Metagenomics and Metatranscriptomics of the Leaf-And Root-Associated Microbiomes of Zostera Marina and Zostera Japonica

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Metagenomics and metatranscriptomics of the leaf- and root-associated microbiomes of Zostera marina and Zostera japonica

by John Michael Adrian Wojahn

A THESIS

submitted to

Oregon State University

Honors College

in partial fulfillment of the requirements for the degree of

Honors Baccalaureate of Science in Microbiology and Biology (Honors BS)

Presented May 10, 2016 Commencement June 2016

AN ABSTRACT OF THE THESIS OF

John M. A. Wojahn for the degree of Honors Baccalaureate of Science in Microbiology and Biology presented on May 10, 2016. Title: Metagenomics and Metatranscriptomics of the leaf- and root-associated microbiomes of Zostera marina and Zostera japonica .

Abstract approved:

______Byron C. Crump

A great deal of research has been focused on the microbiomes of terrestrial angiosperms (flowering plants), but much less research has been performed on the microbiomes of aquatic angiosperms (Turner et al. 2013). Eelgrass beds are extremely productive ecosystems that provide habitat for many marine organisms, such as fish, shelfish, crabs, and algae (Smith et al. 1988). Eelgrass beds contribute to storm surge damping (Spalding et al. 2009), nutrient cycling (Smith et al. 1988), and water clarification (Orth et al. 2006). We examined the metagenomics and metatranscriptomics of the leaf- and root- associated microbiomes of Zostera marina and Zostera japonica. In our study, the phylogenetic composition of plant-associated bacterial communities was not significantly different between plant species for leaf communities (ANOSIM P<0.199) and for root communities (ANOSIM P<0.091). However, leaf-, root-, and water column associated bacterial communities were significantly different from one another (ANOSIM, P<0.001). We found taxa present on leaves that are capable of metabolizing methanol and of producing agarases that cause disease and die-offs in populations of competitive red seaweed, and of producing indoleacetate, a plant hormone. Members of genus Granulosicoccus were found to be particularly abundant in our leaf samples. We also found taxa present on the roots that are capable of metabolizing sulfur compounds, of fixing nitrogen, and of degrading methanol.

Key Words: seagrass, Netarts, Oregon, estuary, ocean, microbiology, molecular biology

Corresponding e-mail address: [email protected]

5 Metagenomics and metatranscriptomics of the leaf- and root-associated microbiomes of Zostera marina and Zostera japonica

by John Michael Adrian Wojahn

A THESIS

submitted to

Oregon State University

Honors College

in partial fulfillment of the requirements for the degree of

Honors Baccalaureate of Science in Microbiology and Biology (Honors BS)

Presented May 10, 2016 Commencement June 2016

6 Honors Baccalaureate of Science in Microbiology and Biology project of John M. A. Wojahn presented on 10 May 2016.

APPROVED:

Byron C Crump, Mentor, representing the College of Earth, Ocean, and Atmospheric Science

Fiona Tomas Nash, Committee Member, representing the Department of Fisheries and Wildlife

Ryan Mueller, Committee Member, representing the Department of Microbiology

Toni Doolen, Dean, Oregon State University Honors College

I understand that my project will become part of the permanent collection of Oregon State University, Honors College. My signature below authorizes release of my project to any reader upon request.

