; Krasper et al., 2016). Most RdhA proteins possess two conserved features, a twin-arginine translocation (Tat) motif (RRXFXK) at the N terminus (Maillard et al., 2003) and two iron-sulfur cluster-binding motifs at the C terminus (CXXCXXCXXXCP and CXXCXXXCP) (Maillard et al., 2003; Maphosa et al., 2010; Hug et al., 2013; Bommer et al., 2014). In addition, most RdhA proteins depend on a corrinoid cofactor for activity (Bommer et al., 2014; Payne et al., 2015; Rupakula et al., 2015). The crystal structures of two RDases, PceA from Sulfurospirillum. multivorans and NpRdhA from Nitratireductor pacificus pht-3B, reveal the presence of cobalamin factors in the active site (Bommer et al., 2014; Payne et al., 2015). The sources of the corrinoid cofactor for organohalide respiring bacteria can be either de novo synthesis or from an exogenous vitamin B12 supply (Rupakula et al., 2015). For example, Dehalococcoides mccartyi strains are dependent on corrinoid salvaging from the environment (Yi et al., 2012; Yan et al., 2013; Men et al., 2014), while Sulfurospirillum multivorans and Desulfitobacterium hafniense are known to possess a complete set of corrinoid biosynthesis genes and are able to synthesize vitamin B12 de novo (Nonaka et al., 2006; Choudhary et al., 2013; Goris, et al., 2014; Keller et al., 2014). However, even with a nearly complete set of corrinoid biosynthesis genes, Dehalobacter restrictus strain PER-K23 still needs an exogenous corrinoid supply due to the truncation of one critical gene (cbiH) (Rupakula et al., 2015).

In this study, we set out to identify the reductive dehalogenase genes in the sponge-associated bacterium D. spongiiphila strain AA1. The genome of D. spongiiphila strain AA1 was sequenced and analysed to detect the gene(s) coding for reductive dehalogenases and the cobalamin synthesis pathway. Transcript analysis was conducted to identify and characterize the expression of reductive dehalogenase genes under different growth conditions. Most rdhA gene products studied to date are involved in dechlorination. The substrate specificity of D. spongiiphila strain AA1, however, is different from that of most well characterized reductive dehalogenating bacteria. Strain AA1 can dehalogenate bromophenols and iodophenols, but not chlorophenols (Ahn et al., 2009). Our findings represent an example of a respiratory debrominase and provide an avenue to explore the role of organohalide respiration in the marine halogen cycle.

Results

Genome characteristics and identification of putative respiratory reductive dehalogenases in Desulfoluna spongiiphila strain AA1

The draft genome sequence of D. spongiiphila strain AA1 consists of 52 scaffolds ranging in length from 1069 bp to 728 457 bp. The total genome size is approximately 6.5 Mbp with a GC content of 57.2%. Based on the annotation by JGI, approximately 5200 protein-coding genes are predicted in the genome, 74% of which are predicted with function (JGI Analysis Project ID: Ga0104423). The annotated protein sequences are also available in NCBI (BioProject Accession: PRJEB15715). The genome of D. spongiiphila contains genes encoding for proteins for dissimilatory sulfate reduction, including genes for sulfate adenylyltransferase (sat), dissimilatory adenylylsulfate reductase (aprAB) and dissimilatory sulfite reductase (dsrAB).

The presence of three putative reductive dehalogenase genes was initially uncovered from a draft genome assembled from SoLiD sequencing data (see Materials and Methods). The presence of these putative reductive dehalogenase genes was further confirmed based on the higher quality Illumina sequence data (labelled here as rdh AA1_02299, JGI Locus Tag: Ga0104423_102299; rdh AA1_07176, JGI Locus Tag: Ga0104423_107176; rdh AA1_16032, JGI Locus Tag: Ga0104423_11632). The rdhA gene labels correspond to the scaffold in which the putative rdhA is located followed with gene number in that scaffold. The size of each putative reductive dehalogenase gene/enzyme are as follows: rdh AA1_02299- 1452 bp, 483 aa; rdh AA1_07176- 1290 bp, 429 aa; and rdh AA1_16032- 1683 bp, 560 aa. Clustal W pairwise alignments of the N- and C-terminal amino acid sequences of the three putative D. spongiiphila AA1 reductive dehalogenases (RdhA) and 20 known RDases identified the twin arginine translocation (Tat) signal motifs (RRXFXK) and two iron-sulfur cluster binding motifs (CXXCXXCXXXCP and CXXCXXXCP; Supporting Information Fig. S1).

