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Eco-physiology of macroscopic pink and green bacterial consortia of the mighty Little Sippewissett salt marsh Elizabeth G Wilbanks1†, Parris T Humphrey2†, Ulrike Jaekel3†, Cristina Moraru2^, Rebekah Ward4^, Victoria J Orphan5,6 1 Microbiology Graduate Group, University of California, Davis. Davis, CA., USA 2 Department of Ecology & Evolutionary Biology, University of Arizona, Tucson AZ., USA 3 Max Planck Institute for Marine Microbiology, Bremen, Germany 4 Department of Biology, Emory University, Atlanta GA., USA 5 Division of Geological and Planetary Sciences, Cal Tech, Pasadena, CA., USA † - These authors contributed equally to this work (students 2010) ^ - TAs for the course (2010) 6 – External faculty project mentor

ABSTRACT

What, if anything, goes on inside of macroscopic microbial consortia that makes them

special? Underlying the idea of a consortium is the notion that certain microbial communities

may profit immensely from living together in close association, if only for a certain time in their

life cycle. Here in the Little Sippewissett Marsh, pink and green microbial aggregates (“berries”)

cover marsh pool sediments like dense carpets. In this report we set out to answer the following

questions: Who is there?, and what, if anything, are they doing? By comparing 16s rRNA clone

libraries from multiple years, we show that community membership in pink berries is stable

despite the likely turnover of the berry crop between sampling points. Using a robust description

of community membership, we tested the hypothesis that sulfur cycling occurs within pink

berries between sulfate reducing bacteria and purple sulfur bacteria (sulfur oxidizers). Using

heavy isotope probing coupled with nano-scale secondary ion mass spectrometry (nanoSIMS),

we establish a direct syntrophic link between resident sulfate reducing bacteria and purple sulfur

bacteria within this naturally occurring microbial consortium. TEM and FISH analyses reveals

the consistent structure of the berry “tissues” and demonstrate the co-localization of sulfate

reducers and purple sulfurs throughout the berry, as well as the localization of additional stable

members (Cytophaga). We couple these findings with whole-berry level stoichiometry and

physiology to build an integrated view of berry eco-physiology. Through demonstrating

community stability and linking structure with physiological function (internal sulfur cycling) we

have helped solidify a basis for using pink berries as a model system to study the ecology and

evolution of syntrophic interactions in microbial consortia.

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INTRODUCTION

Despite the historic importance of pure cultures grown in liquid broth, it appears that

planktonic growth may more of an oddity than the norm in the wild, slimy world of

microorganisms. The vast majority of microbes live as biofilms-- spatially structured, mixed

populations [1]. This coordinated, macroscopic microbial lifestyle has major implications for

diverse fields from medicine to biogeochemical cycling to wastewater treatment. As our culture-

independent toolset expands, we are just beginning to link the taxonomic structure with spatial

organization and ecophysiology of complex microbial consortia.

It is within this framework that we introduce the marvelous pink and green berries of the

Sippewissett salt marsh— a legendary curiosity of the Microbial Diversity Course at MBL.

These microbial aggregates can reach sizes of up to 6 mm in diameter and are found the pools

off the main tidal channels in the marsh. These “berry ponds” are essentially clearings in dense

stands of marsh eelgrass (Spartina alterniflora) and remain covered in 6 inches to a foot of

water at low tide. The berries are found in the fluffy sediment water interface of these ponds

and can be extremely abundant forming magnificent pink, granular carpets.

Seitz et al., who provide the only published description of these macroscopic microbial

wonders [2], established that pink berries are primarily composed of purple sulfur bacteria that

dominate the respiration and metabolism of the aggregates. They hypothesized that sulfate

reducers may also be present in the aggregates as syntrophic consortia members providing

electron donors for the anoxygenic photosynthesis of the purple sulfur bacteria. In addition to

the dense clusters of purple sulfur bacteria, diatoms and rod shaped bacterial cells have been

observed by microscopy [3]. 16S bacterial clone libraries conducted by Udi Banin in the course

during the summer of 1997 laid the foundation of the molecular identification of the pink berry

consortia. Of the modest 10 clones, three were sulfate-reducing bacteria, one was a

Chromaticaea purple sulfur bacteria and four were Cytophaga. Two out of three sulfate reducer

16S clones formed a coherent clade most closely related to Desulfofustis glycolyticus. The

subsequent year, Casto et al. in the course sequenced an addition 10 clones from which they

found, most notably, a sequence nearly identical to the Chromaticaea sequence obtained by Udi

Banin the year previously. The green berries have been less thoroughly examined, though a

16S clone library (69 clones) conducted in 2002 by Margy Gentile in the course found that these

were dominated (84%) by cyanobacteria closely related to Cyanothece. In 2007, Jarrod Scott

conducted several large clone libraries (~170 16S clones) from pink berries [4], the data from

which have been reanalyzed and are presented in our study.

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The sequence based evidence for the presence of sulfate reducers and purple sulfur has

led to the long standing working hypothesis that the pink berries represent a syntrophic bacterial

consortia with a cryptic sulfur cycle: sulfate is reduced to sulfide which is fed to the anoxygenic

phototrophs as an electron donor. The purple sulfur bacteria, in turn, act as the primary

producers in this syntrophic association, fixing and providing carbon to the other community

members. Cytophaga and other heterotrophs are thought to provide an oxygen shield for the

oxygen sensitive physiologies of other berry members. However, despite numerous summers

of berry-magic, there has been no physiological evidence to support this hypothesized sulfur-

based syntrophy. We set out to link the structure of the berry communities with their metabolic

functions and test the hypothesis that the berries are a specific syntrophic association

characterized by carbon fixation and an internal sulfur cycle.

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METHODS

Berry sampling

Green and pink berries were collected fresh from a perennial berry pond in the center of

Little Sippewissett Marsh (Figure 1) <1day prior to incubation experiments, DNA extraction or

embedding and sectioning for FISH/TEM. Marsh water was removed from the same site, filter-

sterilized and made anoxic for use in all incubations. This medium was refreshed every week.

Using a 10cm W x 20cm L x 8cm H plastic sieve, berries were concentrated and panned from

the diffuse sediment-water interface and rinsed in the sieve with standing marsh water several

times before being placed into collection tubes. Once back in the lab, berries were sorted and

washed 3x in seawater base medium and stored in filtered marsh water at room temp until used

downstream.

