Genomic resolution of linkages in carbon, nitrogen, and sulfur cycling among widespread estuary sediment bacteria
© Baker et al.; licensee BioMed Central. 2015
Received: 19 December 2014
Accepted: 12 March 2015
Published: 13 April 2015
Estuaries are among the most productive habitats on the planet. Bacteria in estuary sediments control the turnover of organic carbon and the cycling of nitrogen and sulfur. These communities are complex and primarily made up of uncultured lineages, thus little is known about how ecological and metabolic processes are partitioned in sediments.
De novo assembly and binning resulted in the reconstruction of 82 bacterial genomes from different redox regimes of estuary sediments. These genomes belong to 23 bacterial groups, including uncultured candidate phyla (for example, KSB1, TA06, and KD3-62) and three newly described phyla (White Oak River (WOR)-1, WOR-2, and WOR-3). The uncultured phyla are generally most abundant in the sulfate-methane transition (SMTZ) and methane-rich zones, and genomic data predict that they mediate essential biogeochemical processes of the estuarine environment, including organic carbon degradation and fermentation. Among the most abundant organisms in the sulfate-rich layer are novel Gammaproteobacteria that have genes for the oxidation of sulfur and the reduction of nitrate and nitrite. Interestingly, the terminal steps of denitrification (NO3 to N2O and then N2O to N2) are present in distinct bacterial populations.
This dataset extends our knowledge of the metabolic potential of several uncultured phyla. Within the sediments, there is redundancy in the genomic potential in different lineages, often distinct phyla, for essential biogeochemical processes. We were able to chart the flow of carbon and nutrients through the multiple geochemical layers of bacterial processing and reveal potential ecological interactions within the communities.
Estuaries are dynamic environments where nutrient-rich river water mixes with shallow coastal and nutrient-rich deep ocean water. They are among the most productive environments on the planet . Processes within estuaries also mediate the transfer of carbon from land to sea, release a considerable amount of CO2 to the atmosphere , and sequester carbon in sediments . As this organic carbon is degraded, and oxygen is consumed, a variety of favorable anaerobic respiratory processes are mediated by sediment microorganisms that contribute to sulfur, nitrogen, and iron transformations. Thus, shallow estuary sediments and their microbial communities are a global hotspot for biogeochemical cycling.
Our ability to partition these processes and the underlying metabolic pathways among specific microbial groups have remained limited by the complexity and abundance of uncultured groups present in sediment communities. Metagenomic studies have characterized the genetic potential of marine sediment microbial communities (for example, see Biddle et al. ). However, because sequencing reads are often not assigned to specific taxa, such approaches typically do not link individual community members to metabolic pathways . Further, culture-independent genomic reconstructions of estuary sediment microbial communities are lacking; thus little is known about uncultured communities in this complex microbial ecosystem.
To better understand the metabolic capabilities of uncultured bacteria, we obtained 262 Gbp of genomic sequence from sediment profiles from the White Oak River (WOR) estuary, North Carolina. Our sequencing effort focused on three key redox layers; the sulfate-rich, sulfate-methane transition (SMTZ), and methane-rich zones. This genomic dataset was assembled and binned to obtain partial and near-complete genomes and to reconstruct metabolic pathways of numerous community members. Reconstructing the metabolic capabilities of numerous bacterial groups in the sediment communities enabled us to identify their roles in carbon, iron, nitrogen, and sulfur cycling. This dataset provides a genomic road map of how biogeochemical processes are hypothesized to be partitioned in aquatic sediment bacterial communities.