Pairwise alignment of the three D. spongiiphila strain AA1 RdhA amino acid sequences indicates that they are distinct. Rdh AA1_02299 has a sequence identity of 22.3% with Rdh AA1_16032 and 23.8% with Rdh AA1_07176. Rdh AA1_07176 has an identity of 25.8% with Rdh AA1_16032. They are located in distant branches of the phylogenetic tree of functionally characterized RdhAs (Fig. 1). BlastP analysis of each RdhA against the NCBI database revealed that Rdh AA1_02299 has 39% amino acid identity with an RdhA from Dehalococcoides mccartyi SG1 (Accession: WP_034376939), Rdh AA1_07176 has an identity of 43% with a Dehalobacter spp. CF RdhA (Accession: AFV06381) and Rdh AA1_16032 has an identify of 50% with an RdhA from Shewanella sediminis HAW-EB3 (Accession: WP_012142447). According to the reductive dehalogenase classification system proposed by Hug et al. (2013), the three RdhAs from D. spongiiphila strain AA1 cannot be grouped with any other previously characterized RdhA groups, which are defined by a 90% PID threshold (Supporting Information Fig. S2).

Fig. 1. Phylogeny of strain AA1 and characterized reductive dehalogenases.

Fig. 1.

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Initial tree(s) for the heuristic search were obtained automatically by applying Neighbor-Joining (NJ) and advanced NJ (BioNJ) algorithms to a matrix of pairwise distances estimated using a Jones–Thornton–Taylor (JTT) model, and then selecting the topology with superior log likelihood value. The maximum likelihood tree is drawn to scale, with branch lengths measured in the number of substitutions per site. The analysis involved 34 amino acid sequences. All positions with less than 60% site coverage were eliminated, that is, fewer than 40% alignment gaps, missing data and ambiguous bases were allowed at any position. There were a total of 479 positions in the final dataset. The characterized RdhAs are highlighted with different colours based on their substrates. D. spongiiphila AA1 RdhAs are highlighted in red.

The analysis of the region around the putative rdh genes of D. spongiiphila revealed other possible genes involved in reductive dehalogenation (Fig. 2). All three rdh gene clusters follow the rdhABC model together with transcriptional regulatory genes. Each rdh gene cluster harbours a putative membrane-anchoring gene, rdhB, downstream of rdhA. Putative rdhC genes are named according to cprC of Desulfitobacterium dehalogenans (Smidt et al, 2000) based on the domain similarity. They all contain a FMN-binding domain and Fe-S binding domains. Additional genes annotated with transcriptional regulatory functions were found in all rdh gene clusters. Sigma factor 54 dependent transcriptional activators, which usually assist the initiation of sigma factor 54 dependent transcription, are both found upstream of the rdh AA1_07176 and 16032 gene clusters. The analysis of their promoter region reveals the presence of a sigma factor 54 binding site (CCGGCACGCTTTGTGCT and TTGGCACACCGCTTGCT; Fig. 2). No such sigma-54 dependent protein and binding site are found in the rdh AA1_02299 cluster and its promoter region. A LuxR family regulator is present upstream of rdh AA1_02299 and a MarR-type transcriptional regulator is far downstream of the rdh AA1_02299 gene cluster.

Fig. 2. Reductive dehalogenase gene clusters in strain AA1.

Fig. 2.

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The putative rdhA is in red, putative membrane anchoring gene rdhB is in blue and the putative rdhC-like gene is in green. The genes annotated with potential transcriptional regulation function are coloured in yellow. The genes with other functions or hypothetic proteins are coloured in white. The sequence of sigma factor 54 binding site in promoter region (−12 and −24 element) is indicated for rdh AA1_07176 and rdh AA1_16032. Detailed annotation is as follows: rdh AA1_02299: 02298 – LuxR family regulatory protein; 02305 – MarR family transcriptional regulator. rdh AA1_07176: 07172 – HxlR family transcriptional regulator; 174 – Putative transcriptionary regulatory; 175 - Sigma-54-dependent transcriptional regulator. rdh AA1_16032: 01629 - TetR family transcriptional regulator; 16030 - Sigma-54-dependent transcriptional regulator; 16031 -Tetratricopepetide repeat containing protein; 16037 - Transporter; 16039 - HxlR family transcriptional regulator.