John M. A. Wojahn, Author

Introduction A great deal of research has been focused on the microbiomes of terrestrial angiosperms (flowering plants), but much less research has been performed on the microbiomes (organism-associated microbes) of aquatic angiosperms (Turner et al. 2013). Bacteria associated with terrestrial plant leaves (phyllosphere microbiome) and roots (rhizosphere microbiome) are known to have direct impacts on the health of plants (Bakker et al. 2012). Positive effects of these microbiomes include outcompeting pathogenic soil microbes, modulating plant immunity by priming their defenses for an imminent infection, rendering nitrogen from the air usable to plants (nitrogen fixation) (Galloway et al. 2008), and neutralizing harmful products (e.g. methanol) exuded from leaves (Turner et al. 2013; Abanda-Nkpwatt et al 2006). Rhizosphere microbiomes tend to be very diverse and mainly derived from soil microbiota (Triplett et al. 2002). In contrast, phyllosphere microbiomes are much less diverse and are very different than microbes in the air (Sainis, 2012). The structure of rhizosphere microbiomes is primarily determined by the combination of chemicals exuded by the plant roots (Haichar et al. 2008), whereas the structure of phyllosphere microbiomes is determined by abiotic environmental factors, such as precipitation and light exposure (Turner et al. 2013). We believe a similar contrast likely exists for the microbiomes of submersed aquatic plants, but the factors controlling microbiome community composition and structure may be very different than those of terrestrial plants. Aquatic plant leaves are often submerged in water, and their roots are anchored in saturated sediments that are often anoxic at rhizosphere depths. Seagrasses, marine flowering plants, form the base of extense and productive ecosystems that provide habitat for many marine organisms, such as fish, shelfish, crabs, and algae (Smith et al. 1988). Eelgrass beds contribute to storm surge damping (Spalding et al. 2009), nutrient cycling (Smith et al. 1988), and water clarification (Orth et al. 2006). Also, decomposition of eelgrass detritus fuels a variety of food webs, both local and distal to the beds (Ralph et al. 2002). During the 1920s and 1930s, a large die-off of eelgrass caused by the fungus Labyrinthula macrocystis occurred across much of North America and Europe, leading to the destruction of many eelgrass beds (Ralph et al. 2002). Many estuaries never fully recovered from this die-off. Eelgrass beds have also been lost to human activity, such as chemical contamination, eutrophication, and physical disturbance (Ralph et al. 2002), and are now considered 'canaries in the coal mine' for climate change and associated global and regional changes, such as ocean acidification, warming, or sea-level rise (Boudouresque et al. 2009, Carr et al. 2012). Seagrass beds on the pacific coast of the US were once dominated by Zostera marina, which is a species of eelgrass native to Northern Eurasia and North America. However, since 1957, an invasive species of eelgrass from Japan, Zostera japonica, established itself along the West coast of North America (Lovvorn et al. 1994) (See Figure 1 for a photo of a typical specimens of Z. marina and Z. japonica). Z. japonica has smaller, thinner leaves than Z. marina (<20 cm long and 2 mm wide vs. 50-120 cm long and 12 mm wide (Lovvorn et al. 1994)). Although both species can co-occur, they typically exhibit different reproductive strategies and habitat use. Z. japonica usually

8 colonizes mid to low intertidal mudflats that have been stripped of all vegetation by winter storms, and in autumn, before the mother plants are killed by winter storms, they release large quantities of seeds, some of which will survive to colonize the newly denuded mud the following year (Harrison, 1982). In contrast, Z. marina generally colonizes low to subtidal areas of the mudflats and very rarely flowers, exhibiting both sexual and asexual (vegetative growth of shoots) reproduction, usually maintaining perennial populations (Davidson et al. 1998). Eelgrass-associated microbiomes may serve important roles in the ecosystem their hosts inhabit, including facilitating the growth and survival of their hosts through many of the same mechanisms as microbiomes of land plants (Crump et al. 2008). They may protect the plants from methanol, metabolize sulfides, fix nitrogen, and produce plant hormones. However, they may sometimes also act as phytopathogens (Short et al. 1987). These eelgrass-associated microbes could also benefit from their relationship with their hosts. For example, eelgrass, being angiosperms, may exude methanol and ethanol, which the microbes around them could use as food. The plants also provide a substrate for microbial attachment and stabilize sediments, preventing mixing and loss of sediment habitat. Eelgrass also Figure 1. Z. marina is an aquatic angiosperm that presents with 30 cm enrich the water mature blades, 5 cm hair-like roots, and a segmented rhizome. Z. japonica is smaller than Z. marina and is native to the islands of Japan. and sediment around it with dissolved oxygen. Lastly, the microbes associated with the eelgrass may provide energy and substrate for higher trophic levels, since 99.8% of their mass is lost each growing season (Tornblom 1999). Koch et al. (2001) determined that sulfide is toxic to the eelgrass Thalassia testidium under high salinity and high temperature, and Goodman et al. (1995) found that the phytotoxicity of sulfide was highest under low light conditions. This agrees with Baden et al. (2003), who found that seagrass loss was highest in turbid waters that carry higher concentrations of sulfide. Jensen et al. (2007) found that microbial communities associated with roots of a tropical seagrass were dominated by the classes Epsilonproteobacteria and Gammaproteobacteria, both of which include many organisms capable of oxidizing sulfide. The dominance of these classes suggests rapid metabolism of sulfide, which would benefit plants by removing this toxic molecule.