Results and discussion
Genomic reconstruction and identification
Several of the genomic bins belong to groups that are commonly identified in rRNA gene surveys of marine and estuarine sediments, including Betaproteobacteria, Gammaproteobacteria, Deltaproteobacteria, Chloroflexi, Planctomycetes, Bacteroidetes, Gemmatimonadetes, Nitrospira, Chlamydiae, and Spirochetes [9,10]. Many of the 16S rRNA genes are most similar to sequences from other estuaries (for example, Pearl River  and Yangtze estuary sediments) and marine sediments (for example, South China Sea ) (Figure 2). Genomes recovered from candidate phyla that are commonly identified in a variety of anoxic environments and marine sediments comprise OD1 (Parcubacteria), WS3 (Latescibacteria), TA06, Zixibacteria, and BRC1 (Additional file 1: Table S4). Two of the genomic bins belong to previously unnamed phylum-level lineages of 16S rRNA genes, mostly recovered from marine sediments, referred to as ‘WOR-1 and WOR-3’, for White Oak River groups. These bins (SMTZ1-54-3, DG-78, and DG-78-2) are deeply divergent from all currently recognized phyla based on 16S rRNA gene phylogeny (Figure 2) and concatenated ribosomal proteins (bins SMTZ-42, -60, SMTZ1-77, -54-3, and DG78-2, Figure 1). Based on ribosomal proteins, an additional phylogenetically distinct group (bins SMTZ-29 and SMTZ-72), which we will refer to here at ‘WOR-2’ was identified.
Genomic abundance of community members in the sediment profile
To quantify the genomic abundance of community members in each zone, all reads were mapped to all genes for ribosomal protein S3 in the genome assemblies (Additional file 1: Figure S2). The assembly from shallow, sulfate-rich sediments was dominated by Beta-, Gamma-, and Deltaproteobacteria (including Myxococcales), Bacteroidetes, and Nitrospira. The SMTZ and the deeper methane-rich sediments were dominated by Archaea and Chloroflexi (Additional file 1: Figure S2), consistent with previous qPCR and rRNA slot blot results showing that bacterial dominance in surficial White Oak River estuarine sediments is considerably reduced and even reversed downcore . The candidate bacterial phyla WS3, OD1, TA06, Zixibacteria, WOR-1, WOR-2, and WOR-3 were sufficiently abundant for genome reconstruction only in the deeper sediment layers.
Organic carbon degradation and fermentation
Proteins account for a large proportion of bioavailable carbon and nitrogen for sediment communities . In multiple phyla, these resources are accessed via extracellular peptidases (Additional file 2: Table S5). The greatest numbers of peptidases were identified in the candidate phyla KSB1, WOR-1, WOR-2, and WOR-3, and in the Bacteroidetes and Gemmatimonadetes, suggesting that these community members are substantially involved in protein degradation along with sedimentary benthic Archaea .
Several of the Chloroflexi genomes (SG8-19, DG-18, SMTZ-63, and SMTZ-84) contain the β-oxidation pathway to generate acetyl-CoA from fatty acids and organic acids [8,19]. This capability is also present in genomes from both of the Myxococcales (SG8-38 and SG8-38-1), Gemmatimonadetes (SG8-23 and SG8-28), several Gammaproteobacteria (SG8-11, SG8-30, SG8-31, SG8-50, SM1-46, and SM23-46) and Deltaproteobacteria (SG8-13 and SM23-61), and the shallow sediment-dwelling Betaproteobacteria. Among the candidate phyla that contain the complete β-oxidation pathway are the BRC-1, and one of the KSB1 (SMTZ-57) genomic bins. A majority of genomes that have the β-oxidation pathway (60%) were primarily found in the shallow samples.
