Metatranscriptomic Analysis of Sulfur Oxidation Genes in the Endosymbiont of Solemya Velum

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Citation Stewart, Frank J., Oleg Dmytrenko, Edward F. DeLong, and Colleen M. Cavanaugh. 2011. “Metatranscriptomic Analysis of Sulfur Oxidation Genes in the Endosymbiont of Solemya Velum.” Frontiers in Microbiology 2. doi:10.3389/fmicb.2011.00134.

Published Version doi:10.3389/fmicb.2011.00134

Citable link http://nrs.harvard.edu/urn-3:HUL.InstRepos:14350393

Terms of Use This article was downloaded from Harvard University’s DASH repository, and is made available under the terms and conditions applicable to Other Posted Material, as set forth at http:// nrs.harvard.edu/urn-3:HUL.InstRepos:dash.current.terms-of- use#LAA ORIGINAL RESEARCH ARTICLE published: 20 June 2011 doi: 10.3389/fmicb.2011.00134 Metatranscriptomic analysis of sulfur oxidation genes in the endosymbiont of Solemya velum

Frank J. Stewart 1*, Oleg Dmytrenko2, Edward F. DeLong3 and Colleen M. Cavanaugh2

1 School of Biology, Georgia Institute of Technology, Atlanta, GA, USA 2 Department of Organismic and Evolutionary Biology, Harvard University, Cambridge, MA, USA 3 Department of Civil and Environmental Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA

Edited by: Thioautotrophic endosymbionts in the Domain Bacteria mediate key sulfur transformations Donald A. Bryant, The Pennsylvania in marine reducing environments. However, the molecular pathways underlying symbiont State University, USA metabolism and the extent to which these pathways are expressed in situ are poorly Reviewed by: Donald A. Bryant, The Pennsylvania characterized for almost all symbioses. This is largely due to the difficulty of culturing sym- State University, USA bionts apart from their hosts. Here, we use pyrosequencing of community RNA transcripts Christiane Dahl, Rheinische (i.e., the metatranscriptome) to characterize enzymes of dissimilatory sulfur metabolism Friedrich-Wilhelms-Universität Bonn, in the model symbiosis between the coastal bivalve Solemya velum and its intracellular Germany thioautotrophic symbionts. High-throughput sequencing of total RNA from the symbiont- *Correspondence: Frank J. Stewart, School of Biology, containing gill of a single host individual generated 1.6 million sequence reads (500 Mbp). Georgia Institute of Technology, Ford Of these, 43,735 matched Bacteria protein-coding genes in BLASTX searches of the ES&T Building, Room 1242, 311 Ferst NCBI database. The taxonomic identities of the matched genes indicated relatedness to Drive, Atlanta, GA 30332, USA. diverse species of sulfur-oxidizing Gammaproteobacteria, including other thioautotrophic e-mail: [email protected]. edu symbionts and the purple sulfur bacterium Allochromatium vinosum. Manual querying of these data identified 28 genes from diverse pathways of sulfur energy metabolism, includ- ing the dissimilatory sulfite reductase (Dsr) pathway for sulfur oxidation to sulfite, the APS pathway for sulfite oxidation, and the Sox pathway for thiosulfate oxidation. In total, reads matching sulfur energy metabolism genes represented 7% of the Bacteria mRNA pool. Together, these data highlight the dominance of thioautotrophy in the context of sym- biont community metabolism, identify the likely pathways mediating sulfur oxidation, and illustrate the utility of metatranscriptome sequencing for characterizing community gene transcription of uncultured symbionts.

Keywords: chemosynthesis, endosymbiosis, sulfide, thiosulfate, gene expression

INTRODUCTION biochemical transformations in a handful of thioautotrophic sym- Symbioses between thioautotrophic bacteria and invertebrates bioses (Woyke et al., 2006; Newton et al., 2007; Grzymski et al., perform important steps in the marine sulfur cycle. In these 2008; Robidart et al., 2008). For example, a comparative genomics associations,bacteria living intra- or extra-cellularly with a eukary- approach has been used to characterize the probable sulfur oxida- otic host oxidize reduced sulfur compounds as an energy source tion pathways in two endosymbionts of deep-sea vent clams (fam- for autotrophic CO2 fixation, providing a substantial source of ily Vesicomyidae; (Harada et al., 2009). However, DNA sequences fixed carbon for the host (Cavanaugh et al., 2006; Dubilier et al., do not reflect the actual metabolic activity of microorganisms. 2008). Such symbioses have been described in 7 host phyla, from In contrast, methods that measure gene transcription (transcrip- ciliates to giant tubeworms (Stewart et al., 2005), and are rou- tomics) and translation (proteomics) can provide more direct tinely discovered in new species as marine environments become descriptions of symbiont function and metabolism, particularly better explored (Losekann et al., 2008; Fujiwara et al., 2010; if administered under in situ conditions (Markert et al., 2007; Rodrigues et al., 2010). In reducing habitats (e.g., anaerobic sed- Harada et al., 2009; Wilmes and Bond, 2009). iments, hydrothermal vents, hydrocarbon seeps), these symbioses Notably, technical advancements in RNA amplification and may dominate the biomass, playing critical roles in local primary parallel pyrosequencing have made it possible to obtain hun- production and sulfur transformations (Sievert et al., 2007). dreds of thousands of sequences from the microbial community Despite their ubiquity and ecological importance, thioau- RNA pool, i.e., the metatranscriptome. Such methods enable totrophic symbioses are poorly described molecularly. Character- inferences of the relative metabolic activity of hundreds of ization of the genes and proteins mediating symbiont metabolism diverse metabolic pathways using transcript abundance as a proxy has been hindered by the inability (as of yet) to culture most sym- for gene expression (Frias-Lopez et al., 2008; Stewart et al., bionts outside their hosts. Genomic and metagenomic sequencing 2011b). While metatranscriptomics has been increasingly used has helped reverse this trend, clarifying the genetic basis for diverse to study community metabolism in free-living microorganisms