Expression of reductive dehalogenase genes

D. spongiiphila strain AA1 dehalogenates ortho bromophenols and iodophenols, but not chlorophenols. We conducted a set of experiments to determine whether reductive debromination activity in D. spongiiphila was inducible. Strain AA1 was cultivated with sulfate as the electron acceptor and then transferred into fresh medium amended with 2,6-dibromophenol (2,6-DBP) and streptomycin to inhibit further protein synthesis. In the presence of streptomycin, debromination activity was minimal over 48 h. In the absence of streptomycin, debromination of 2,6-DBP was observed after a lag phase of 6 h (Fig. 3) with transient formation of 2-bromophenol (2-BP) and the accumulation of phenol over the next 60–80 h, indicating that dehalogenation activity in strain AA1 is induced by 2,6-DBP.

Fig. 3. Induction of debromination activity.

Fig. 3.

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The concentration of 2,6-DBP in D. spongiiphila strain AA1 culture with or without streptomycin (1 mg/ml) to inhibit protein synthesis. Data points are the means of triplicate cultures.

After demonstrating induction of debromination activity, we attempted to identify which of the three putative reductive dehalogenase genes (rdh AA1_02299, rdh AA1_07176 and rdh AA1_16032) were transcribed and expressed during bromophenol respiration. After growth on sulfate, dehalogenation of 2,6-DBP by strain AA1 was observed after a 3 h lag phase with sequential production of 2-BP and phenol as debromination products over the next 9 h of incubation (Fig. 4A). The expression of the three putative rdhA genes was analyzed by reverse transcription at time points during this incubation. Transcripts were amplified with two sets of specific primers designed for each gene and the cDNA product loaded onto an agarose gel. As shown in Fig. 4B, clear bands of rdh AA1_07176 and rdh AA1_16032 cDNA PCR products were observed, while no band was found in control cultures without 2,6-DBP amendment. For rdh AA1_02299, there was no difference found between induced and control cultures. These results indicate that the expression of rdh AA1_07176 and rdh AA1_16032, but not rdh AA1_02299 was induced by the addition of 2,6-DBP.

Fig. 4. Debromination activity in 2,6-DBP induced D. spongiiphila AA1 culture over time and the expression of each rdhA gene.

Fig. 4.

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A. Concentration of 2,6-DBP and its debrominating product, 2-bromophenol and phenol in induced cultures over time. Points and error bars are means and standard deviations of biological triplicates. </p/> B. PCR amplification of cDNA from strain AA1 culture after 6 h incubation with primers targeting rdh AA1_16032 (a), rdh AA1_07176 (b) and rdh AA1_02299 (c). For each rdh gene, two primer sets (S and L) were used in order to confirm the results. Lanes S/L1–3: Control culture triplicates; Lane S/L 4–6: Induced culture triplicates; S/L 7: Positive control; S/L 8: Negative control. 100 bp DNA ladder (New England Biolabs) was loaded on the left of the gel.

C. Relative expression of rdh AA1_16032 and rdh AA1_07176 over incubation time. The expression level of the genes was normalized to the expression of the 16S rRNA gene. The y-axis indicates the expression fold of 2,6-DBP induced cultures compared to untreated controls. Error bars represent the standard deviation of three biological triplicates, each of which contains two or three RT-PCR reaction replicates.

D. Structure of 2,6-DBP, aeroplysinin-1 and dienone, two examples of brominated compounds found in Aplysina aerophoba sponges (Teeyapant et al., 1993) and expression of strain AA1 rdhAs in the presence of sponge extracts after 4 h incubation.

The gene rdh AA1_16032 exhibited a more pronounced upregulation compared to rdh AA1_07176 with an approximately fivefold increase in expression at the initial time point, taken 20 min after 2,6-DBP addition (Fig. 4C). Expression of rdh AA1_16032 increased approximately 200-fold over the first 12 h. A lower fold increase in the expression of rdh AA1_07176 was observed after 2,6-DBP addition. These results suggest that rdh AA1_16032 is the major gene responsible for both 2,6-DBP and 2-BP debromination, which is upregulated in response to 2,6-DBP and 2-BP.

Extracts of an Aplysina aerophoba sponge also induced pronounced upregulation of rdh AA1_16032 after 4 h of incubation compared to the other two rdhAs (Fig. 4D). This indicates that the dehalogenase encoded by rdh AA1_16032 may be also responsible in debrominating natural sponge-derived organohalides in vivo.