16s and 18s rRNA community analysis

DNA extraction DNA was extracted using the MoBio PowerSoil kit. Berries were

placed into an eppendorf tube with ~100 μL sterile milliQ water and pulverized with a mortar and

pessle. The disrupted berry suspension was added to the MoBio PowerBead tubes and gently

vortexed. Following addition of 60 μL C1 solution, tubes were beadbeaten for 30 seconds and

then incubated 5 minutes at 70°C. The remainder of the MoBio PowerSoil protocol was

completed without modifications.

454-metagenomic sequencing DNA samples from 10 pink berries and 10 green

berries was submitted for one plate of 454-metagenomic sequencing along with three other

genomic samples from the course. 454-sequencing was conducted on a GSFLX Titanium at

Penn State University. Dr. Libusha Kelley from the course conducted quality filtering and

assembly using the Newbler assembler.

16S bacterial PCR amplification The diversity of the bacterial members of the green

and pink berry communities was investigated by constructing 16S clone libraries. Universal

bacterial primers 8F and 1492R were used at a final concentration of 0.2 mM to amplify the 16S

gene. To determine the appropriate concentration of DNA, PCR was performed on a dilution

series (direct, 1:10 and 1:100 dilutions) of pink berry DNA (at ~12ng/μL). 16S PCR reactions

were set up as shown in Table 1.

Table 1. Recipe for 16S bacterial PCR reactions of 25μL final volume.

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Reagent Volume added

Promega Master Mix 2x 12.5 μL

10 μM 8F primer 0.5 uL

10 μM 1492R primer 0.5 μL

DNA 2.0 μL

Nuclease-free water 9.5 μL

PCR reaction was performed at 95°C for 5 minutes followed by 30 cycles of 95°C for

30seconds, 46°C for 30seconds and 72°C for 1 minute 30 seconds and a final 72°C extension

for 5 minutes.

16S archaeal PCR amplification Due to time constraints, archaeal 16S PCR and

cloning was attempted only from the pink berries. Initial attempts to amplify archaeal 16S

sequence employed the 2FA/1391R primer pair at 2 μM concentrations with 30 cycles and an

annealing temperature of 58°C, as described in previously [4]. Several alternate universal

archaeal primer sets were tested: (1) 571FA with 1391R (2) 571FA with 1492R and (3) 2FA

with 1391R. All primers were used at 10 μM to minimize primer-dimers. Twelve replicate tubes

were incubated in a gradient PCR as follows, 95°C for 5 minutes, followed by 35 cycles of 95°C

for 1 minute, gradient of 40°C to 60°C for 1 minute, 72°C for 2 minutes, and a final 10 minute

extension at 72°C.

18S eukaryal PCR amplificiation Eukaryotes in the pink and green berries were

targeted using the degenerate eukaryotic 360FE primer (see Appendix I for sequence and

primer information) with the universal 1492R primer, as employed by Dawson and Pace [5].

Primers were used at 0.2 mM in an identical 25μL PCR cocktail to that described above for the

bacterial 16S reaction. Twelve replicate tubes were incubated in a gradient PCR in [5]: 95°C for

12 minutes, followed by 35 cycles of 94°C for 1 minute, gradient of 45°C to 65°C for 1 minute,

72°C for 2 minutes, and a final 10 minute extension at 72°C.

Cloning, transformation and sequencing All PCR products were gel purified and

cloned using the TOP10 electrocompetant “One Shot Cells”. 96 colonies were picked and

prepared for Sanger sequencing from each clone library. Sequence data was trimmed, quality

filtered and sequences below 400 bp in length were discarded from future analysis.

Phylogenetic analysis All SSU rDNA sequences were aligned using the SILVA

database [6] and imported to ARB [7]. Overview classification of bacterial and archaeal 16S

SSU rDNA sequences was conducted with the RDP classifier [8]. Overview of eukaryal 18S

sequences was provided by examining the position of ARB parsimony insertion of SILVA

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aligned sequences. For phylogenies of focused groups, maximum likelihood trees were

conducted using RAxML with either bacterial or eukaryotic filter position variable bacterial / euk

position variability by parismony102 masks. Rapid bootstrap analysis was conducted with 50

replicates. Formatting of subsequent newick trees was conducted in FigTree [9]. Rarefaction

curves were generated in MOTHUR [10] and plotted using custom R scripts. Unweighted

UniFrac comparisons were conducted to determine whether select sequence libraries were

significantly different [11]. The input trees for UniFrac analysis were calculated according to the

methodology of Lozupone and Knight [12] to handle non-overlapping sequences (except that

SILVA aligner was used in place of NAST).

Fluorescence in situ hybridization (FISH)

Fixation and cutting Berries were washed several times with sterile filtrated

Sippewissett pond water and then carefully moved with some water into Petri dishes (60mm

diameter), one berry per dish. The water was removed using Pasteur pipettes without

destroying the aggregate. The aggregate was washed with sterile 1x PBS for 10 min on a slowly

moving oscillator. After careful removal of the PBS with a Pasteur pipette, the berries were fixed

(4% PFA, 0.5% glutaraldehyde in 1xPBS) in parafilm sealed Petri dishes for 1 hour at room

temperature. The fixative was removed and the berries were washed three times with 1x PBS

for 10 min on a slow moving oscillator. The berries were thereafter incubated in a sucrose

solution (7.5% sucrose in 1x PBS) for 1 hour on a slowly moving oscillator. The sucrose was

removed carefully and OCT (Optical Cutting Solution, Tissue-Tek, Sakura) was added to the

Petri dish, so that the berries were completely covered. The petri dishes with the berries were

incubated for 2 hours on a slowly moving oscillator. The berries were carefully moved into cryo-

freezing molds and snap-frozen in liquid nitrogen. The molds were moved into a cryotome

chamber to equilibrate to -20°C overnight. The frozen berries were cut using the cryotome into

50µm sections which were placed onto coated glass slides (Fischer Scientific, Superfrost®) and

kept at 4°C until further use.

CARD-FISH The structural organization of phylotypes found to be overrepresented in

the 16SrRNA clone libraries of the pink berries were investigated by hybridizing the sectioned

pink berries with group specific probes. The Deltaproteobacteria-probe (Delta 495a-c +

competitor a-c) was checked in ARB to target the sequences closest related to the Desulfofustis

spp. (Figure 6), the Gammproteobacteria-probe (Gam42a) was checked to target the

Halochromatium spp. related sequences (Figure 5) and the Cytophaga-probe (CF319a probe)

was checked to target the sequences related to the Cytophaga spp.. In addition, the sectioned

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pink berries were hybridized with a Eubacterial probe (EUB 338 probe) as a positive control for

successful hybridization and a Nonsense probe (Non338) as a control for unspecific binding.