9 Jensen et al. (2007) also found members of the phylum Actinobacteria attached to the roots, which include many endophytic diazotrophs (nitrogen fixing organisms). However, the mere presence of taxa capable of sulfide metabolism and nitrogen fixation associated with eelgrass does not corresponding in situ activities. In our study we aim to resolve this discrepancy by analyzing the metatranscriptomes of eelgrass leaves and roots, to determine if genes for sulfide metabolism are being expressed, and by whom. Crump et al. (2008) found that the Alphaproteobacteria Roseobacter of were the dominant organisms on the leaves of Z. marina, Potamogeton perfoliatus, and Stuckenia pectinata. This is significant because members of Roseobacter are often capable of breaking down methanol, a phytotoxic compound secreted by eelgrass leaves. If they also dominate the samples in our study, their dominance may indicate that they are successfully degrading methanol. Once again, our analysis of the eelgrass metatranscriptome will complement this idea, by revealing if any methanol metabolizing genes are being expressed. Ethanol, though not directly toxic to the plant, may be indirectly toxic: according to Widdel (1988), ethanol released from plant roots during anaerobic fermentation increases the concentration of sulfide-producing microbes in the surrounding soil. This happens because the ethanol-consumers are capable of metabolizing ethanol and will use it if it is available. This may open up a niche for non- sulfide-producing ethanol-metabolizing plant associated microbes to exist on the surface of the roots, metabolizing the ethanol before it can reach the soil and ultimately the sulfide producers. This function of eelgrass-associated microbes may be revealed if our study finds epiphytic taxa capable of ethanol metabolism, and the expression of ethanol metabolizing genes.

Materials and Methods Samples were collected from tidal mudflats in Netarts Bay near Tillamook, Oregon on 28 July 2014 and on 8 September 2014 from two pools chosen for their mixture of Z. marina and Z. japonica (Figure 2). Samples were taken in pairs, so that there were two samples of the leaves and roots of each species from each pool, totaling 16 samples. Whole plants were removed from the sediment by sliding gloved fingers under the rhizome, gently moving fingers to loosen mud from the delicate roots, and lifting the plant onto a rinsed cooler top. To remove mud, samples were then gently sprayed with sterile seawater collected from near the mouth of the estuary (salinity 34 PSU, 12.7°C, sterilized by passage through Sterivex-GP 0.22 micron filters). Roots Figure 2. Approximate location of sampling site within and leaves were separated from Netarts Bay (USGS) rhizomes using gloved fingers, and placed in separate 50 ml Falcon tubes. Samples were then rinsed 3 to 5 times by filling the Falcon tube with enough sterile seawater to cover the sample and gently inverting. Samples were stablized by adding