Corrinoid biosynthesis genes in the genome of strain AA1

Most RdhA proteins depend on a corrinoid cofactor for dehalogenation activity. The source of cobalamin for dehalogenating bacteria can be from de novo biosynthesis or scavenging from the environment (Rupakula et al., 2015). D. spongiiphila strain AA1 has been grown in medium with approximately 50 μg/l cobalamin from the time it was isolated. Cobalamin biosynthesis and uptake genes are present in the genome of strain AA1 (Supporting Information Table S1) and when strain AA1 was sequentially transferred and grown in cobalamin-free medium and then exposed to different cobalamin concentrations, there was no substantial difference in debrominating activity (Supporting Information Fig. S3). This indicates that strain AA1 does not require an exogenous cobalamin supply for dehalogenation. Experiments to determine whether propyl iodide, an inhibitor of corrinoid enzymes (Ghambeer et al., 1971), would inhibit dehalogenation activity were inconclusive (data not shown). Whether the RdhA cofactor indeed is a corrinoid or a heme, as in Desulfomonile tiedjei DCB-1 (Ni et al., 1995) will require additional confirmation.

Discussion

Because of their metabolic diversity, non-obligate organohalide respiring bacteria usually have a larger genome size and fewer reductive dehalogenase genes than obligate ORB. For example, the obligate ORB Dehalobacter sp. strain 12DCB1 has 39 rdhA homologs in its 2.9 Mb genome, while the facultative Desulfitobacterium hafniense TCE-1 has only one rdhA gene in its 5.74 Mb genome (Kruse et al., 2016). D. spongiiphila strain AA1 is a typical non-obligate ORB with a genome size of around 6.5 Mb containing three putative rdhA genes.

A classification for orthologous group RhdA proteins was proposed by Hug et al. (2013) to categorize dehalogenases based on a 90% cutoff of amino acid sequence identity. None of the three RdhAs of D. spongiiphila strain AA1 can be clustered with any of the current existing groups. The highest identity of the AA1 dehalogenases to any RdhAs in the database is approximately 50%, which is much lower than the 90% cutoff (Supporting Information Fig. S2). The substrate specificity of a new RDase cannot be predicted based on its sequence similarity with characterized RDases (Jugder et al., 2016). The determination of substrate specificity requires the isolation and purification of the functional enzymes, which remains a major constraint in many cases. The substrates of most well characterized RDase’s so far are chlorinated compounds, including chlorinated ethenes, benzenes and phenols (Fig. 1). The substrate specificity of D. spongiiphila, however, is different from these characterized RDases in that strain AA1 can dehalogenate bromophenols and iodophenols, but not chlorophenols (Ahn et al., 2009). In addition, reductive debromination in D. spongiiphila AA1 occurs in the presence of sulfate (Ahn et al., 2009) in contrast with many other dehalogenating bacteria and cultures, whose dehalogenation activity are inhibited by sulfate (see Zanaroli et al., 2015).

Many brominated pollutants, such as brominated biphenyls and tetrabromobisphenol A are ubiquitously present in the environment and reductive dehalogenation is considered crucial in their biodegradation (e.g., Bedard and Van Dort, 1998; Voordeckers et al., 2002; Liu et al., 2013). Reductive debromination has been observed in sediment microcosms (e.g., Monserrate and Häggblom, 1997), and some bacteria are reported with reductive debromination ability, for example, a highly enriched Dehalococcoides culture was able to debrominate polybrominated diphenyl ethers, tetrabromobisphenol A and other phenolic bromoaromatics (He et al., 2006; Lee et al., 2011; Cooper et al., 2015; Yang et al., 2015), and Desulfovibrio strains and Desulfobacterium chlororespirans are reported to debrominate brominated phenolics (Boyle et al., 1999; Fennell et al., 2004; Cupples et al., 2005). Sulfurospirillum multivorans and Desulfitobacterium hafniense PCE-S are able to debrominate brominated ethenes (Ye et al., 2010). Nonetheless, compared with reductive dechlorinases, there is limited knowledge on reductive debrominases (Adrian and Löffler, 2016). Two characterized reductive debrominases (BhbA from Comamonas sp. 7D-2 and NpRdhA from Nitratireductor pacificus pht-3B) are not involved in organohalide respiration (Payne et al., 2015) and belong to a different dehalogenase enzyme class than the RdhAs in strain AA1 and other organohalide-respiring bacteria. Alternatively, a transcriptional approach coupled to metabolic analysis can be used to link a putative rdhA gene with a measured gene function.