Several of the bacteria possess genes for Ni,Fe-hydrogenases, which can be involved in H2 production or consumption. These genes are ubiquitous in Gemmatiomonas, Myxococcales, Delta-, and Gammaproteobacteria (SG-11, SG-13, SG-15, SG-30, and SG-31) and are likely used for consumption of H2 by respiratory processes (for example, sulfate reduction and denitrification). The Nitrospira bin (SG8-35-4, related to Thermodesulfovibrio spp., Figure 1) contains genes for Ni,Fe-hydrogenases that are likely to participate in both sulfate reduction with hydrogen as the electron donor and the fermentative production of H2, which have been demonstrated in Thermodesulfovibrio spp. . Several bacterial groups that are capable of organic carbon degradation and fermentation have Ni,Fe-hydrogenase genes, including Planctomycetes, Spirochetes, Chloroflexi, WOR-2, WOR-3, and WOR-1 (Figure 4). The extensive distribution of these hydrogenases among both fermenting and respiring bacteria indicates that H2 is a highly dynamic electron carrier produced and consumed by a wide range of sediment microbes, as it is in other anoxic environments [22,23]. No genes for Fe,Fe-hydrogenases were identified, which are thought to primarily produce H2 .
Dissimilatory sulfur and nitrogen cycling
Aquatic sediments are characterized by redox gradients, as oxidized compounds are gradually reduced by respiration. To determine the respiratory repertoire of the bacterial community members, the estuary genomic bins were surveyed for key genes of respiration pathways. Several of the genomic bins contain dissimilatory sulfite reductase (dsr) genes, indicative of microbial sulfate and sulfite reduction. To account for the possibility that these genes belong to a phylogenetically distinct group of genes (rdsr) that mediate the reverse reaction , we generated a phylogenetic tree of all the dsr-like genes recovered (Additional file 1: Figure S3). The Deltaproteobacteria, which constituted the most abundantly detected microorganisms in the sulfate-rich zone (Additional file 1: Figure S2), have reducing-type dsr genes and complete sulfate reduction pathways (with the exception of the two Myxococcales bins). The bin SG8-35-4, a sister lineage to Thermodesulfovibrio spp. within the Nitrospira phylum, is capable of sulfate reduction based on the presence of dsr genes.
A complete sulfate reduction pathway was identified in the Gemmatiomonas-like bin SG8-17. The dsrAB genes from this bin fall within a phylogenetically deeply branched unknown clade (Additional file 1: Figure S3). This clade (designated DSR-J ) includes dsrAB sequences from an intertidal sand flat, Hydrate Ridge, deep-sea, and estuary sediments, suggesting these sulfate reducers are widespread in coastal and marine sediments. Genes of this clade were hypothesized to have been horizontally transferred . No dsr genes were identified in any of the other Gemmatiomonadetes-like bins. Since all of the sequences for this clade had previously been recovered from large-insert (fosmid) clones that lacked 16S rRNA genes, their taxonomic affiliation had been uncertain. In this dataset the dsrAB genes are located on a 9.6 kb contig that is confidently binned, with both the tetranucleotide and coverage signatures, consistent with assignment to SG8-17. This genomic bin contains additional genes for sulfate reduction on other contigs, including aprAB, SAT, and dsrC. Thus, this member of the Gemmatimonadetes appears to be capable of sulfate reduction.
Up to 95% of the sulfide and thiosulfate generated by sulfate reduction is re-oxidized to sulfate in marine sediments . Interestingly, the genomes of the most abundant Gammaproteobacteria (SG8-11, SG8-15, SG8-45, SG8-47 SG8-50, SMTZ1-46, and SMTZ-46) include genes for sulfur oxidation (rdsr, apr, and SAT) and pathways for thiosulfate oxidation (soxABDZY genes). Four bins contain sulfide quinone oxidoreductase genes (sqr) required for the oxidation of sulfide (SG8-15, SG8-30, SG8-47, and SG8-50). These sqr-containing Gammaproteobacteria are phylogenetically distinct and are related to Thioalkalivibrio sp. (Figure 1); unless relevant genomes have been missed, sulfide oxidation in the WOR sediments appears to be mediated by this group. All of these Gammaproteobacteria genomes contain genes for nitrate reduction.
Dissimilatory nitrate reduction to ammonia (DNRA) has been shown to co-occur with denitrification in estuary sediments . To look for the presence of DNRA genes in the genomes, we constructed a phylogenetic tree of all the formate-dependent nitrite reductase (nrfA) genes identified in genomic bins (Additional file 1: Figure S4). Most (72%) of these do not fall into a clade that has been described as true nrfA genes [34,35]. Two Deltaproteobacteria bins (SG8-35 and SG8-35-2) were confidently identified as being involved in DNRA.