10 enough RNAlater to completely cover the samples. Once stablized, the samples were promptly placed on dry ice and stored at -20°C until extraction. For DNA extractions, thawed root and leaf samples were transferred to new 50ml Falcon tubes, and soaked in sterile ultrapure water at 4 degrees C for 20 minutes to remove RNAlater. The samples were then cut into small pieces with sterile razor blades and cutting boards, and DNA was extracted with the PowerBiofilm DNA Isolation Kits (MO BIO Laboratories, Inc.) following manufacturers instructions for the vortex adapter option. DNA from water column microbes collected on Sterivex filters was isolated using a phenol:chloroform extraction. DNA was extracted using methods adapted from Zhou et al. (1996) and Crump et al. (2003). Bacterial community composition was determined with PCR amplicon sequencing of 16S rRNA genes following Kozich et al. (2013), with dual-barcoded versions of the PCR primers from Caporaso et al. (2012). The V4 hypervariable region of 16S rRNA genes were PCR amplified with 250 nM primers (final concentration) and HotMasterMix (5 Prime) under the following conditions (94°C for 3 min; 30 cycles of 94°C for 45 sec, 50°C for 60 sec, 72°C for 90 sec; 72°C for 10 min). Three technical PCR replicates were performed for each sample. Each of these amplicons were pooled, and quantified using Picogreen. Amplicons were then pooled at equimolar concentrations, cleaned using a MoBio Ultraclean PCR Clean-Up Kit, quantified using Picogreen, and sequenced at the Oregon State University Center for Genome Research and Biocomputing (CGRB) with Illumina MiSeq 2x150 bp paired-end reads. Metagenomes sequences were collected for two leaf samples (EM28: marina leaf, and EM34: japonica leaf) at CGRB on the same Illumina MiSeq run as the amplicon sequences (1/3 of sequences for amplicons, 2/3 for metagenomes). Metatranscriptomes sequences were collected for four samples (EM1: marina leaf, EM4: japonica leaf, EM5: marina root, and EM8: japonica root). For RNA extraction, thawed leaf and root samples were sliced into small pieces, and placed in new sterile 50ml Falcon tubes. The RNAlater was then pushed through a Sterivex-GP 0.22-micron filter to capture dislodged cells. The filter material was then removed from the capsule, sliced into pieces, and placed in 50ml Falcon tubes along with the plant pieces. RNA was isolated with the MoBio PowerSoil Total RNA Isolation Kit following manufacturers instructions. DNA was removed from RNA extractions using the Ambion TURBO DNA-free kit following the manufacturer’s instructions. The rRNA was removed from total RNA with sequential use of two Ribo-Zero Gold kits (Bacterial rRNA, Plant rRNA) according to the methods presented in Doyle et al. 2011. The depleted RNA was run on a Wafergen Apollo 324 robot using the PrepX RNA-Seq for Illumina library prep kit and sequenced with HiSeq 3000 at the CGRB. Amplicon sequences were paired using make.contigs from the MOTHUR package (v.1.32.1; Schloss et al. 2014), converted to QIIME format with split.groups from MOTHUR and add_qiime_labels.py from QIIME (Caporaso et al. 2010). Sequences were quality filtered with an expected error rate of 0.5, dereplicated (derep_fulllength), and abundance sorted (sortbysize) using USEARCH (v.7.0.1001_i86linux64; Edgar 2013). Singleton sequences were removed in the latter step to prevent them from seeding clusters when clustering OTUs. Reads were then clustered (cluster_otus) at 97% similarity. A de novo chimera check is inherent in the cluster_otus algorithm and chimeric sequences were removed during OTU clustering. Reference-based chimera

11 filtering was performed (uchime_ref) with the Gold Database (http://www.genomesonline.org/) as reference. Reads (including singletons) were subsequently mapped back to OTUs using UPARSE (usearch_global) and an OTU table created. Taxonomy of the representative sequences was assigned in QIIME (assign_taxonomy.py) using the RDP classifier trained to the SILVA database (v.111 database clustered to 97% OTUs). Metagenome and metatranscriptome sequences were each co-assembled with Megahit (Li et al. 2015) and CDS sequences were identified and annotated through the Microbial Genome Annotation Pipeline of the Integrated Microbial Genome (IMG) online system (Huntemann et al. 2015). Raw paired-end sequences from each sample were quality controlled (trimfq command in seqtk v.1.0-r72-dirty) and mapped to CDS sequences with Bowtie2. Gene and transcript abundances for each CDS were counted as the sum of paired-end alignments (both sequences match CDS) and unpaired alignments (only one sequence matches CDS). Transcripts assigned to eukaryotes and viruses were excluded from analysis. Transcripts assigned to Cyanobacteria and Fusobacteria included a large proportion of photosynthesis gene transcripts, and so were also excluded because it was unclear whether these were mis-assigned chloroplast transcripts. The abundances of gene and transcripts sequences that mapped to KEGG-annotated CDS sequences were normalized as transcripts per million (TPM) and genes per million (GPM) following Wagner (2012) to account for variations in sequence length and template length, and were explored in MEGAN V.5 (Huson et al. 2011).