Hybridization was performed according to Blazejak et al., [13] with the following exceptions: The

sections were covered in 0.2% low melting agarose (Metaphor) by immersing the entire slide

into a Petri dish filled with the warm but not hot agarose solution and dried at 35°C for 30 min at

the beginning of the procedure. This step was repeated after permeabilization and inactivation

in order to prevent the sections from coming off the slides. Samples were not incubated in

RotiHistol®. The sections were dehydrated in an ascending ethanol series (50%, 70%, 80%,

100% ethanol, 3 minutes each), then air dried at RT. After the dehydration step permeabilization

was performed by incubating the slides in 20mg/ml Lysozyme for 90 minutes at 35°C, followed

by washing the slides twice with mQH2O. Bleaching of endogenous peroxidases was performed

by incubating the slides in 0.01M HCl for 20 min at RT, followed by washing them twice with 1x

PBS, 5 minutes each and once with mQH2O for 1m. The slides were thereafter dipped in 100%

ethanol and air dried. Hybridization was performed on separate slides for each probe for 2 hours

at 46°C instead of 3 hours at 35°C, washing was done at 48°C instead of 37°C. CARD was

done at 46°C instead of 37°C. Pictures were taken using an Axio IMAGER MZ Microscope

equipped with a color camera (AxioCam HRc, Zeiss) in order to be able to distinguish between

autofluorescence of the purple sulfur bacteria and the probe signal (Alexa 488).

Transmission electron microscopy (TEM)

Fixation All fixation washes and incubations were done 50 mL falcon tubes on an

oscillating shaker. Pink berries were incubated for 30 minutes in a solution of 4%

paraformaldehyde / 0.5% glutaraldehyde 0.1M PBS. Berries were then washed three times in

0.1M sodium cacodylate buffer at pH 7.4 for 3, 7, and 10 minutes. Post-fixation was performed

for 1 hour in a 2% osmium tetraoxide PBS solution, then washed 3 times in 0.1M sodium

cacodylate buffer at pH 7.4 for 10-20 minutes.

Dehydration Dehydration washes from 10 to 70% were conducted in 50 mL Falcon

tubes, while the 90 and 100% washes were conducted in medium sized petri dishes. Unless

stated otherwise all dehydration was done at room temperature and without shaking. A

dehydration gradient was performed with incubations of 15 minutes each of 10%, 20%, 50%

ethanol in PBS. Berries were incubated at 70% ethanol in PBS overnight at 4 degrees (due to

time constraints, ideally we might proceed directly through with a 15 minute incubation at room

temperature). Note that the berries became very fragile upon addition to 70% ethanol and many

were lost. It appears that light pink berries were the most friable (compared to darker purple

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and green berries). N Following the overnight incubation berries were incubated at 90% ethanol

in PBS for 15 minutes followed by three washes in 100% ethanol for 15 minutes each.

Embedding Embedding was performed with EMBed-812 resin from Electron

Microscopy Sciences (EMS #14120) which was pre-mixed according to manufacturer’s

instructions. Dehydrated berries were incubated in 50 mL Falcon tubes an oscillating shaker in

the concentration ratios of EMBed-812 to ethanol shown in Table 2. The EMBed-812

concentration was increased by adding the appropriate amount to each tube, rather than

transferring berries to avoid breaking the samples. For transfer to pure EMBed-812, berries

were gently scooped up with spatula and transferred to a fresh tub e. Following these

incubations, the berries were then placed into molds and cured at 70°C for 24 hrs.

Table 2. EMBed-812 incubation schema.

Mixed EMBed-812 : Ethanol ratio Time incubated

1:4 30 minutes

1:3 1 hr

1:2 1 hr

1:1 1 hr

2:1 2 hrs

3:1 3 hrs

4:1 5 hrs

Pure EMBed-812 without accelerator 7 hrs

EMBed-812 with accelerator

(DMP-30)

6.5 hrs

Sectioning Sample blocks were trimmed and thin sectioned (10-150 nm) using the

ultramicrotome. Sections were deposited on formvar coated Electron Microscopy Sciences

copper grids and stained with 4% uranyl acetate (Electron Microscopy Sciences) for 5 minutes

and then Reynolds Lead Citrate (Electron Microscopy Sciences # 17800) for 1 minute. Then

dried and viewed at 80 kV on a Zeiss 10CA transmission electron microscope with a

Hamamatsu Orca-ER digital camera.

Heavy C,N and S isotope labeling and nanoSIMS analysis

Incubation Conditions Stable isotope probing of carbon, nitrogen and sulfate was

conducted to investigate the eco-physiology of the pink berry consortia. 13C sodium

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bicarbonate (Isotech 372382-1G) was used to monitor and track carbon fixation, while 15-N2

gas was used to monitor nitrogen fixation. To investigate our hypothesis that there exists a

cryptic sulfur cycle within the berries, we added 34-S sodium sulfate (Isotech 718882-SPEC).

An outline of the experimental design in depicted in Figure S1. Briefly, five berries were

incubated in 3 mL of filter-sterilized, MOPS-buffered berry pond water that was spiked to a final

concentration of 0.5 mM with sodium sulfide to stimulate sulfur granule formation. For labeling

experiment, filtrate was enriched with 28 mM 34S sodium sulfate, 10 mM 13C sodium

bicarbonate and 50% of the headspace was replaced with 15N2 gas. For the unlabeled

background control, the same additions of the corresponding unlabelled chemical were added to

make sure incubation conditions were identical except for the heavy isotope label. Isotope

labeling experiments were also conducted with the addition of 10 and 20 mM sodium molybdate

(a known inhibitor of sulfate reducers), to explore the importance of the sulfate reducing bacteria

in the berry consortia. Once incubations were set up, they were stopper capped and incubated

at 30°C for 5 days on a 14hr light, 10 hour dark cycle. The exact protocol for this experimental

set up is presented in Appendix II.

Fixation and sectioning Upon termination of the incubation, berries were washed

with 1x PBS for 10 minutes on an oscillating platform then fixed in 4% paraformaldehyde / 0.5%

gluteraldehyde PBS solution for 1 hour (oscillating). Following fixation berries were washed

three times in 1x PBS for 10 minutes oscillating. Berries were then washed in a 7.5% sucrose

solution on an oscillating platform for 2 hours, followed by a 2 hour incubation oscillating in

TissueTek OCT. Berries were then placed into molds and snap frozen with liquid nitrogen.