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of the open ocean (Poretsky et al., 2009; Hewson et al., 2010; 770–1180 μm glass beads. Total gill RNA (symbiont + host) was Stewart et al., 2011a), transcriptomic analysis of marine symbioses extracted with a chloroform–ethanol method using miRNeasy has been restricted to only a few taxa, and has focused primar- Mini Kit (Qiagen) according to the protocol for purification of ily on host gene expression (Nyholm et al., 2008; Bettencourt total RNA, including small RNA, from animal cells. An aliquot et al., 2010). Here, we use pyrosequencing to analyze the in situ of total RNA was then enriched for symbiont RNA using the metatranscriptome of a model thiotrophic symbiont, the bacte- MICROBEnrich™ kit (Ambion) according to the manufacturer’s rial endosymbiont of the coastal bivalve Solemya velum, focus- instructions. The kit employs capture oligonucleotides and sub- ing specifically on transcripts encoding proteins of dissimilatory tractive hybridization to selectively remove eukaryotic polyadeny- sulfur metabolism. lated mRNAs,enriching for non-polyadenylated prokaryotic RNA. Studies of the protobranch S. velum and its endosymbiont were This kit is also designed to remove eukaryotic 18S and 28S rRNA. some of the first to describe the physiology and ecology of thioau- However, because capture oligonucleotides for rRNA removal are totrophic symbioses (see review in Stewart and Cavanaugh, 2006). optimized for use with mammalian 18S/28S sequences (Ambion S. velum lives in shallow, sulfur-rich sediments along the Atlantic internal documentation), the removal efficiency of bivalve host Coast (Florida to Canada), obtaining the bulk of its nutrition rRNA was anticipated to be low. (>97% of host carbon) from a dense population of autotrophic bacteria living within specialized cells of the host gills (Cavanaugh, 1983; Krueger et al., 1992). Early molecular evidence (16S rRNA RNA AMPLIFICATION AND cDNA SYNTHESIS gene sequence) suggested that the symbiont population consists of Symbiont-enriched RNA was linearly amplified and converted to a single, unique bacterial phylotype within the Gammaproteobac- cDNA as described in Frias-Lopez et al. (2008), Shi et al. (2009). ™ teria (Eisen et al., 1992). Sulfur-based autotrophy in the symbiont Briefly, using the MessageAmp II-Bacteria kit (Ambion), total population was initially demonstrated through physiological and RNA was polyadenylated, converted to double-stranded cDNA molecular studies showing (1) the activity and corresponding gene via reverse transcription, and then transcribed in vitro to pro- sequence for ribulose 1,5-bisphosphate carboxylase–oxygenase duce large quantities (tens of micrograms) of single-stranded (RubisCO), the primary CO -fixing enzyme of the Calvin cycle, antisense RNA. Amplified RNA was then converted to double- 2 ® in host gills (Cavanaugh, 1983; Schwedock et al., 2004), (2) an stranded cDNA using the SuperScript III First-Strand Synthesis increase in symbiont CO fixation and oxygen consumption in System (Invitrogen) with priming via random hexamers for first- 2 ™ response to sulfide and thiosulfate addition (Cavanaugh, 1983; strand synthesis, and the SuperScript Double-Stranded cDNA Anderson et al., 1987), and (3) the activity of two enzymes in the synthesis kit (Invitrogen) for second-strand synthesis. cDNA was APS pathway mediating sulfite oxidation, APS reductase, and ATP then purified with the QIAquick PCR purification kit (Qiagen), sulfurylase (Chen et al., 1987). While these data confirm that S. digested with BpmI to remove poly(A) tails, and used directly for velum symbionts can oxidize reduced sulfur for energy, the full pyrosequencing. biochemical pathway through which this occurs, and the extent to which this pathway is expressed, remains uncharacterized. SEQUENCING Here, pyrosequencing of the symbiont-enriched RNA frac- Purified cDNA was used for the generation of single-stranded tion from S. velum sampled from the environment revealed a DNA libraries and emulsion PCR via standard protocols (454 strong representation by genes of dissimilatory sulfur metabo- Life Sciences, Roche). Amplified library fragments were sequenced lism in the total transcript pool. These results underscore the via a single full plate run on a Roche Genome Sequencer FLX importance of thioautotrophy in the symbiosis, identify the likely instrument using Titanium series chemistry. Sequencing yields pathways by which sulfur oxidation occurs, and illustrate the util- are shown in Table 1. ity of metatranscriptome sequencing for characterizing symbiont community metabolism across thousands of genes.

MATERIALS AND METHODS Table 1 | Metatranscriptome read statistics. SAMPLE COLLECTION Total reads 1,591,449 Individuals of S. velum were collected at low tide from Bluff Hill Mean length (bp) 316 − Cove, Point Judith Pond, Rhode Island (RI; 41.380˚N, 71.502˚W) Mean GC% 50.1 on 5 December 2009. Bluff Hill Cove is connected to the ocean by Non-rRNA reads1 203,641 a narrow inlet and bordered by extensive mud flats that support a Protein-coding reads2 70,427 stable and abundant population of S. velum. At the time of collec- Bacteria (%) 62.1 tion the average sediment temperature was 8˚C. Individuals were Eukarya (%) 37.3 dissected in the field upon collection, with symbiont-containing Other (%) 0.6 gill tissue placed immediately into RNAlater® and stored frozen until RNA extraction. 1Reads not matching ribosomal RNA genes. 2Reads with significant matches (bit score > 50) to protein-coding genes in SYMBIONT RNA EXTRACTION AND ENRICHMENT NCBI-nr; designation as Bacteria or Eukarya according to NCBI annotations of Symbiont-containing gill tissue of a single host individual was top BLASTX matches; “other” = Archaeal (0.08%), viral (0.03%), or unclassified lysed using a FastPrep FP120 tissue homogenizer (Savant) with (0.48%) genes.