While the microbial cycling of iron in marine sediments has been commonly documented by geochemical approaches , well-documented biochemical pathways for iron oxidation and reduction now enable environmental genetic studies of these processes as well. For example, periplasmic and outer-membrane-anchored c-type cytochromes and a beta-barrel protein within the metal reduction (Mtr) respiratory pathway are essential for respiratory electron transport across the outer cell membrane to iron minerals in Shewanella oneidensis [37,38]. Homologues of these enzymes catalyze lithotrophic iron oxidation in Sideroxydans lithotrophicus  and phototrophic iron oxidation in Rhodopseudomonas palustris . Genes with homology to mtr genes are present in seven different WOR genomic bins. Betaproteobacterial genomes reconstructed from the sulfate-rich zone (SG8-39, SG8-40, and SG8-41) have mtrABC genes with sequence identity (40% to 50%, 23% to 35%, and 26% to 28% at the protein level, respectively) to those found in Shewanella and Geobacter spp., suggesting that they are capable of iron reduction. Gemmatimonadetes bin SG8-38-2 has a putative mtrABC gene cluster and may be involved in iron cycling as well. Consistent with Zixibacteria genomes obtained from groundwater, bin SM-73-2 contains mtrAB but lacks the genes for extracellular cytochromes implicated in iron reduction . The Gammaproteobacteria bin SG8-47 contains both the genes homologous to mtrABC and a cytoplasmic membrane-associated c-type cytochrome (cymA) required for iron reduction . The closely related bacteria SG8-11 and SG8-30 have all these genes except mtrC and cymA, respectively. Bacteria potentially capable of iron reduction have multiple multi-heme cytochromes ; Gammaproteobacteria bins SG8-11 and SG8-30 also each have genes encoding six unique types of these cytochromes.
The highly resolved genomic reconstruction of estuary sediment microbial populations revealed potential physiological pathways of individual community members, including several recently defined (for example, KD3-62, TA06, Zixibacteria, and BRC1) and three newly described (WOR-1, WOR-2, and WOR-3) uncultured candidate phyla. The 12 genome bins belonging to WOR-1, WOR-2, WOR-3, KD3-62, and TA06 are the first to be constructed from these phyla. Based on their genome sequences, several of these groups appear to be capable of hydrolysis and fermentation of a variety of organic compounds, expanding the range of bacterial phyla known to hydrolyze and ferment biopolymers (sugars and proteins) to low molecular weight substrates. An average of 14 carbohydrate hydrolases was found per genome, with WOR-1 and WOR-2 having the most with 20 and 39, respectively. Planctomycetes and Bacteroides are also among the most versatile carbohydrate degrading bacteria in the White Oak River sediments with an average of 30 and 80 hydrolytic genes per genome, respectively.
This study identified potential new bacterial capabilities in sulfur cycling. One uncultured Gemmatimonadetes bacterium was linked to a previously taxonomically unassigned dsr gene clade, suggesting that this group is capable of sulfate reduction, a process that is commonly catalyzed by Deltaproteobacteria. Sequences for this group have been recovered from sediments throughout the world, suggesting a cosmopolitan Gemmatimonadetes lineage of sulfate-reducing bacteria. The oxidation of sulfide and thiosulfate is essential to sulfur cycling in marine and coastal sediments ; the ongoing microbial census of these processes is uncovering new key populations, such as uncultured Gammaproteobacteria mediating sulfur oxidation in coastal sediments . Several of the most abundant Gammaproteobacteria in the sulfate-rich zone described here have the genetic potential for coupling anaerobic sulfur or hydrogen oxidation to reduction of nitrate to nitrous oxide, as documented in Beggiatoa spp. in marine sediments as well . Interestingly, the genomic potential for the next denitrification step (N2O to N2) in the White Oak River estuary sediments is found in different organisms, the Gemmatiomadetes and Myxococcales. This apparent splitting of the denitrification pathway over multiple taxa, and concurrent leakage of the intermediate N2O, provide an explanation as to why estuaries function as a significant source of N2O to the atmosphere .