Sectioning was completed with a cryotome to generate 10 μm sections which were deposited

onto nanoSIMS rounds which had been combusted at 260°C for 6 hours. Dr. Victoria Orphan

conducted the nanoSIMS analysis at her lab at Caltech.

Acetylene reduction assay for N2 fixation

We assessed the potential for pink and green berries to fix nitrogen by assaying for

acetylene reduction. This reaction, (C2H2 + 2H+ + 2e- C2H4) takes advantage of the lack of

substrate specificity of many nitrogenase enzymes and provides a product of reaction (ethylene)

that can be readily detected using gas chromatography. 5ml of anoxic, filtered marsh water was

added to each of twelve 30ml serum vials anaerobically. Media in each bottle was amended

with 28mM sulfate, 10mM carbonate and 500µM sulfide added from anoxic stocks. We added

five pre-washed, fresh berries (collected <1d prior to setup) from anoxic seawater base into

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each of four bottles per berry type. For all experiments, sulfide was added last to sealed, blue-

capped serum bottles using 1ml syringes.

Acetylene was generated by adding 500µl H2O via syringe to a capped, fully evacuated

150ml serum bottle containing 500mg calcium carbide (CaC2). 4ml of headspace was replaced

via syringe with the same volume of acetylene, resulting in a 10% Acetylene/90% CO2/N2 mix.

Two bottles remained berry-free as controls to establish baseline abiotic acetylene reduction. In

addition, two serum bottles of each berry type were incubated without acetylene to control for

the background evolution of ethylene from the berries. Acetylene reduction was assayed by

analyzing 100µl from the headspace of each bottle with gas chromatography every 12h for three

days.

Berry-level stoichiometry

We determined the total % carbon and nitrogen as well as C:N and isotopic ratios for

green and pink berries. Ten pink and green berries were recovered from the acetylene reduction

assay control condition (no acetylene), washed 3x with seawater base media and dried at 60C

overnight in an uncovered 96-well plate (NUNC). Six samples per berry type were then

analyzed using a Europa 20-20 elemental analyzer interfaced with a Europa ANCA-SL mass

analyzer under contract with the Stable Isotope Lab, Ecosystem Center, Marine Biological Lab.

13Carbon fixation rates in pink berries We tested whether the rate of carbon

fixation in whole pink berries was enhanced under supplementation with external H2S or

inhibited by sodium molybdate. Using 5mM 13carbonate instead of 12carbonate, 5ml pink berry

incubations were setup as above (see Acetylene reduction) at variable sulfide concentrations

(0,0.1,0.25,0.5µM) with and without sodium molybdate (10mM). Fifteen berries for each

condition were incubated at 30C in a full-spectrum light incubator for six hours. Three berries

from each of three replicates per treatment were removed form the incubator after 6h, washed

3x with sterile miliQ water, and placed in a 96-well plate for overnight drying. Three berries per

replicate per treatment were pooled for C:N and isotope ratio analysis.

Sulfide and sulfate consumption assay

To measure the flux of sulfate and sulfide from pink berries, we conducted a time course

incubation measuring the change in concentration of sulfate and sulfide from the incubation

solution. 50ml of filtered, unbuffered berry pond water was added to 120ml serum bottles each

containing 50 pink berries. One bottle contained only the pond water and berries, another was

spiked to a final concentration of 10 mM sodium molybdate and a third was spiked to 0.5 mM

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final concentration sulfide. Two abiotic controls were also set up: one with just filtered pond

water and a second with pond water spiked with 0.5 mM sulfide. An overview of the

experimental design is provided in Figure S2. All experimental conditions were set up in

triplicate.

The experiment was set up at ~11am and To was taken. From then forth, time points were

taken at ~9:30 PM just before the light cycle shut off for the day and at 7:45AM just prior to the

light cycle switching on. A total of 5 time points were sampled. At each time point, sulfide

concentration was measured using the Cline assay and samples were collected for future ion

chromatography measurements of sulfate concentration. For the sulfate samples, 0.6ml of 0.2

μm-filtered incubation solution was added to 0.6 ml of 1% iron chloride solution and frozen at -

20°C. Samples were shipped on dry ice to the Max Planck Institute in Bremen, Germany for

analysis using ion chromatography.

Preliminary metagenomic analysis

We briefly analyzed 454® metagenomic data generated for pink and green berries by

BLASTing quality-trimmed contigs >500bp against a database of functional genes for nitrogen

fixation (nifH), sulfide oxidation (sox genes), sulfite reductase (dsrA) and sulfur globule

formation (sgpA). The purpose was to examine whether the sequencing depth picked up genes

known to play a role in the physiology of the dominant organisms discovered in 16s clone

libraries. Gene calling and annotation jobs have been submitted to both CAMERA2

(http://legacy.camera.calit2.net/) and MG-RAST (http://metagenomics.nmpdr.org/). Additional

sequencing (likely mate-pair Solexa sequencing of 6-10kb libraries) will be required to fill out

coverage for an in-depth metagenomic analysis (in progress as of this writing).

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RESULTS

DNA extraction The efficiency of DNA extraction was assessed by measuring DNA yield

obtained with the MoBio Powersoil kit from 1, 5 or 10 homogenized pink berries. For each berry

quantity, the DNA extraction was performed both with and without a 5 minute incubation at 70°C

to assess the impact of heat treatment on DNA yield. DNA concentration and 260/280 ratio

were determined via Nanodrop. Using five berries or more was found to provide the best quality

and appropriate yields (Table 3). DNA extraction from a single berry was found to provide low

concentrations of DNA with a low 260/280 ratio indicating an undesireable DNA:protein ratio.

Heat treatment did not affect the yield or quality of the DNA extracted, though we were unable to

assess whether the heat treatment provided a better representation of the berry community

members. For DNA extraction from berries, we recommend working with 5 berries and the

addition of a pulverizing step and heat treatment to the standard MoBio PowerSoil kit.

Table 3. DNA extraction efficiency from variable pink berry biomass with and without heat

treatment.

Number of berries Heat treatment? [DNA] ng/μL 260/280

1 2.9 1.37

1 X 4.9 1.32

5 13.4 1.66

5 X 10.1 1.69

10 21.8 1.63

10 X 22.5 1.68

16S/18S PCR amplification 16S bacterial PCR products were obtained for the 1:10 and

1:100 dilutions of pink berry DNA, while the concentrated DNA provided no detectable product.

Subsequent archaeal and eukaryal PCR was conducted with 1:10 dilutions. For the archaeal

PCR reactions, detectable PCR product was obtained only for the 571FA – 1391R primer set at

annealing temperatures above 54°C. Eukaryal 18S PCR was successful for the pink berries at

annealing temperatures from 45°C to 52°C.