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DATA ANALYSIS RESULTS Duplicate sequence reads (reads with 100% nucleotide similar- READ STATISTICS ity and identical length), which may arise as artifacts of the Pyrosequencing generated ∼1.6 million sequence reads (∼500 pyrosequencing method, were identified as in Stewart et al. (2010) Mbp) from the symbiont-enriched gill metatranscriptome using the program CD-HIT (Li and Godzik, 2006) and removed (Table 1). Reads matching ribosomal RNA represented 87% of from the dataset. Non-duplicate reads matching ribosomal RNA the dataset. Of 284,553 ribosomal reads with top matches to the genes were identified by BLASTN searches against a custom prokaryotic (Bacteria or Archaea) small subunit rRNA (16S rRNA) database of prokaryotic and eukaryotic small and large sub- gene, 278,192 (98%) matched the published S. velum symbiont unit rRNA nucleotide sequences (5S, 16S, 18S, 23S, 28S rRNA, 16S rRNA sequence at 97% nucleotide identity or greater, with compiled from microbial genomes and sequences in the ARB 233,904 (82%) matching at ≥ 99% identity. These results suggest a SILVA databases, and including the published 16S rRNA gene genetically homogenous microbial population within the S. velum sequence of the S. velum symbiont, http://www.arb-silva.de) and gill. However, this pattern does not rule out the co-occurrence removed from the analysis. Protein-coding gene sequences were of distinct symbiont lineages (strains) within the same host or identified among the remaining non-duplicate, non-rRNA reads of non-symbiont microorganisms associated with the gill surface. by BLASTX searches against the NCBI non-redundant protein Our dataset therefore reflects a “meta” transcriptome representing database (NCBI-nr, as of May 2010). The top reference gene(s) the transcriptional activity of the entire gill-associated microbial matching each read above a bit score cutoff of 50 was used community. to designate each protein-coding read. For reads having multi- ple top matches with equal bit score, each matching reference TAXONOMIC IDENTITIES OF PROTEIN-CODING READS gene was designated a top hit, with its representation scaled Removal of ribosomal RNA reads from the dataset left a total of proportionate to the number of genes sharing an equal bit 203,641 non-rRNA sequences, of which 70,427 (34.7%) matched score. Read counts per gene were tabulated and normalized as protein-coding genes in the NCBI-nr database (above bit score 50). a proportion of the total number of protein-coding reads (or Of these, 62.1% matched genes from Bacteria and 37.3% matched protein-coding reads from the Domain Bacteria) in each sam- genes from Eukarya, with the remainder matching taxonomically ple. The taxonomic identity of protein-coding genes was inferred unclassified or Archaeal/viral genes (Table 1). The relative abun- from the annotation affiliated with each gene, and tabulated dance of Bacteria reads suggests that the symbiont-enrichment according to the NCBI taxonomy using the program MEGAN protocol used here (see Methods), while not 100% efficient, likely (Huson et al., 2007). did increase the proportion of symbiont mRNA in the total tran- Detailed analysis of all functional gene categories in the script pool. The proportion of total mRNA in the metatranscrip- dataset is beyond the scope of this paper, and is the topic of tome could likely have been further enriched by implementing a separate study (Dmytrenko et al., in preparation). Here, we sample-specific methods to remove symbiont and host rRNA focus only on transcripts matching genes of dissimilatory sulfur reads, e.g., subtractive hybridization with taxon-specific probes metabolism, as detected via BLASTX against all protein-coding (Stewart et al., 2010). genes in NCBI-nr. BLASTX results were manually queried to The annotations of NCBI-nr genes can be used to infer the infer the relative abundance of genes encoding proteins of the taxonomic relatedness of symbiont and host sequences to refer- primary pathways of dissimilatory sulfur metabolism, includ- ence organisms. Approximately 80% of all protein-coding tran- ing: the dissimilatory sulfite reductase (DsrABCEFHMKLJOP- scripts from Bacteria matched genes of Gammaproteobacteria as NRS) pathway catalyzing sulfur oxidation to sulfite; the sul- their closest sequence relative (BLASTX searches of NCBI-nr). fur oxidation (SoxABCDEFGHRSVWXYZ) pathway responsible Among the Gammaproteobacterial reads, nearly half matched for thiosulfate oxidation; the APS pathway mediating the con- species of the Order Chromatiales (Figure 1), with the freshwater version of sulfite to sulfate via an APS intermediate, via the purple sulfur bacterium Allochromatium vinosum DSM 180 con- enzymes adenosine-5-phosphosulfate (APS) reductase (AprAB), stituting over 17% of the Bacteria protein-coding reads (>10% APS reductase membrane anchor (AprM), quinone-interacting of the total protein-coding reads; Figures 1–3). Other highly membrane-bound oxidoreductase (QmoABC), and sulfate adeny- represented Gammaproteobacteria included the haloalkaliphilic, lyltransferase (SAT); and the sulfide oxidation step involving sulfur-oxidizing species Thioalkalivibrio sp. HL-EbGR7 (Chroma- flavocytochrome c sulfide dehydrogenase (FccAB) and sulfide– tiales) and the sulfur-oxidizing symbionts of marine invertebrates, quinone reductase (Sqr), which initiate electron flow from sulfide notably Endoriftia persephone,the endosymbiont of the hydrother- to the transport chain. When specified, sequence read counts mal vent tubeworm Riftia pachyptila (Figure 2). The non-Bacteria per gene were normalized based on a best approximate gene protein-coding reads in the dataset matched eukaryotes from length (estimated based on full-length open reading frames from diverse phyla, suggesting a lack of close relatives to S. velum in sequenced genomes). the database (Figure 2). SEQUENCE DATA All transcript sequences generated in this study have been SULFUR OXIDATION GENES deposited in the Community Cyberinfrastructure for Advanced BLASTX results revealed a dominant role for transcripts matching Microbial Ecology Research and Analysis (CAMERA) Portal for genes of dissimilatory sulfur oxidation pathways in the symbiont access by the broader scientific community. metatranscriptome. The putative functions of proteins encoded