Results The community structure of plant-associated bacterial communities was not significantly different between plant species for leaf communities (ANOSIM P<0.199) and for root communities (ANOSIM P<0.091) (Figure 3). However, leaf-, root-, and water column associated bacterial communities were significantly different from one another Figure 3. Multidimensional scaling diagram (ANOSIM, P<0.001) (Figure 3). The bacterial showing betadiversity of plant-associated community associated with plant leaves was bacterial communities. Communities were composed of approximately equal proportions of not significantly different between plant Granulosiccoci, Alphaproteobacteria, species for leaf communities and for root communities, however, leaf-, root-, and water Bacteroidetes, and other Gammaproteobacteria, column associated bacterial communities whereas the roots were dominated by were significantly different from one another Deltaproteobacteria, Bacteroidetes and other Gammaproteobacteria, with smaller proportions of Spirochaetes, Arcobacter, Firmicutes, and Flexibacter. Bacterioplankton communities in water samples were dominated by Bacteroidetes, Alphaproteobacteria, and other Gammaproteobacteria, with smaller proportions of Actinobacteria and other Betaproteobacteria (Figure 4).

Indicator species: Leaves The top indicators differentiating eelgrass leaf communities from root

12 communities were from the families Methylophilaceae and Piscirickettsiaceae, and the genera Granulosicoccus and Simiduia. Microbes of genus Granulosicoccus were particularly abundant, representing 14% on average of phyllosphere communities. One dominant indicator, OTU 69, (average 3.2% of phyllosphere communities), was initially classified to the class level Gammaproteobacteria, but further BLAST analysis revealed it to be 93% similar to the methylotrophic organism Methylobacter marinus. OTU_126 (average 1.61% of phyllosphere communities) was initially classified to the bacterial family Piscirickettsiaceae, which includes the fish pathogen Piscirickettsia salmonis, but further BLAST analysis revealed this organism to be more closely related to methylotrophic organisms in the Methylophaga genus, and was 93% similar to M. lonarensis (Shetty et al. 2015).

Figure 4. Bacterial community composition of all samples based on 16S rRNA amplicon sequences.

Indicator Species: Roots The top indicators differentiating eelgrass root communities from leaf communities were from the species Spongiibacter marinus, the family Rhodobacteraceae, the species Thiomicrospira chilensis, the species Arcobacter nitrofigilis, and the genus Sedimenticola. For example, OTU 6 (average 11.6% of rhizosphere communities), was initially classified as Gammaproteobacteria, and further BLAST analyses revealed it to be 93% similar to Spongiibacter marinus, and 100% similar to Spongiibacter marinus DSM 19753 H573DRAFT_scaffold00009.9_C, whole genome shotgun sequence. OTU 91 (average 0.07% of rhizosphere communities), was classified to the family Rhodobacteraceae, and further shown by BLAST analysis to be 99% similarity Oceanicola batsensis, and 100% similar to Uncultured Rhodobacteraceae bacterium clone 1LM16-8, which was found associated with the seagrass Halodule beaudettei in Laguna Madre, TX. OTU 30, (average 3.00% of rhizosphere communities), was initially classified to Gammaproteobacteria, and further BLAST analysis found it to be 92% similar to the sulfur-oxidizer Thiomicrospira chilensis DSM 12352. OTU 61 (average

13 3.0 % of rhizosphere communities) was classified to the genus Arcobacter, and was 99% similar to the nitrogen fixing heterotroph Arcobacter nitrofigilis, which was originally isolated from roots of the marsh grass Spartina alterniflora. OTU 90, (average 2.2 % of rhizosphere communities), was classified to genus Sedimenticola, and 96% similar to the sulfur oxidizing Gammaproteobacterium Thioprofundum lithotrophicum.