COMMUNITY STRUCTURE

SSU rDNA sequence analysis of berry microbes. Sequencing from all five clone libraries we

compared was very successful, yielding at least 80 useable sequences out of 96 for all libraries.

Rarefaction curves from the bacterial and eukaryal suggest that these communities have been

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surprisingly well covered by our clone library sequencing (Figure 2, A-D). The eukaryal

communities are substantially less diverse than the bacterial and archaeal communities. In

addition to the clone libraries we constructed and sequenced, we were also able to obtained

16S bacterial sequence data generated during the summer of 2007 and these data were

included in the pink berry rarefaction curve [4]. Archaeal rarefaction curve shows no sign of

leveling off, indicating that, unlike for the bacterial and eukaryal sequences, pink berry archaeal

diversity has not yet been adequately sampled (Figure 2,E).

An overview of the taxonomic classification of the pink berry archaeal clone library

sequences is provided in Figure 3. The majority of the clones obtained were from the

euryarchaeota and clustered most closely with uncultivated archaea. Further work is needed to

more thoroughly investigate the patterns of diversity within this archaeal library, as time ran out

for more detailed analyses.

Figure 4, A-B provides a comparison of the phylum-level bacterial diversity in pink and

green berries. An unweighted UniFrac analysis of the 2010 green berry sequences and

2010+2007 pink berry sequences determined that these communities were significantly different

(Bonferroni corrected p-value < 0.01). Notably, the cyanobacteria comprise a more substantial

proportion of the green berry sequence abundance (39%) while they appear to be only minor

constituents of the pink berry community (3% of 16S sequences). Within the green berry

cyanobacterial sequences, approximately 50% were assigned as chloroplasts while the others

were labeled “unclassified cyanobacteria.” Further investigation in ARB suggests that these

taxa are closely related to Cyanothece and Gleothece. (For more interesting discussions of

green berry cyanobacteria, see James Henrickson’s report from 2010 discussing fluorescence

confocal microscopy of green berries.)

Proteobacterial taxa were by far the dominant sequence recovered from the pink berry

16S clone libraries (77% of sequences), compared with only 48% abundance in green berries.

The classification proteobacterial sequences from green and pink berries were also quite

different. The pink berries were dominated by the gamma (58%) and delta (40%)-

proteobacteria with a small minority of alpha proteobacteria, while the green berry communities

were evenly split between the alpha, delta and gamma proteobacteria. Closer examination of

the pink berry gamma proteobacteria from both 2010 and 2007 revealed that these clones

cluster into a single, unresolved polytomy of very closely related sequences within the purple

sulfur bacterial family Chromatiacaea (Figure 5). Likewise, the pink berry delta proteobacterial

clones from 2010 and 2007 clustered into a single, unresolved polytomy that was closely related

to Desulfofustis glycolicus (Figure 6).

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In contrast to the berry bacteria, the eukaryotic communities of the green and pink

berries were very similar (Figure 7). Unweighted UniFrac analysis of these two communities

revealed marginally significant difference between the green and pink berry eukaryotes

(Bonferroni corrected p-value = 0.04). Members of the stramenopiles, a major lineage of

eukaryotes that includes diatoms, dominate the 18S sequences of both green and pink berries.

A more detailed maximum likelihood phylogeny of the stramenopile 18S sequences found that

representatives from green and pink berries were interspersed throughout the tree, showing no

clear clustering by berry color (Figure 8). Thus, we suspect that if UniFrac were repeated using

this tree, no significant differences would be recovered. From a cursory first inspection, the

well-supported relatives of these stramenopile taxa show considerable morphological similarity

to diatoms observed via phase contrast and fluorescence microscopy.

Microscopic structure of pink berries: FISH & TEM

Microscopic investigation of pink berry structure reveals brain-like contortions at low-

magnification made up of EPS-embedded cell tubes of an apparent fixed width (Figure 9). FISH

revealed that these tubes that give the berries their shape and structure are gamma-

proteobacteria (purple sulfur bacteria, PSB; Chromatiales) (Figure 9, B). The PSB are visible by

the orange color due to their autofluorescence (Figure 9, A,C). In addition, sulfate-reducing

bacteria (SRB; delta-proteobacteria) were very prevalent in and on the tubular Chromatiales

backbone (Figure 9, C). To support this finding, TEM images revealed rod-shaped bacteria

interspersed between individual Chromatiales cells that have the same superficial morphology

as the delta-proteobacteria highlighted with FISH (Figure 10, C-D). Cytophaga FISH probe

illuminated dense microbial population confined to the EPS-rich external margins of the

Chromatiales tubes (Figure 9, D-F). In many cases, Cytophaga cells are observed in dense

groups and display nearly filamentous morphologies.

Transmission electron microscopy showed that the large, round PSB contained ~ 20

sulfur granules per cell (only the empty sheaths were actually visible since the elemental sulfur

was removed during TEM preparation) with an average percent ratio of S0 granule volume to

cell volume of 0.07% (calculations were done by measuring cell and S0 granule diameters from

TEM pictures and calculating corresponding sphere volumes from pictures of 3 PSB cells). TEM

pictures furthermore showed that different rod-shaped cells were located in the near vicinity of

the bigger, round PSB cells (Figure 10, A-B). The surrounding matrix can be seen in Figure 10,

D, even though it cannot be clearly stated that this space is made up of a polysaccharide, it can

15

be speculated that this might be the exo-polysaccharide (EPS) matrix which structurally holds

the cells of the berry aggregates together.

FUNCTION

Nano-scale isotopic enrichment analysis

Using nano-scale secondary ion mass spectrometry (nanoSIMS), we detected 13C, 15N

and 34S accumulation within pink berry consortia (Figures 11). Background isotopic values

were orders of magnitude below that detected in the berry slice from the heavy isotope

treatment. Heat maps generated by nanoSIMS reveals enrichment patterns consistent with the

size (~2µM diamter) and shape (spherical, ovoid) of individual purple sulfur bacterial cells within

the thin-sections (Figure 12). This suggests that in the pink berries, PSBs in the Chromatiales fix

carbon and nitrogen, as well as assimilate elemental sulfur.

The putative mechanism by which PSB could amass 34S in intra-cellular compartments

is through depositions of elemental sulfur (S°), a storage byproduct of H2S oxidation by PSB.