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reads matching NCBI-nr Bacterial taxa 0 5000 10000 15000 20000 25000 30000 35000 Alphaproteobacteria

Betaproteobacteria Gammaproteobacteria

Deltaproteobacteria

Epsilonproteobacteria Gammaproteobacteria only

uncultured marine proteobacterium ANT32C12

uncultured proteobacterium Enterobacteriaceae Other uncultured proteobacterium RedeBAC7D11 unclassified (misc) Actinobacteria Vibrionales Aquificae OMG group Bacteroidetes Pseudomonadales Chlorobi

Chlamydiae

Lentisphaerae Methylococcaceae

Verrucomicrobia Chromatiales Chloroflexi

Cyanobacteria Thiotrichales

Deferribacteres

Deinococcus-Thermus

environmental samples

Acidobacteria Alteromonadales

Fibrobacteres

Firmicutes

Fusobacteria Oceanospirillales Gemmatimonadetes

Nitrospirae

Planctomycetes Spirochaetes Other sulfur-oxidizing symbionts Synergistetes Legionellales Xanthomonadaceae Tenericutes Aeromonadaceae Thermotogae Pasteurellaceae Candidatus Thiobios zoothamnicoli candidate division WS3 Acidithiobacillus candidate division WS6 Reinekea Thermobaculum Cardiobacteriaceae environmental samples Magnetococcus sp. MC-1 uncultured gamma EBAC20E09 Mariprofundus ferrooxydans PV-1 Thiohalophilus thiocyanatoxydans

unclassified Bacteria (miscellaneous)

FIGURE 1 |Taxonomic breakdown of all identified protein-coding transcripts assigned to the Domain Bacteria. Inset pie chart shows breakdown within the Gammaproteobacteria, which represented 79.4% of all protein-coding transcripts from the Domain Bacteria. by these transcripts is inferred here based on the well character- cycle: the SoxAX complex responsible for the fusion of thiosul- ized pathways from model sulfur-oxidizing bacteria, notably the fate to the carrier complex, the carrier complex SoxYZ, and SoxB, Dsr pathway of the purple sulfur bacterium A. vinosum and the a putative thiol esterase mediating the hydrolytic release of sulfate Sox pathway of the Alphaproteobacterium Paracoccus pantotro- from sulfur-bound SoxYZ (Figure 4B). Two additional sox genes, phus (for detailed descriptions of these pathways, see (Friedrich soxH, encoding a beta lactamase domain-containing protein, and et al., 2001, 2005; Dahl et al., 2005; Frigaard and Dahl, 2009; Ghosh soxW, encoding a periplasmic thioredoxin, were detected at low and Dam, 2009). abundance. In P. pantotrophus, neither soxH or soxW appears nec- Our analysis revealed 2888 transcript sequences matching a essary for autotrophic growth on thiosulfate (Rother et al., 2001; total of 28 genes from the primary sulfur oxidation pathways Bardischewsky et al., 2006). (Figure 4). Of these, 65% (1886 reads) matched 14 genes encod- Several additional sulfur energy metabolism genes were well ing proteins of the Dsr pathway, including the dissimilatory sulfite represented in the transcriptome (Figure 4). These included four reductase enzyme (DsrAB), the DsrAB-interacting flavoprotein genes of the APS pathway for sulfite oxidation to sulfate: aprAB DsrL, the transmembrane electron transport complex DsrKMJOP, and aprM, encoding APS reductase (adenylylsulfate reductase) and the putative sulfur relay molecules DsrEFH and DsrC, with and the APS reductase membrane anchor, respectively, and SAT, the latter constituting the single most abundant sulfur gene in the encoding ATP sulfurylase (sulfate adenylyltransferase). Together, transcriptome when read counts were corrected for variation in these four genes were represented by 672 sequences (23% of gene length (circles, Figure 4A). the reads matching sulfur genes). Finally, the analysis detected Seven genes of the Sox system were detected, including five transcripts encoding two primary enzymes for sulfide oxidation: genes encoding the core proteins of the thiosulfate oxidation the membrane-bound sulfide–quinone reductase (Sqr),mediating

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% of reads matching NCBI-nr 024681012

Allochromatium vinosum DSM 180 (66%)

Endoriftia persephone 'Hot96_1+Hot96_2' (69%)

Thioalkalivibrio sp. HL-EbGR7 (65%)

Branchiostoma floridae (59%) Cephalochordata, “Florida lancelet”

Solemya velum (83%) Protobranchia, “Atlantic awningclam”

Solemya velum gill symbiont (96%)

Saccoglossus kowalevskii (60%) Hemichordata, “acorn worm”

Thiobacillus denitrificans ATCC 25259 (65%) Betaproteobacteria

Strongylocentrotus purpuratus (60%) Echinodermata, “purple sea urchin”

Ciona intestinalis (62%) Urochordata, “vase tunicate”