Placing potential metabolic capabilities of individual populations within the framework of other community members provided a wiring diagram of the potential biogeochemical interactions at a system level. This study demonstrates how key pathways of carbon degradation and sulfur, nitrogen, and iron cycling may be distributed over a previously unexplored range of bacterial phyla within the estuarine sediment community; the genomic analysis provides a new perspective on the functioning of these pathways in nature, characterized by a high degree of functional redundancy among different lineages and by metabolic plasticity within specific organisms . The comprehensive genomic reconstruction of these sediment communities provides a wide spectrum of specific links between metabolic potential and diversity and reveals potential ecological interactions within the communities; it begins to chart the possible flow of carbon and nutrients through the multiple layers of microbial processing, assimilation, and remineralization in the estuarine environment.
Sample collection and processing
Six 1-m plunger cores were collected from approximately 1.5-m water depth in three mid-estuary locations (two cores per site) of the White Oak River, North Carolina in October 2010 (site 1 at 34°44.592 N, 77°07.435 W; site 2 at 34°44.482 N, 77°07.404 W, and site 3 at 34°44.141 N, 77°07298 W). Cores were stored at 4°C overnight and processed 24 h after sampling, as detailed in Lazar et al. . Each core was sectioned into 2-cm intervals. From each site, one core was subsampled for geochemical analyses and the others were subsampled for DNA extractions. DNA was extracted using the UltraClean Mega Soil DNA Isolation Kit (MoBio, Carlsbad, CA, USA), using 6 g of sediment, and stored at −80°C until use. Sulfate, sulfide, and methane profiles of these cores at the three sites can be seen in Lazar et al. . The SMTZ is located at around 26, 24, and 16 cm in sites 1, 2, and 3 (respectively); therefore, the samples above that depth represent the sulfate-rich zones and those below the methane-rich zones.
Genomic assembly, binning, and annotation
Illumina (HiSeq 2000 PE100) shotgun genomic reads were screened against Illumina artifacts (adapters, DNA spike-ins) (Illumina Inc., San Diego, CA, USA) with a sliding window kmer size of 28 and a step size of 1. Reads with 3 or more N’s or with average quality score of less than Q20 and a length <50 bps were removed. Screened reads were trimmed from both ends using a minimum quality cutoff of 5 using Sickle (https, //github.com/najoshi/sickle). Trimmed, screened, paired-end Illumina reads were assembled using IDBA-UD  with the following parameters (--pre_correction --mink 55 --maxk 95 --step 10 --seed_kmer 55). To maximize assembly, reads from different sites were co-assembled.
The shallow assembly was a combination of high-quality reads (474,179,948 with an average read length 148 bp) from sites 2 (8 to 12 cm) and 3 (8 to 10 cm). The SMTZ assembly was generated from a combination of high-quality reads (698,574,240, average read length 143 bp and average insert 274 bp) from sites 2 (30 to 32 cm) and 3 (24 to 28 cm). The deep assembly was generated from high-quality reads (378,027,948, average read length 124 bp and average insert 284 bp) of site 1 (52 to 54 cm). Since we were not able to co-assemble all three of the samples from the SMTZ due to computational limits, an additional assembly was generated from the third sample (site 1, 26 to 30 cm) from high-quality 345,710,832 reads (average length of 129 bp and average insert 281 bp). The contigs from this sample were co-binned with the assembly of the other two samples (sites 2 and 3, described above). This resulted in some closely related but unique bins, for example, the Gammaproteobacteria bins SMTZ1-46 (from site 1) and SMTZ-46 (from the site 2 and 3 assembly). Contigs with genes of particular interest were checked for chimeras by looking for dips in coverage within read mappings.