In this study, we used known structural information about rdh genes and gene clusters to identify putative rdh gene clusters in strain AA1. For example, the pceABCT cluster is commonly present in the genomes of many organohalide respiring bacteria (Jugder et al., 2015). The rdhB gene encodes for a putative membrane anchoring protein while rdhC encodes for a putative membrane bound transcriptional regulatory protein (Smidt et al., 2000). The three rdh gene clusters of D. spongiiphila strain AA1 follow the rdhABC model. An rdhC-like gene encodes a FMN binding domain and 4Fe-4S binding domain-containing protein, which may function as transcriptional regulators. All three rdh gene clusters of strain AA1 contain this rdhC-like gene downstream of rdhB. The rdhT gene encodes a trigger factor-like protein, which may be involved in RdhA folding (Smidt et al., 2000). However, rdhT-like genes are not found in the rdh gene clusters of strain AA1.

Although the debromination of 2,6-DBP exhibited a short lag phase, the up-regulation of rdh AA1_16032 was immediately detected after addition of 2,6-DBP, while rdh AA1_07176 was only weakly upregulated during debromination of 2,6-DBP to 2-BP. Considering that rdh AA1_07176 and rdh AA1_16032 don’t show the same response pattern to 2,6-DBP, their regulatory mechanisms may be distinct. These two clusters both contain a transcriptional regulatory gene belonging to the HxlR family, at a sequence identity of 50%. HxlR is a putative transcriptional regulator with a winged helix-turn-helix (wHTH) structure similar to the MarR type wHTH. It was first elucidated as a transcriptional activator of the hxlAB operon in Bacillus subtilis (Yurimoto et al., 2005).

The regulatory mechanisms differ among organohalide respiring bacteria of different phylogeny (Kruse et al., 2016). In Desulfitobacterium and Dehalobacter, the rdh clusters often contain an rdhK gene, which encodes for a transcriptional regulator belonging to the CRP/FNR family. CprK could act as a transcriptional activator and bind to a dehalobox motif in the promoter regions in the presence of substrate (Futagami et al., 2006; Gábor et al., 2006; Kemp et al., 2013). None of the strain AA1 rdh gene clusters possesses CRP/FNR family-like regulators, suggesting a different regulatory mechanism for strain AA1 rdh gene regulation.

Interestingly, sigma-54-dependent transcriptional regulators are found upstream of the rdh AA1_07176 and rdh AA1_16032 gene promoter regions. Although sharing low pairwise identity (30.8%), these two genes contain a regulatory domain, a sigma factor 54 interaction domain and a DNA binding domain. The sigma-54-depenedent regulators are also called enhancer-binding-protein (EBP), which play an important role in sigma factor 54 dependent transcription (Bush and Dixon, 2012). Unlike sigma factor 70, sigma factor 54 recognizes a different but more conserved consensus sequence, YTGGCACGrNNNTTGCW, where binding occurs at −24 (GG) and −12 (TGC) elements (Studholme and Dixon, 2003; Bush and Dixon, 2012). The initiation of sigma factor 54 dependent transcription requires the assistance of EBP to open the closed complex. Examining the promoter region of these two rdhA genes, we found conserved sigma factor 54 binding sites in their promoters. Further molecular investigations are needed to confirm the function of the sigma factor 54 dependent regulators in these gene clusters.

The MarR-type regulator is known to act as a repressor in Dehalococcoides species, which contains a winged helix-turn-helix (wHTH) motif that involves in the interaction between regulator and palindrome site on binding DNA (Wagner et al., 2013; Krasper et al., 2016). However, most MarR-type regulatory genes in the rdh clusters of Dehalococcoide spp. are very close and in opposite orientation to the rdhA gene. The MarR-type regulator in the rdh AA1_02299 cluster may not function as in Dehalococcoides species. Another transcriptional regulator belonging to the LuxR family is closely upstream of rdhA in the rdh AA1_02299 cluster. This contains aHTH DNA binding structure, and may also be involved in rdh AA1_02299 gene cluster regulation.