*Methylobacter tundripaludum SV96 (64%)

Beggiatoa sp. PS (64%) Bacteria Halothiobacillus neapolitanus c2 (70%) Eukarya

*Nitrosococcus halophilus Nc4 (65%)

Danio rerio (63%) Chordata, “zebrafish”

Methylophaga thiooxidans DMS010 (67%)

Thioalkalivibrio sp. K90mix (68%)

Alkalilimnicola ehrlichii MLHE-1 (65%)

Ixodes scapularis (64%) Arthropoda, “deer tick”

Nematostella vectensis (61%) Cnidaria, “starlet sea anemone”

FIGURE 2 |Top 20 taxa from the Domains Bacteria and Eukarya identified bars) are labeled according to phylum or subphylum designation. Numbers in as best matches to protein-coding transcripts. All Bacteria taxa (red bars) parentheses are the amino acid identities averaged across all sequence reads belong to the Gammaproteobacteria and are putatively capable of matching a given taxon as a top BLASTX hit (above bit score 50). Identities chemolithotrophic growth on reduced sulfur compounds, unless otherwise reflect only the high-scoring segment pair (HSP) region for each BLAST result; marked (∗=no evidence of sulfur-based chemolithotrophy). Eukarya (blue gaps were not included in identity calculations. electron transport to quinone,and flavocytochrome c sulfide dehy- genes identified here (n = 28; Figure 4) represented 6.6% of the drogenase (FccAB), mediating transport to a c-type cytochrome Bacteria protein-coding reads in the dataset (1 in 15 sequences). (Dahl and Friedrich, 2008). Several genes encoding enzymes for sulfide and thiosulfate oxi- dation, including the dsr genes and SAT (ATP sulfurylase), were DISCUSSION among the top most abundant reference genes (i.e., unique This study provides the first molecular evidence that diverse genes accession numbers) detected in the study (Figure 3). Many of of sulfur energy metabolism are present and actively transcribed these were most closely related to homologs from known sulfur- in the S. velum symbiont population under in situ conditions. oxidizing Gammaproteobacteria (Figures 1–3), in particular A. The metatranscriptome contained Bacteria transcripts encoding vinosum and Endoriftia persephone, the symbiont of the tube- genes of several primary sulfur oxidation pathways, including the worm Riftia pachyptila, consistent with the phylogenetic place- reverse dissimilatory sulfite reductase (Dsr) complex for sulfur ment of the Solemya symbiont within this group (Eisen et al., oxidation to sulfite, the adenosine-5 -phosphate (APS) reductase 1992; Distel, 1998). Strikingly, a single reference gene, the pre- pathway for sulfite oxidation, and the sulfur oxidation (Sox) path- viously characterized cbbL gene encoding the large subunit of way mediating thiosulfate oxidation. In total, the sulfur oxidation the CO2 fixation enzyme RubisCO (ribulose 1,5-bisphosphate

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cbbL, RubisCO, Solemya velum gill symbiont 4

dsrL, Allochromatium vinosum DSM 180

ATP sulfurylase, Riftia pachyptila symbiont 3 dsrA, Allochromatium vinosum DSM 180

dsrH, Allochromatium vinosum DSM 180 dsrO, Allochromatium vinosum DSM 180 2 dsrA, Candidatus Thiobios zoothamnicolis aprB, Thioalkalivibrio sp. HL-EbGR7 dsrK, Allochromatium vinosum DSM 180 dsrM, Allochromatium vinosum DSM 180 dsrK, Allochromatium vinosum DSM 180 1 aprA, Thioalkalivibrio sp. HL-EbGR7 aprA, Thiobacillus denitrificans ATCC 25259

% of reads matching NCBI-nr Bacterial matching genes % of reads soxB, Oceanospirillum sp. MED92 dsrB, Allochromatium vinosum DSM 180 dsrE, Allochromatium vinosum DSM 180 0 pdb|1YX3|A pdb|1JHD|A gb|ACI42952.1| gb|AAT01432.1| gb|AAT01431.1| gb|AAT01430.1| gb|EEZ79872.1| gb|AAC35401.2| dbj|BAG16735.1| ref|YP_315815.1| ref|YP_865642.1| ref|YP_345019.1| ref|YP_692091.1| ref|YP_315819.1| ref|YP_314333.1| ref|YP_095768.1| ref|YP_315818.1| ref|YP_315816.1| ref|YP_390734.1| ref|YP_741437.1| ref|YP_314206.1| ref|YP_316560.1| ref|YP_390778.1| ref|YP_864145.1| ref|YP_436094.1| ref|YP_315817.1| ref|YP_316040.1| ref|NP_902774.1| ref|ZP_05060561.1| ref|ZP_02536538.1| ref|ZP_07655044.1| ref|ZP_02536594.1| ref|ZP_01452851.1| ref|ZP_02001504.1| ref|ZP_02003567.1| ref|ZP_02536657.1| ref|ZP_01999538.1| ref|ZP_02537192.1| ref|ZP_02536433.1| ref|ZP_01225470.1| ref|ZP_02534108.1| ref|ZP_04603189.1| ref|ZP_07653501.1| ref|ZP_01167148.1| ref|ZP_01225456.1| ref|YP_003444308.1| ref|YP_003444306.1| ref|YP_003443223.1| ref|YP_003443224.1| ref|YP_003444006.1| ref|YP_002512898.1| ref|YP_002515068.1| ref|YP_003444061.1| ref|YP_002514370.1| ref|YP_003263299.1| ref|YP_003444542.1| ref|YP_003443230.1| ref|YP_003444836.1| ref|YP_003444844.1| ref|YP_003524333.1| ref|YP_003443222.1| ref|YP_002512437.1| ref|YP_003443226.1| ref|YP_002512410.1| ref|YP_003443232.1| ref|YP_002515057.1| ref|YP_003809368.1| ref|YP_003443384.1| ref|YP_002798411.1| ref|YP_003263853.1| ref|YP_002515067.1| ref|YP_003913036.1| ref|YP_002512523.1| ref|YP_002515262.1| ref|YP_003442037.1| ref|YP_003444295.1| ref|YP_003444316.1| ref|YP_003444959.1| ref|YP_003444314.1| ref|YP_004067505.1| ref|YP_002512436.1| ref|YP_003443229.1| ref|YP_003442050.1| ref|YP_002515295.1| ref|YP_003443228.1| ref|YP_002512435.1| ref|YP_003444292.1| ref|YP_003524293.1| ref|YP_003527882.1| ref|YP_001970870.1| ref|YP_003444297.1| ref|YP_003442309.1| ref|YP_003264033.1| ref|YP_003443382.1| ref|YP_003443724.1| ref|YP_002513067.1| ref|YP_003444362.1| ref|YP_003443197.1| sp|Q673V5|RBL_SOVGS sp|Q3SH79|GLGA_THIDA