We incubated pink berries with 34-sulfate, which serves as a terminal electron acceptor of

sulfate reducing bacteria in the pink berries (Desulfofustis spp.) and gets reduced and expelled

as H2S within the berry. Therefore, the accumulation of 34-S inside PSB is direct evidence that

PSB in pink berries utilize reduced H2S generated by nearby SRB. Figure 12C shows a patchy

distribution of 34S enrichment within the field corresponding to PSB cells; at the sub-2µm scale,

smaller enrichment spikes can be observed within the 2µm hotspots potentially corresponding to

intracellular sulfur globules. Compare nanoSIMS isotopic ratio heat maps (Figure 12) with TEM

images at similar magnification (Figure 10) for comparison of PSB size and shape, as well as

distribution and size of intracellular sulfur globules.

Acetylene reduction activity in the Berries

Green berries reduced significantly more acetylene compared to pink berries (Figure

13). The amount of ethylene produced by pink berries was not distinguishable from abiotic

acetylene reduction. After four days incubating, neither green nor pink berries evolved any

detectable ethylene in the acetylene-free treatment, confirming that biological reduction was

responsible for the excess ethylene produced in the green berry incubations. This result is

inconsistent with nanoSIMS data, which shows that pink berries strongly enriched for 15N at the

scale of 2µm clusters corresponding to PSB cells (Chromatiales) (Figure 12, B). Acetylene

reduction therefore is not a sensitive measure of nitrogenase activity in pink berry consortia.

16

Whole-berry stoichiometry

Superficially, green and pink berries were of similar diameter pre-drying and were in fact

selected for analysis on the basis of apparent size uniformity. However, the biomass of the pink

berries was significantly lower (Figure 14, A). Although we did not quantify the apparent volume

of green or pink berries prior to drying, these data suggest pink berries may be considerably

less dense per unit size than green berries, a factor that may affect the establishment of

chemical gradients within the consortium.

We observed a higher %N (t-test, p=0.005) and a lower C:N ratio (t-test, p=0.01) in pink

berries than green berries (Figure 14, C-D). Overall, the stoichiometry of pink berries suggests

they are less nitrogen limited, despite the fact that we failed to detect nitrogen fixation in pink

berries through assaying for acetylene reduction (Figure 13). These results should be

interpreted with caution because the C:N ratio of the two batches of pink berries sent through

the elemental analyzer were significantly different (Figure 16). This could indicate that culture

conditions strongly dictate C:N ratios, or that the first batch measurements were prone to error

given the smaller relative biomass of the pink berries in batch 1 vs. batch 2 (~100µg vs. ~1mg).

Samples with more mass tend to give more accurate readings (M. Otter, personal

communication).

Red berries are naturally more depleted in 13C than green berries, indicated by the

more negative ∂13C values (Figure 15A). Natural isotope abundances can indicate the extent to

which organisms obtain their carbon from various sources; both green and pink berries can fix

carbon, yet the means by which purple sulfur bacteria achieve this is different than for

cyanobacteria (e.g. Calvin cycle vs. reductive TCA cycle). Both green and pink berries were

collected from the very same water sample, so differences in background dissolved inorganic

carbon fractionation could not explain the fractionation differences. These data point towards

inherent differences in fractionation between the carbon fixing machinery of the primary

producers in the green and pink berries.

Sulfide vs. Carbon Uptake

We observed a statistically insignificant trend towards increasing carbon fixation with

sulfide availability (Figure 15B). Low replication (n=3 per treatment) prevents us from rejecting

the null hypothesis that sulfide does not affect carbon fixation; however, the 500µM sulfide

treatment is much more similar to the 0µM treatment than either +100µM or +250µM suggesting

that sulfide levels above 500µM may inhibit carbon fixation in pink berries. The addition of

molybdate to the +0µM H2S had no affect on carbon fixation (data not shown). The background

17

sulfide concentration in the base media is unknown; however, the +0µM H2S condition

demonstrated significantly enriched ∂13C with respect to the background isotope enrichment

(∂13C of -24). Hydrogen sulfide is required for reducing equivalents shunted towards carbon

fixation. Whether the background H2S concentration is sufficiently high to fuel carbon fixation at

this rate, or whether sulfide is internally enriched in the berries due to local production by sulfate

reducing bacteria is the focus of experiments described below.

Sulfide and sulfate consumption by pink berries

Using the Cline assay, we detected significant H2S consumption in pink berries over a

46h incubation (Figure 17). Throughout the course of the experiment, both abiotic controls

showed no change in sulfide concentration. All experimental conditions without added sodium

sulfide had no detectable concentrations of sulfide at any time points. We observed a notable

consumption on sulfide in the berry incubation spiked with 500µM H2S throughout the time

course. No data is available at present on the sulfate concentrations throughout the experiment,

though these measurements will be made soon.

Preliminary metagenomic analysis

Green and pink berry metagenomes show the same % contigs with BLAST hits to nifH

with e-values ≤10E-4 (Table 4). Further sequencing and assembly will be able to link gene to its

genomic context and to identify the taxonomic distribution of functional genes such as nifH.

Table 4. Metagenomic reads that map to nifH alleles recovered from GenBank.

Berry Type # Contigs >500bp # contig hits to nif % contigs hits to nif

Green 3945 221 0.056

Red 6322 392 0.062

DISCUSSION

Berry phylogenetic community structure

The diversity of bacterial and eukaryal members of the pink and green berry

communities appears to be well sampled by our SSU rDNA clone libraries, suggesting a

relatively low level of diversity in these samples. The persistence of a lineage of Chromatiales

and Desulfofustis in the pink berries from 2007 to 2010 strongly suggests that the bacterial

members of the berry consortia are involved in a stable and specific consortium, not simply a

random assemblage of planktonic mucilage. Furthermore, the significant difference between

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green and pink berry bacterial communities lends some support to the hypothesis that while

these aggregates represent distinct consortia, rather than different stages in the lifecycle of a

single aggregate. The failure to detect in the green berries any sequences related to the pink

berry Chromatiales cluster further supports the idea that pink and green berries do not arise

simply from shifts in the abundance of community members, though it is possible that these taxa

could be present but below our sampling detection threshold. We hypothesize that the pink and

green berries present an example of convergent evolution in the Sippewissett, indicating a

distinct fitness advantage of macroscopic granular aggregation. Based on the work of Gijs

Kuenen and colleagues on granular sludge (consortial aggregates of nitrification and anammox

bacteria from wastewater treatment upflow reactors) [10], upflow and hydrology of the system

can provide a powerful selective pressure (one among many) for the formation of millimeter

scale microbial aggregates. Further investigation of the hydrology of Sippewissett berry-ponds

would be very informative in addressing this theory.