The pceABCT gene cluster in D. hafniense strain Y51 and pceA in strain TCE1 are not regulated at the mRNA level (Prat et al., 2011; Peng et al., 2012). However, for D. hafniense TCE1 a significant PceA expression difference was found at the protein level between cultures during long-term cultivation with or without PCE (Prat et al., 2011). Unlike other well-studied reductive dehalogenase gene clusters, the pceABCT gene cluster in strains Y51 and TCE1 are located on mobile genetic elements and do not contain any obvious regulatory elements (Maillard et al., 2005; Futagami et al., 2006). The pceABCT gene cluster in these strains may gradually be lost in the absence of organohalides, which could explain the expression difference between mRNA and protein level in these studies (Prat et al., 2011).

The sponge Aplysina aerophoba, the host of D. spongiiphila, is known to produce a variety of brominated compounds (Norte et al., 1988; Teeyapant et al., 1993; Hentschel et al., 2003). Because of their antimicrobial activity, these brominated metabolites, for example, aeroplysinin-1 and dienone may be produced for a defense purpose and can be toxic to some bacteria. The response of rdh AA1_16032 to Aplysina aerophoba extracts reveals that this rdhA can be induced by natural organohalides produced in sponges (Fig. 4D). In the sponge environment, D. spongiiphila strain AA1 may thus take advantage of both brominated compounds and sulfate as electron acceptors for respiration. Interestingly, debromination by D. spongiiphila strain AA1 occurs concurrently with sulfate reduction (Ahn et al., 2009; unpublished data) in contrast to the inhibitory effect of sulfur oxyanions observed for many dechlorinating bacteria (for review see Zanaroli et al., 2015).

A corrinoid is a key cofactor found in most characterized reductive dehalogenases (Adrian and Löffler, 2016). Cobalamin or vitamin B12 along with cobalt is found at the centre of the corrinoid enzyme (Bommer et al., 2014). A few ORB have been shown to be dependent on an external source of vitamin B12, while others have been shown to have de novo corrinoid biosynthesis pathways (Nonaka et al., 2006; Yan et al., 2013; Rupakula et al., 2015). Most potential corrinoid biosynthesis and uptake proteins were found in the genome of D. spongiiphila strain AA1 (Supporting Information Table S1). However, corrinoid biosynthesis ability can be eliminated by a minor truncation in one single gene, as shown for Dehalobacter restrictus (Rupakula et al., 2015). Thus, a nearly complete corrinoid biosynthesis pathway from genome annotation cannot guarantee a functional metabolic pathway. Our growth and dehalogenation experiments indicated that D. spongiiphila does not rely on an exogenous cobalamin supply. However, experiments to determine whether propyl iodide, a light-reversible inhibitor of corrinoid enzymes (Ghambeer et al., 1971), would inhibit dehalogenation activity were inconclusive (data not shown). For comparison, the 3,5-dichlorophenol reductive dehalogenase from Desulfitobacterium frappieri PCP-1 was inhibited by propyl iodide, suggesting the involvement of a cobalamin cofactor in the enzyme (Thibodeau et al., 2004). Pure PCE-RDase of Dehalobacter restrictus was also inhibited by propyl iodide and reactivated once illuminated (Maillard et al., 2003). However, reductive dehalogenation in Desulfomonile tiedjei DCB-1 was not inhibited by propyl iodide (Louie and Mohn, 1999) and the RdhA cofactor is a heme (Ni et al., 1995). Further investigations are needed to identify the cofactor of D. spongiiphila strain AA1 dehalogenases.

Conclusions

Considering the abundant organohalide content, marine environments provide favourable habitats for organohalide respiring bacteria. The remarkable distribution of rdhA homologs in marine sediments reveals that organohalide respiration is a significant process of the organohalide cycle (Futagami et al., 2009; Futagami et al., 2013). As the only pure ORB culture isolated from a marine sponge, the genomic and metabolic properties of D. spongiiphila strain AA1 enrich our knowledge about the organohalide cycle. Our studies uncovered three distinct reductive dehalogenase genes in strain AA1, which show low similarity with rdhAs from other sources. The transcriptional upregulation of these rdhA genes indicate their potential roles in reductive dehalogenation. The rdh gene clusters of D. spongiiphila strain AA1 contain transcriptional regulatory genes, whose function needs to be further studied. Future work will aim to unravel the rdh gene transcription regulation mechanism of strain AA1 and how strain AA1 responds to organohalides.