FIGURE 3 |Top 100 most abundant reference genes (unique accession numbers) in the Domain Bacteria detected in the metatranscriptome, as a percentage of the total sequence reads matching Bacteria genes in the NCBI-nr database (accession numbers at bottom). Red bars indicate genes involved in sulfur-based energy metabolism. carboxylase–oxygenase) of the S. velum symbiont, accounted for globules, while common in other thioautotrophic symbionts (e.g., over 4% of all protein-coding reads (Figure 3). The high abun- Endoriftia persephone), has not been observed in Solemya, suggest- dance of transcripts for enzymes of sulfur compound oxida- ing a potentially rapid turnover of elemental sulfur compounds by tion and carbon fixation suggests that thioautotrophy dominated an active Dsr system. While the exact roles of Dsr enzymes remain symbiont community metabolism at the time of collection in to be confirmed in the Solemya symbiont, our data indicate a early winter. prominent role for sulfur oxidation to sulfite in this symbiosis. Genes of the reverse Dsr pathway were among the most highly Consistent with this finding,transcripts encoding theAPS path- expressed in the symbiont metatranscriptome (Figures 3 and 4). In way for sulfite oxidation were abundant in the dataset. Use of sulfate-reducing Bacteria and Archaea, dissimilatory sulfite reduc- the APS pathway by Solemya symbionts was established previ- tase (DsrAB) mediates sulfite reduction to sulfide (Klein et al., ously by enzyme assays showing the activity of APS reductase 2001; Wagner et al., 2005). DsrAB homologs have been isolated (AprAB) and ATP sulfurylase (SAT; Chen et al., 1987). Together, from a wide variety of sulfur-oxidizing taxa (Loy et al., 2009), in these enzymes catalyze the AMP-dependent oxidation of sul- which the enzymes are thought to operate in the oxidative direc- fite to APS and the subsequent ATP-generating conversion of tion in conjunction with a diverse set of accessory proteins, includ- APS to sulfate (Figure 4). Here, AprAB and SAT-encoding tran- ing the transmembrane complex DsrMKJOP, the sulfur shuttle scripts represented a combined 1.5% of all protein-coding Bacte- molecules DsrEFH and DsrC, and the cytoplasmic flavoprotein ria reads. Additionally, transcripts encoding the transmembrane DsrL (Dahl et al., 2005; Ghosh and Dam, 2009), all of which were anchor AprM were detected at low abundance. In other Bacte- detected in the Solemya symbiont transcriptome (Figure 4). It has ria taxa, electron transport from the cytoplasmic APS reductase been suggested that sulfide for oxidation by the DsrAB complex is to the membrane quinol/quinone pool is mediated via interac- released via the reduction of a polysulfur carrier molecule, as cat- tions with either AprM, which is more common in Gammapro- alyzed by the NADH: (acceptor) oxidoreductase activity of DsrL teobacterial sulfur-oxidizers (e.g., A. vinosum), or the func- (Dahl et al., 2005), which has been shown to be critical for sulfur tionally associated membrane-bound redox complex QmoABC, oxidation in A. vinosum (Lubbe et al., 2006). In this species, the common in green sulfur bacteria of the Order Chlorobiales carrier molecule may be an organic perthiol involved with bring- (Meyer and Kuever, 2007). Here, detection of aprM but not ing stored elemental sulfur (sulfur globules) from the periplasm qmoABC transcripts is consistent with the placement of the to the cytoplasm (Frigaard and Dahl, 2009). Stored sulfur globules Solemya symbiont among the Gammaproteobacteria. Together, in A. vinosum are produced during growth on sulfide by enzymes these data suggest a functional sulfite oxidation complex in including FccAB and Sqr or via the activity of the Sox pathway this symbiosis. during growth on thiosulfate (Frigaard and Dahl, 2009; Ghosh The detection of transcripts matching genes of the Sox path- and Dam, 2009). Interestingly, the deposition of sulfur storage way also confirmed an active role for thiosulfate oxidation in

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A ) ) )

xB (65 xZ (42) o oxW (1 oxY (12 AT (341) rpoB (98) dsrA (365)dsrB (264)dsrC (217)dsrE (75) dsrF (75)dsrH (134)dsrJ (2) dsrK (137)dsrL (337)dsrM (126)dsrO (98)dsrP (31)dsrR (18)dsrS (7) soxA (16)s soxH (3) s soxX (52)s so aprA (201)aprB (126)aprM (4) fccA (40) fccB (32) sqr (66) S