Initial binning of the assembled fragments was done using tetra-nucleotide frequencies signatures using 5 kb fragments of the contigs, as detailed in Dick et al. . ESOM maps were manually delineated and curated based on clusters within the map (as shown in Additional file 1: Figure S1). This binning was enhanced by incorporating coverage signatures for all of the assembled contigs into the ESOM maps [49,50]. Coverage was determined by recruiting reads (from each individual library/sample) to scaffolds by BLASTN (bitscore >75) and then normalized to the number of reads from each library, determining which genomic bin each of the 5 kb sub-portions of the contigs were assigned then assessed the accuracy of the binning. Contigs larger than 15 kb were assigned to the bin where the majority of the 5 kb sub-portions were assigned. The completeness of the genomes within bins was then estimated by counting single-copy genes using CheckM . Some of these bins were then shown to contain multiple (2-5) closely related genomes based on the presence of multiple copies of single-copy genes. Those bins were further separated by plotting differential coverage of scaffolds between two libraries. Distinct clusters of scaffolds on the coverage plots were manually delineated into new bins. Binning was also manually curated based on GC content, top blast hits, and mate-pairings.
Genes were called and putative function was assigned using the JGI IMG/MER system . The functions of predicted genes (including all those discussed in detail here) were manually curated and revised by comparison of homology to a variety of databases including KEGG, pfam, NCBI, and COG. The CAZy database was used to identify carbohydrate active genes ; a subset was selected which has been shown to be involved in specific carbohydrate degradation pathways (Additional file 3: Table S6) [22,53].
The concatenated ribosomal protein tree was generated using syntenic genes that have been shown to have undergone limited lateral gene transfer (rpL2, 3, 4, 5, 6, 14, 15, 16, 18, 22, 24 and rpS3, 8, 10, 17, 19) . The reference data sets were derived from the Phylosift database , with additional sets from the Joint Genomic Institute IMG database as detailed in Castelle et al. . Scaffolds containing <50% of these 16 genes were not included in the analyses. Only two bins (DG-61 and DG-78) remained below this cutoff. We searched NCBI to include additional closely related reference sequences. Amino acid alignments of the individual genes were generated using MUSCLE  and manually curated. The curated alignments were then concatenated for phylogenetic analyses. The ribosomal protein tree included 182 taxa and 2,411 unambiguously aligned positions. The phylogeny shown in all the figures were generated using maximum likelihood using Phyml . Bootstrap values were generated from 100 replicates of UPGMA tree building method and Jukes-Cantor distance modeling.
Availability of supporting data
The full metagenomic assemblies presented in this study are available in IMG with the following IMG Taxon IDs. They are Taxon ID: 3300001855 (sulfate-rich assembly, ‘SG’ genomes), 3300002052 (SMTZ site 1 assembly, ‘SM1’ genomes), 3300001753 (SMTZ sites 2 and 3 assembly, ‘SM23’ genomes), and 3300001854 (methane-rich assembly, ‘DG’ genomes). The genomic bins supporting the results of this article are being made available in NCBI Genbank under the BioProjectID PRJNA270657.
This article is funded by grants from the Gordon and Betty Moore Foundation (GBMF2609) to GJD, European Research Council ‘DARCLIFE’ grant 247153 (granted to Kai-Uwe Hinrichs, University of Bremen, who is gratefully acknowledged for funding for Cassandre Lazar), and by a University of Michigan Rackham Pre-doctoral fellowship to Brett Baker. The sequencing was funded by the U.S. Department of Energy Joint Genome Institute is supported by the Office of Science of the U.S. Department of Energy under Contract No. DE-AC02-05CH11231 to BJB. We thank Drs. Karen G. Lloyd and Cody Sheik for helpful suggestions and comments on an earlier draft of this manuscript.
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