Experimental procedures

Growth of Desulfoluna spongiiphila strain AA1

Strain AA1 was grown anaerobically in a minimal salts medium as described by Fennell et al. (2004) with minor modification at room temperature. The NaCl concentration was modified to 25 g/L, which is the optimal salinity for strain AA1 growth (Ahn et al., 2009). Lactate (30 mM) and sulfate (20 mM) were utilized as electron donor and acceptor.

Genome sequencing and annotation

The genome of D. spongiiphila strain AA1 was initially sequenced on the SOLiD™ 3 Analyzer platform (Applied Biosystems, Foster City, CA) in 2010 and later on the Illumina platform in 2015.

For SOLiD sequencing, 3 l of strain AA1 grown under sulfidogenic conditions was collected by filtration and total DNA was extracted using the QIAGEN QIAamp® DNA mini kit.

For Illumina sequencing, strain AA1 grown under sulfidogenic conditions was collected by centrifugation. Genomic DNA was extracted and purified by following a DOE Joint Genomic Institute (JGI) genomic DNA phenol-chloroform extraction protocol. A 300 bp insert standard shotgun library was constructed and sequenced using the Illumina HiSeq–2000 1TB platform which generated 5 262 106 reads totalling 789.3 Mbp. All raw Illumina sequence data was filtered using BBDuk, which removes known Illumina artefacts and PhiX. Reads with more than one ‘N’ or with quality scores (before trimming) averaging less than 8 or reads shorter than 51 bp (after trimming) were discarded. Remaining reads were mapped to masked versions of human, cat and dog references using BBMAP and discarded if identity exceeds 93%. Sequence masking was performed with BBMask. Following steps were then performed for assembly: (1) artefact filtered Illumina reads were assembled using Velvet (version 1.2.07) (Zerbino and Birney, 2008); (2) 1–3 kbp simulated paired end reads were created from Velvet contigs using wgsim (version 0.3.0); (3) Illumina reads were assembled with simulated read pairs using Allpaths–LG (version r46652) (Gnerre et al., 2011). The D. spongiiphila strain AA1 genome is available as JGI Analysis Project ID: Ga0104423 (GOLD Project ID: Gp0127298). The annotated protein sequences are available in NCBI (BioProject Accession: PRJEB15715).

Identification of rdhA genes

The initial identification of rdh genes was conducted on SoLiD sequencing data. The scaffold sequences were analyzed through TrAnsFuSE (Harel et al., 2012), a protein sequence-scanning program that targets proteins containing EC1 (transition metal-utilizing redox domains); LocTree2 (Goldberg et al., 2012) to predict the subcellular localization and type of protein (membrane-bound or cytoplasmic); and Blast2GO (Conesa et al., 2005) used to annotate and identify functional proteins. This process uncovered three putative reductive dehalogenase genes in the D. spongiiphila strain AA1 genome.

N- and C-terminal amino acid sequence alignment of potential reductive dehalogenases of D. spongiiphila and functionally characterized reductive dehalogenases (Jugder et al., 2015) was done using Clustal W pairwise alignment with Cost Matrix BLOSUM in MEGA 7 (Kumar et al., 2016).

Phylogenetic analyses of reductive dehalogenases were conducted in MEGA7 (Kumar et al., 2016). Initial tree(s) of strain AA1 and characterized reductive dehalogenases for the heuristic search were obtained automatically by applying Neighbor-Joining (NJ) and advanced NJ (BioNJ) algorithms to a matrix of pairwise distances estimated using a Jones–Thornton–Taylor (JTT) model, and then selecting the topology with superior log likelihood value.

Induction and expression experiments

For analysis of rdhA gene expression strain AA1 was grown in sulfidogenic medium and then inoculated into fresh anaerobic medium containing 1 mM lactate as electron donor and 200 μM 2,6-dibromophenol (2,6-DBP, Aldrich Chemical Co., Milwaukee, Wis) as an electron acceptor (10% transfer for the induction experiment and 50% for the expression experiment). Debromination of 2,6-DBP was monitored via high performance liquid chromatography (HPLC) as described below. For inhibition of protein synthesis 1 mg/ml streptomycin was added. In the expression experiment, the control contained 200 μM sulfate instead of 200 μM 2,6-DBP. Samples for total RNA extraction and analysis rdhA gene expression were taken periodically.