2- B sox complex S O + 2 3 2e- + H SoxYZ SoxYZ -S-S-SO -HS SoxAX

- 2- R-Sn+1 H SoxB H O 3 flavocytochrome c ? 2 or H-Sn+1 sulfide dehydrogenase + - SO 2- + H R-Sn H 4 FccAB or H-S n SoxYZ -S-SH DsrJ n HS- HS- DsrO n Periplasm APS reductase DsrM Q DsrP AprM Sqr QH2 Cytoplasm sulfide:quinone reductase NADP + DsrK AprB + acceptor H O 6e- + 6H+ PP ATP 2 AprA i DsrL 2- 2- + SO APS SO NADPH + H H 2 S 3 4 + DsrB AMP 2e- SAT acceptor DsrB sulfate adenylyltransferase HS SH S S HS SH (ATP sulfurylase) DsrE DsrE DsrA DsrA DsrC DsrC DsrH DsrH dissimilatory HS SH DsrF DsrF HS SH sulfite reductase DsrC DsrC

FIGURE 4 | Relative abundance of transcripts encoding key enzymes of matching the same gene from different organisms have been pooled, in sulfur energy metabolism in the Solemya velum symbiont contrast to the data in Figure 3. (B) Putative metabolic function of metatranscriptome. (A) Abundances of transcripts with top matches corresponding enzymes, according to Dahl et al. (2005) and Frigaard and Dahl (BLASTX) to dissimilatory sulfur metabolism genes, shown relative to the (2009). dsr = dissimilatory sulfite reductase gene cluster, sox = sulfur abundance of transcripts matching RNA polymerase subunit B (rpoB), which oxidation gene cluster; aprAB = adenosine-5’-phosphosulfate (APS) typically occurs in a single copy per Bacteria genome. Numbers = read counts reductase; aprM, APS reductase membrane anchor; fccAB, flavocytochrome per gene. Circle area = read counts per gene normalized to the approximate c sulfide dehydrogenase; SQR, sulfide–quinone reductase; SAT, sulfate gene length (in kilobases) inferred from published genomes. Sequences adenylyltransferase.

S. velum symbionts. The Sox (sulfur oxidation) pathway medi- Alphaproteobacterium Paracoccus pantotrophus,inwhich15genes ates the oxidation of thiosulfate to sulfate in a variety of both soxRSVWXYZABCDEFGH comprise the sox operon (Friedrich non-photosynthetic and photosynthetic sulfur-oxidizing bacte- et al., 2001, 2005; Rother et al., 2001). Of these, seven genes ria (Frigaard and Dahl, 2009; Ghosh and Dam, 2009; Sakurai (soxABCDXYZ) encode the core periplasmic sox proteins, SoxB, et al., 2010). The pathway has been extensively studied in the SoxXA, SoxYZ, and SoxCD, with SoxYZ acting as the primary

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carrier molecule at every step in the pathway (Friedrich et al., convergence of a Y-shaped burrow (Levinton, 1977) where it can 2001; Figure 4). Analysis of the S. velum symbiont metatran- access anoxic water from lower in the sediment. This water is likely scriptome revealed transcripts matching two peripheral sox genes enriched in sulfide but may also contain thiosulfate,perhaps result- (soxHW ) at low abundance and five of the seven core sox genes ing from chemical sulfide oxidation during burrow ventilation (soxABXYZ) at higher abundances (Figure 4). Transcripts encod- (Howarth et al., 1983; Elsgaard and Jorgensen, 1992). Alternatively, ing the sulfur dehydrogenase SoxCD were not detected in our thiosulfate may also be available via sulfide oxidation by host mito- analysis, consistent with the absence of these genes from the chondria, as has been demonstrated in the Pacific congener S. reidi genomes of a variety of green and purple sulfur bacteria, includ- (Obrien andVetter,1990). Though both sulfide and thiosulfate can ing A. vinosum and the endosymbionts of deep-sea clams and fuel symbiont autotrophy, sulfide has a much stronger effect (sev- tubeworms (Frigaard and Dahl, 2009; Harada et al., 2009). Alter- enfold) on carbon fixation rates in S. velum symbionts (Scott and natively, it is possible that soxC and soxD are present in the Cavanaugh, 2007), suggesting that the symbiosis is adapted to use symbiont genome, but were not transcribed (above the level sulfide most efficiently. of detection) at the time of collection. In organisms lacking Our transcriptome data, showing the relative abundance of soxCD, the sulfur atom of the sulfane intermediate (SoxYZ- transcripts in the Dsr and Sqr/Fcc pathways (Figure 4), potentially S-SH, Figure 4) is transferred to other acceptor substrates indicates a dominant role for sulfide metabolism in the S. velum − − − (RSnH and HSn in Figure 4) and eventually into sulfur stor- symbiosis. However, transcript abundance may not be tightly cou- age globules, or enters other sulfur oxidation pathways (Sauve pled to protein abundance or enzyme activity (Taniguchi et al., et al., 2007; Frigaard and Dahl, 2009). The deposition of ele- 2010) and should be interpreted as a proxy for metabolic activ- mental sulfur globules has not yet been demonstrated for S. ity without supporting biochemical data. In other endosymbioses, velum. However, the potential for globule storage under vary- namely those involving deep-sea hydrothermal vent clams, diverse ing concentrations of thiosulfate or sulfide has not yet been sulfur pathways (Sox, Dsr, APS) have been shown to be consti- exhaustively explored. tutively expressed under variable environmental conditions (e.g., oxygen concentration; (Harada et al., 2009). However, compared IMPLICATIONS to gene expression in the relatively stable environment of the Our metatranscriptome data indicates the use of diverse pathways deep sea, expression of S. velum symbiont genes, and the relative for both sulfide and thiosulfate oxidation by S. velum symbionts reliance on diverse reduced sulfur sources, may be more stochastic under environmental conditions, consistent with prior experi- given the potential for diel and seasonal variation in the coastal ments showing that both substrates stimulate symbiont carbon environment. fixation (Cavanaugh, 1983; Scott and Cavanaugh, 2007). The uti- Metatranscriptomics can now provide a comprehensive mol- lization of multiple sulfur oxidation pathways is not unusual ecular framework for interpreting physiochemical measure- among thioautotrophic bacteria and is likely related to substrate ments of metabolism in uncultured symbionts. Such analy- availability (Ghosh and Dam, 2009). For the endosymbionts of ses can be easily integrated into studies exploring the links deep-sea clams, which share a complement of sulfur oxidation between sulfur availability, environmental conditions, and sym- genes similar to that in S. velum symbionts (Kuwahara et al., 2007; biont gene expression. Our study confirms that genes of sul- Newton et al., 2007), it has been argued that the ability to oxi- fur energy metabolism are a dominant component of the tran- dize both thiosulfate (Sox system) and sulfide (Dsr, Sqr/Fcc) is scriptionally active symbiont gene pool in S. velum. Future linked to the relative abundance of these compounds in the clam experiments will help determine the extent to which genes microhabitat (basaltic cracks and fissures in zones of diffuse-flow of thioautotrophy co-vary with other metabolic pathways in venting; Harada et al., 2009). In contrast, the endosymbiont of the transcriptome (Dmytrenko et al., in preparation) and with the tubeworm Riftia pachyptila putatively lacks the Sox pathway the biochemical transformations of sulfur mediated by this (Markert et al., 2007; Robidart et al., 2008), which may reflect an model symbiosis. adaptation to a greater reliance on sulfide in the zones surround- ing black smokers where this symbiosis occurs (Nelson and Fisher, ACKNOWLEDGMENTS 1995; Harada et al., 2009). We thank Rachel Barry for preparing cDNA samples for pyrose- For S. velum, the relative abundance of reduced sulfur species quencing. Generous support for this study came from the Gordon available for symbiont oxidation has not been directly character- and Betty Moore Foundation (EFD) and the National Science ized. The host bivalve lives in coastal muds, positioning itself at the Foundation (CMC, OCE-0453901).