Discussion The recent explosion of microbiome research has demonstrated the close relationships between microbes and larger organisms. Microbes directly interact with organisms as phylogenetically diverse as other bacteria (Husnik, 2013), terrestrial plants (Bakker et al. 2012), and even human beings (Huttenhower, 2012). However, much less is known about microbiomes of aquatic plants. Plants suffer from the presence of harmful chemicals in their surroundings (Abanda-Nkpwatt et al. 2006); these chemicals may be a result of their own metabolism or the metabolism of co-occurring organisms (Koch et al. 2001, Abanda-Nkpwatt et al. 2006). Plants can also suffer from light competition, which starves them of food (Heijs et al. 1985). Bacteria associated with leaves and roots may assist plants by performing four services that help ameliorate the above problems: removal of waste products, neutralization of poisonous mud exudates, fixation of nitrogen, and disruption of epiphytic algal growth. Our study provides compelling evidence that eelgrass microbiomes benefit plants in multiple ways. We found bacteria associated with the leaves that are closely related to organisms that are capable of metabolizing methanol, of releasing plant hormones such auxins and cytokinins which stimulate plant growth, of producing agarases that cause disease and die-off in populations of red seaweed (which trophically compete with the eelgrass), of storing carbon in polyhydroxybutyrate granules, and of degrading gelatin. We found bacteria on the roots related to organisms that are capable of metabolizing sulfide and other sulfur compunds, of fixing nitrogen, of storing carbon in poly-ß-hydroxybutyrate granules, and of metabolizing methanol. Our findings may help demonstrate the complex relationship between seagrasses and their microbiomes.

Leaf taxa with possible interactions with Z. marina and Z. japonica There were several taxa present on the leaves of both species of eelgrass that have potential beneficial interactions with the plants. Microbes of family Methylophilaceae are capable of degrading methanol (Kurilenko et al. 2010), a compound which is exuded from angiosperm leaves as a waste product of cell wall synthesis (Abanda-Nkpwatt et al. 2006). Methanol has been shown to inhibit germination and retard the growth of angiosperm seedlings, an effect that is mitigated in strawberry plants by Methylobacterium extorquens, a microbe of the family Methylophilaceae (Abanda- Nkpwatt et al. 2006). Methanol consuming bacteria are common in marine environments, and include Methylophaga lonarensis (Antony, 2012), and Methylobacter marinus (Flynn et al. 2016). This is significant because both species of eelgrass, being angiosperms, release methanol from their leaves as a byproduct of cell wall synthesis. In our metatranscriptomic analysis we found expression of the genes mtaA, mtaB, and mtaC, which code for a protein complex that irreversibly transforms methanol into methyl-CoM (Sauer et al. 1999). The expression of this protein complex in our samples

14 suggests that some of the taxa in our study, possibly even one of the above methanotrophs, may be degrading methanol. Some of the methanotrophs present on angiosperms have been found to release plant hormones—such as auxins and cytokinins—which stimulate plant growth (Abanda- Nkpwatt et al. 2006). In our metatranscriptomic analysis we detected expression of genes that produce the auxin Indoleacetate. These genes are MAO, which transforms Tryptophan to Tryptamine (Shih et al. 1999), and DDC, which transforms Tryptamine into Indole-3-acetaldehyde (Chassande et al. 1994), and aldehyde dehydrogenase (NAD+), which transforms Indole-3-acetaldehyde into Indoleacetate (Racker, 1949). The expression of these genes suggest that one or more of the taxa in our study, potentially one or more of the above methanotrophs associated with the plant leaves, may be producing Indoleacetate and influencing the plants’ metabolism. Genus Simiduia is associated with agarose hydrolysis (Li et al. 2013); agar is produced by Red Seaweeds, which commonly grow as epibionts on eelgrass leaves (Heijs et al. 1985). Agarases produced by these agrolytes cause disease and die-off in populations of red seaweed (Schroeder et al. 2003). Epibionts negatively impact the health of the basibonts on which they live because they compete with them for light (Wahl, 1989). We found some expression of beta-agarase, an enzyme that degrades agarose (Duckworth et al. 1969), suggesting some of our taxa may be capable of degrading agarose. Since Simiduia is capable of agarose hydrolysis, and we found genes for agarose hydrolysis on the eelgrass leaves, it is possible that Simidua was preventing Red Seaweed from growing on the eelgrass leaves. In conclusion, there are taxa present on the leaves that are capable of metabolizing methanol, of releasing plant hormones such as auxins which stimulate plant growth, and of producing agarases that cause disease and die-off in populations of red seaweed which trophically compete with the eelgrass.