Sulfur cycling in pink berries revealed by nanoSIMS

Nano-scale isotopic analysis clearly demonstrates the incorporation of 34S in PSB

through reduction of 34-sulfate by SRB within pink berry consortia. Sulfur shuffling between

SRB and PSB within the berry suggests the precedent for syntrophy to be a driving force in the

maintenance of community stability of the pink berry reported in this study through 16s rRNA

libraries (Figures 5,6). We show that community membership is stable over at least two years

despite the likely turnover of the berry population between sampling points. Recovery of

Desulfofustis clones as early as 1997 adds weight to the claim that pink berries retain

constitutive membership [3]. Using this phylogenetic description of community membership, we

have hypothesized a putative sulfur cycle and demonstrated its occurrence within a naturally

occurring microbial consortium. Whether reduced 34-sulfur eventually gets passed as sulfate

back to SRB remains to be determined (to complete the sulfur “cycle” formally).

Additional samples need to be run to verify the results observed so far. We ran one berry

slice per condition (unlabeled vs. labeled) but clear differences were observed (see Results). In

addition, data from a molybdate-incubated berry are intermediate between the unlabeled and

labeled treatments (Figure 11). This suggests that molybdate may reduce total activity of purple

sulfur bacteria through an unknown mechanism, whether through direct inhibition of PSB

metabolism or cutoff of the direct H2S source from the SRB through the postulated mechanism

of SRB inhibition. Additional berry sections from each of two additional berries are in queue for

additional nanoSIMS analysis to corroborate the results reported herein.

19

Additional tracer experiments could be done to examine the fate of fixed labeled carbon

and nitrogen throughout the berry to understand which components are providing the biomass

and who is eating whom (e.g. coupled FISH-nanoSIMS).

Nitrogen Fixation in the Berries

Whether members of green and pink berry consortia are capable of procuring nitrogen

through diazotrophy will strongly impact our model of berry-level physiology. We predicted that

nitrogen fixing by the berries would, along with carbon fixation and an internal sulfur cycle,

enable higher productivity and allow the consortia to maintain a degree of self-sufficiency. The

nitrogen content of marsh sediments like the Sippewissett is made up mostly of ammonium, with

nitrite being limiting except in sources of freshwater inflow. High rates of denitrification have

been observed in tidal marsh sediments, but the relative balance of nitrification and

denitrification depends on salinity changes brought about by cyclical tides. Internal nitrogen

fixation may enable berry members to maintain relative autonomy from the vicissitudes of life in

a tidal marsh and promote the stability and heritability of the community.

Acetylene reduction may not be an appropriate way to assay for nitrogenase activity in

all cases. We report a negative result with acetylene reduction in the pink berries; however, the

pink berry thin sections analyzed with nanoSIMS show a hot signal for the incorporation of 15N2

in the purple sulfur bacteria (Figure 12). The fine resolution and sensitivity of nanoSIMS carries

far more weight than the acetylene reduction assay; therefore, we conclude that at least one

member within the pink berry (Chromatiales) consortium can fix nitrogen.

Berry Stoichiometry

Heavy carbon content of an organism depends on where they get their carbon and the

degree to which their assimilation machinery discriminates between heavy and light isotopes.

Both green and pink berries harbor carbon-fixing organisms with apparently different

fractionation patterns. The carbon isotope profile of dissolved inorganic carbon (DIC) in the

surrounding seawater at Little Sippewissett is unknown. Literature values for marine

environments range from -24 to +15 depending on the nature of the microbial loop and whether

biogenic methane—notoriously depleted in 13C--is cycled substantially [14]. Atmospheric ∂13C

tends to be around -7.8; that for C3 and C4 plants are -27 and -13, respectively, and DIC in the

marsh will be affected by the relative contribution of primary producers [14]. The difference can

result if either the carbon fixers in green and pink berries are drawing on different inorganic

20

carbon sources or there are differences in fractionation patterns between their respective

carbon assimilation pathways. Future work can focus on the carbon cycle within the

Sippewissett marsh system and what if any role the pink and green berries play in affecting the

∂13C of DIC and DOC in this tidal marsh trophic network.

What could be making up the biomass of the green berries that is neither C nor N? If

there is more phosphorus per unit biomass, berry-level productivity should be higher. This can

be tested by digesting whole berries and using an acid persulfate colorimetric assay to quantify

phosphorus content (E K Hall, personal communication). Identifying the limiting nutrients in pink

and green berry consortia will help us predict their production rates in situ and provide a scaffold

on which to estimate system-level inputs into the tidal marsh system.

Drawing conclusions about inherent C:N differences between green and pink berries is

premature given the significant variation between days in C:N among many pink berries (Figure

16). Whether C:N is strongly condition-dependent in one or both berry types could be an

interesting avenue for future research. Bulk berry C:N will be driven by the biomass makeup of

different community members; therefore, temporal or spatial variation in consortium

membership may lead to berry-level variation in stoichiometry. At least for pink berries,

community membership seems to be extremely stable. As such, C:N shifts may have more to

do with physiological plasticity than membership differences. The least exciting option is that

C:N changes are complete artifacts of low sample biomass in pink berries from batch 1 (Figures

14, 16). In this case, C:N would be equivalent between pink and green berries on the whole.

Does high sulfide inhibit pink berry carbon fixation?

Results outlined in Figure 15B suggest that H2S is a blessing and a curse. The addition

of sulfide marginally increased the degree of 13C uptake by pink berries after a 6h incubation.

However, at high supplementation (+500µM), carbon incorporation dropped to a level

indistinguishable from the no-addition treatment (Figure 15B). The dissolved H2S content of the

marsh likely fluctuates dramatically with light, temperature and tidal cycles; during dead low tide

at high noon, sulfide production rates are detectably higher (PT Humphrey, personal

observation) than at night or during high tide, potentially reaching 1-10mM at the sediment-

water interface (J Pavissich, personal communication). Temporal variation in sediment-level

sulfide production may therefore dictate the times during when of pink berry consortia can

allocate resources towards biomass. An intriguing alternate hypothesis is that berry structure

itself can act as a physical diffusion buffer against the more toxic side effects of sulfide. Under

21

this model, sulfur cycling would be accomplished at some basal rate within the berry by the

action of SRBs evolving H2S and direct consumption by closely associated PSBs. We have

demonstrated concrete evidence of sulfur cycling in the pink berries; however, the links between

environmental sulfide and internal sulfur cycling remain to be quantified. Future work could more

intricately describe variation in sulfide production in the marsh and couple these data with a

closer look at the factors contributing to variation in carbon fixation rates (and therefore berry

growth rate).