Expression of rdhAs in the presence of sponge extracts

Approximately 1.5 g (dry weight) of Aplysina aerophoba sponge tissue (collected in 2001 and stored at −65°C) (Ahn et al., 2003) was extracted with 55 ml of methanol by shaking for two days. Extracts (10 ml) was pipetted into autoclaved serum bottles and the methanol evaporated in a fume hood. A culture of D. spongiiphila AA1 grown on lactate and sulfate as described above was transferred to the serum bottles containing the dried extracts and examined for rdhA gene expression. In total, three treatments (control without sponge extracts; with sponge extracts; with both sponge extracts and 200 μM 2-BP) in duplicate were prepared and analyzed for rdhA gene expression.

RNA extraction, reverse transcription and qPCR

Total RNA was extracted from 5 ml of culture samples. Cells were pelleted by centrifugation and then extracted using TRIzol (Ambion, Life Technologies) reagent according to the manufacturer’s instructions. The RNA pellet was dissolved in 30 μl nuclease-free water and further treated by DNA-free™ DNA removal kit (Ambion, Life Technologies) to remove gDNA contamination in RNA. The integrity and purity of RNA was validated by analysis on an agarose gel. 0.5 μl of RNA was used to synthesize cDNA in a 20 μl reaction by using the iScript™ Reverse Transcription Supermix (Bio-Rad Laboratories, Inc.) according to manufacturer’s instructions. The obtained cDNA was amplified with primer sets specifically designed to each rdhA gene and to the 16S rRNA gene using both regular PCR and qPCR (Supporting Information Table S2). The specificity of the rdhA gene primers was examined by sequencing of the PCR products and comparison to the genome. For qPCR, a serial dilution of a cDNA sample of 2,6-DBP induced cells was made to generate a standard curve of each gene. RT-qPCR was performed using an IQ™ SYBR Green Supermix (BIO-RAD) in a 20 μl reaction. Thermocycling conditions for RT-qPCR were was as follows: 2 min at 90°C, followed by 40 cycles of 30 s at 94°C, 30 s at 59°C and 70 s at 72°C. The melting curve data collection started from 100°C for 10 s with a decreasing rate of 0.5°C/cycle for 130 cycles.

Transcription levels of the rdhA genes were calculated by relative quantitation using relative standard curve methods normalized to the 16S rRNA gene. Normalized expression in cultures treated with 2,6-DBP was divided by normalized expression in control cultures to indicate the expression level.

Effect of exogenous cobalamin on debromination

D. spongiiphila strain AA1 was previously grown in medium containing 50 μg/l of cobalamin. To remove cobalamin from the culture, strain AA1 culture was successively transferred into cobalamin-free sulfidogenic medium. The transfers represented a 1:3000 dilution, yielding a cobalamin concentration below 0.02 μg/l. The culture was then exposed to different cobalamin concentrations (0, 1, 10, 50, 200 μg/l) in medium amended with 2,6-DBP to determine debrominating activity.

Analytical methods

The concentration of 2,6-DBP, 2-BP and phenol was measured via high performance liquid chromatography (HPLC) on a Shimadzu system equipped with an auto injector (SIL-10A, Shimadzu), a system controller (SCK-10A, Shimadzu), a diode array detector (SPD-M10A, Shimadzu). A Sphereclone C-18 column (250 mm × 4.6 mm, particle size 5 μm; Phenomenex) was used with a mobile phase consisting of methanol:water:acetic acid (methanol varied from 70% to 80% depending on sample batch; 1% acetic acid) at an isocratic flow rate of 1 ml/min. The detection wavelength was 280 nm.

Supplementary Material

1

Acknowledgements

This work was funded in part by a grant from the National Science Foundation (OCE-451708) and the New Jersey Agricultural Experiment Station. The strain AA1 genome was sequenced as part of the ‘Genomic Encyclopedia of Archaeal and Bacterial Type Strains, Phase II (KMG-II): from individual species to whole genera’ study. The work conducted by the U.S. Department of Energy Joint Genome Institute, a DOE Office of Science User Facility, is supported by the Office of Science of the U.S. Department of Energy under Contract No. DE-AC02–05CH11231. We thank Randall Kerstetter, Mark Diamond, Ariella S. Sasson, Anirvan M. Sengupta and Joachim Messing (Waksman Institute of Microbiology, Rutgers University) for the initial sequencing of the D. spongiiphila strain AA1 genome on the SoLiD platform.

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