Root taxa with possible interactions with the eelgrass There were several taxa present on the roots of both species of eelgrass that have possible beneficial interactions with eelgrass. Thiomicrospira chilensis is an obligatory aerobe that is capable of metabolizing sulfur, sulfide, thiosulfate, and tetrathionate (Brinkhoff et al. 1999). The genus Sedimenticola also contains organisms that can metabolize these sulfur compounds (e.g., Sedimenticola thiotaurini; Narasingarao et al. 2006, Bailey et al. 2015). In our metatranscriptomic analysis we found expression of dsrA/dsrB dissimilatory sulfite reductase which converts sulfite into bisulfite (Hatchikian et al. 1983), and the gene fccB sulfide dehydrogenase which converts hydrogen sulfide into elemental sulfur (Fukumori et al. 1978). Since these genes are being expressed, the above root taxa may be involved in sulfite reduction and sulfide oxidation. Sulfide is highly toxic to eelgrass photosynthetic pathways (Goodman et al. 1995), and increases in sediment sulfide concentration lead to a decrease in the ATP available to the plant’s cells (Koch et al. 2001). Therefore, since there were taxa capable of sulfide oxidation present in the eelgrass rhizosphere, and there was expression of fccB, a sulfide oxidation gene, it may be possible that taxa on the eelgrass roots were removing sulfide from the plants’ immediate vicinity. Arcobacter nitrofigilis is a symbiont of the roots of the marsh grass Spartina alterniflora, where it fixes nitrogen (Pati et al. 2010). Nitrogen fixation is necessary in

15 the nitrogen-limiting environment of estuaries to provide eelgrass with the much-needed element nitrogen (Capone et al. 1982). In our metatranscriptomic analysis we detected expression of nifD, nifE and nifH, which code for proteins involved in nitrogen fixation (Loo et al. 1974). Since these genes are being expressed, the above taxa may be fixing nitrogen in the eelgrass rhizosphere. We found expression of alcohol dehydrogenase in our samples. Alcohol dehydrogenase can turn ethanol into acetate (Nakayama, 1960). Ethanol, though not directly toxic to the plant, may be indirectly toxic to it: according to Widdel (1988), ethanol released from plant roots during anaerobic fermentation increases the concentration of sulfide-producing microbes in the surrounding soil. This happens because the ethanol-consumers are capable of metabolizing ethanol and will use it if it is available. This may open up a niche for non-sulfide-producing ethanol-metabolizing plant associated microbes to exist on the surface of the roots, metabolizing the ethanol before it can reach the soil (and ultimately the sulfide producers). Many of the methanol-degrading organisms associated with leaves were also found associated with roots, as was expression of genes involved in methanol consumption. This suggests that methanol is released by both leaves and roots of eelgrass, and is consumed by the eelgrass microbiome. In conclusion, there are taxa present on the roots that are capable of metabolizing sulfur compounds, of fixing nitrogen, of degrading ethanol, and of degrading methanol.

Other Taxa Several taxa were found to be associated with eelgrass roots and leaves that have no clear association with the plants. One of the most abundant taxa associated with eelgrass leaves was the Gammaproteobacteria genus Granulosiccocus. There are currently 30 rRNA sequences in GenBank for organisms in the genus Granulosiccocus, and one species, Granulosicoccus coccoides, is capable of hydrogen sulfide production, gelatin liquefaction, indole production, denitrification of storing its carbon in hydroxybuterate granules (Kurilenko et al. 2010). Oceanicola batsensis, which was an indicator taxon for root communities, is a heterotrophic aerobic marine microbe (Jang-Cheon et al. 2004) that, like the eelgrass-associated microbe Granulosicoccus coccoides, stores carbon in poly-ß-hydroxybutyrate granules (Jang-Cheon et al. 2004) (Kurilenko et al 2010). S. marinus, another root indicator, is a heterotrophic marine epibiont of sponges (Graeber et al. 2008).

Conclusion In our study, the phylogenetic composition of plant-associated bacterial communities was not significantly different between plant species for leaf communities and for root communities. However, leaf-, root-, and water column associated bacterial communities were significantly different from one another. We found taxa present on leaves that are capable of metabolizing methanol and of producing agarases that cause disease and die- offs in populations of competitive red seaweed, and of producing indoleacetate, a plant hormone. Members of genus Granulosicoccus were found to be particularly abundant in our leaf samples. We also found taxa present on the roots that are capable of metabolizing sulfur compounds, of fixing nitrogen, of degrading ethanol, and of degrading methanol.

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