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Stewart, F. J., and Cavanaugh, C. patterns of protein sequence con- Wilmes, P., and Bond, P. L. (2009). potential conflict of interest. M. (2006). Bacterial endosym- servation in natural microbial Microbial community proteomics: bioses in Solemya (Mollusca: communities. 12, R26. elucidating the catalysts and Received: 16 May 2011; paper pending Bivalvia): model systems for stud- Stewart, F. J., Ulloa, O., and DeLong, E. metabolic mechanisms that published: 03 June 2011; accepted: 09 June ies of symbiont-host adaptation. F.(2011b). Microbial metatranscrip- drive the Earth’s biogeochemi- 2011; published online: 20 June 2011. Antonie Van Leeuwenhoek 90, tomics in a permanent marine oxy- cal cycles. Curr. Opin. Microbiol. 12, Citation: Stewart FJ, Dmytrenko O, 343–360. gen minimum zone. Environ. Micro- 310–317. DeLong EF and Cavanaugh CM (2011) Stewart, F. J., Newton, I. L. G., biol. (in press). doi: 10.1111/j.1462- Woyke, T., Teeling, H., Ivanova, N. Metatranscriptomic analysis of sulfur and Cavanaugh, C. M. (2005). 2920.2010.02400x N., Huntemann, M., Richter, M., oxidation genes in the endosymbiont of Chemosynthetic endosymbioses: Taniguchi,Y., Choi, P.J., Li, G. W., Chen, Gloeckner, F. O., Boffelli, D., Ander- Solemya velum. Front. Microbio. 2:134. adaptations to oxic-anoxic H. Y., Babu, M., Hearn, J., Emili, son, I. J., Barry, K. W., Shapiro, doi: 10.3389/fmicb.2011.00134 interfaces. Trends Microbiol. 13, A., and Xie, X. S. (2010). Quan- H. J., Szeto, E., Kyrpides, N. C., This article was submitted to Frontiers in 439–448. tifying E. coli proteome and tran- Mussmann, M., Amann, R., Bergin, Microbial Physiology and Metabolism, a Stewart, F. J., Ottesen, E. A., and scriptome with single molecule sen- C., Ruehland, C., Rubin, E. M., specialty of Frontiers in Microbiology. DeLong, E. F. (2010). Development sitivity in single cells. Science 329, and Dubilier, N. (2006). Symbio- Copyright © 2011 Stewart, Dmytrenko, and quantitative analyses of a uni- 533–538. sis insights through metagenomic DeLong and Cavanaugh. This is an open- versal rRNA-subtraction protocol Wagner, M., Loy, A., Klein, M., analysis of a microbial consortium. access article subject to a non-exclusive for microbial metatranscriptomics. Lee, N., Ramsing, N. B., Stahl, Nature 443, 950–955. license between the authors and Frontiers ISME J. 4, 896–907. D. A., and Friedrich, M. W. Media SA, which permits use, distribu- Stewart, F. J., Sharma, A. K., Bryant, J. (2005). Functional marker genes Conflict of Interest Statement: This tion and reproduction in other forums, A., Eppley, J. M., and Delong, for identification of sulfate-reducing research was conducted in the absence provided the original authors and source E. F. (2011a). Community prokaryotes. Environ. Microbiol. 397, of any commercial or financial rela- are credited and other Frontiers condi- transcriptomics reveals universal 469–489. tionships that could be construed as a tions are complied with.

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