Pink berry fitness, ecology and evolution

What are the major determinants of and constraints on berry-level fitness? Are they

reproductive, and if so, is the community itself heritable? Does the berry consortium lifestyle

contribute meaningfully to the evolutionary fitness of the component species separate from the

berry life-style? Our data help to shed light on the community structure, metabolic network and

environmental sensitivities of pink berries. Linking the phylogenetic structure of berry

communities with isotope, carbon and nitrogen fixation information and specific syntrophic sulfur

transfer is a major step in unveiling the berry mystery.

We have established a direct syntrophic link between resident sulfate reducing bacteria

and purple sulfur bacteria in this system. What metabolic properties do the other major

component species (i.e. Cytophaga) demonstrate, and how (if at all) do they contribute to berry-

level eco-physiology? These questions are the target of broader meta-genomic analyses

underway by the authors. The gold standard for any physiological hypothesis, however, is

empirical demonstration. Future students will be charged with devising clever experiments to get

to the bottom of the origin of the EPS that holds the things together, as well as the roles of

Cytophaga spp. and Stramenopiles in the system.

Now for the wacky stuff

We end with the following hypothesis, that large berry size allows the berries to escape

gape-limited predators in the marsh (e.g. hermit grabs, other invertebrates, fish, filter feeding

mollusks, etc.). High berry productivity may be a result of a well-oiled set of internal, syntrophic

processes. If berry-size is a group-level phenomenon, driven perhaps by the interaction of a few

crucial members, berry predator escape represents a potential example of community-level

selection fostering the reciprocation between diverse microbial species with distinct but

compatible physiologies (“euberryotes,” coined by E K Hall). We encourage many future

students to peer down the berry rabbit hole, shake off the vertigo and jump right in.

22

ACKNOWLEGEMENTS We would like to thank all of the berry-obsessed MBL students, past+present (and

future) for their cumulative insights upon which this project was built. TEM, FISH nanoSIMS sample sectioning support was graciously provided by Cristina Moraru and Rebekah Ward. Chunk Pepe-Ranney was fundamental to solving ARB-related crises among other bioinformatic and clone library malarchy. Gargi Kulkarni saved the nitrogen supply to anaerobic box on several occasions and helped us avoid oxidative disaster. Marshall Otter at the Ecosystem Center graciously prioritized our samples for elemental analysis. We would like to recognize Steve Zinder and Dan Buckley for guidance and for securing/procuring the financial support that enabled this project. Special thanks to Victoria Orphan for guidance in experimental design for the nanoSIMS experiment and keeping the machines running in the face of adversity (earthquakes. No, really). We’d also like to recognize FedEx and the MBL Shipping/Receiving office for making science happen and for supplying copious dry ice for our overseas berry traveling shenanigans. Lastly, thanks to all of our fellow students for providing a high-octane, compassionate and supportive environment this summer. Mussel snot (makes it hot).

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REFERENCES

1. Stoodley P, Sauer K, Davies DG, Costerton JW (2002) Biofilms as complex differentiated

communities. Annual Review of Microbiology 56: 187-209. 2. Seitz AP, Nielsen TH, Overmann J (1993) Physiology of Purple Sulfur Bacteria Forming

Macroscopic Aggregates in Great Sippewissett Salt-Marsh, Massachusetts. Fems Microbiology Ecology 12: 225-236.

3. Banin U (1997) Identification of the bacteria forming the "berries"- purple macroscopic aggregates in the Great Sippewissett salt Marsh. Microbial Diversity Course, MBL.

4. Scott JJ (2007) Phylogenetic relationship and organization of within berry bacterial consortia. Microbial Diversity Course, MBL.

5. Dawson SC, Pace NR (2002) Novel kingdom-level eukaryotic diversity in anoxic environments. Proc Natl Acad Sci U S A 99: 8324-8329.

6. Pruesse E, Quast C, Knittel K, Fuchs BM, Ludwig W, et al. (2007) SILVA: a comprehensive online resource for quality checked and aligned ribosomal RNA sequence data compatible with ARB. Nucleic Acids Res 35: 7188-7196.

7. Ludwig W, Strunk O, Westram R, Richter L, Meier H, et al. (2004) ARB: a software environment for sequence data. Nucleic Acids Research 32: 1363-1371.

8. Wang Q, Garrity GM, Tiedje JM, Cole JR (2007) Naive Bayesian classifier for rapid assignment of rRNA sequences into the new bacterial taxonomy. Appl Environ Microbiol 73: 5261-5267.

9. Deza H, Fonio de Gijon T, Puig J, Remis JA (1987) [Benign granular cell tumor of the esophagus: apropos of a case]. Acta Gastroenterol Latinoam 17: 119-126.

10. Schloss PD, Westcott SL, Ryabin T, Hall JR, Hartmann M, et al. (2009) Introducing mothur: open-source, platform-independent, community-supported software for describing and comparing microbial communities. Appl Environ Microbiol 75: 7537-7541.

11. Lozupone C, Hamady M, Knight R (2006) UniFrac--an online tool for comparing microbial community diversity in a phylogenetic context. BMC Bioinformatics 7: 371.

12. Lozupone CA, Knight R (2007) Global patterns in bacterial diversity. Proc Natl Acad Sci U S A 104: 11436-11440.

13. Blazejak A, Erseus C, Amann R, Dubilier N (2005) Coexistence of bacterial sulfide oxidizers, sulfate reducers, and spirochetes in a gutless worm (Oligochaeta) from the Peru margin. Applied and Environmental Microbiology 71: 1553-1561.

14. Bouton, T.W., 1991. Stable carbon isotope ratios of natural materials. In: Coleman, D.C., Fry, B. (Eds.), Carbon Isotope Techniques. Academic Press, New York, pp. 155-171.

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Appendix 1. Eukaryotic 18S primer information. 360FE primer – Sequence 5′-CGGAGARGGMGCMTGAGA-3′ Appendix II. 34-sulfate, 13-carbonate 15-N2 labeling experiment Please refer to Figure S1 for experimental design. Schematic Overview of Conditions: CONDITION NAME PINK PURPLE CONDITIONS A 1 11 34-sulfate + 13-bicarb + 0.5 mM sulfide B 2 12 34-sulfate + 13-bicarb + 0.5 mM sulfide + 10 mM molybdate C 3 13 34-sulfate + 13-bicarb + 0.5 mM sulfide + 20 mM molybdate D 4,5 14,15 32-sulfate + 12-bicarb + 0.5 mM sulfide E 1 11 34-sulfate + 13-bicarb + 3 mM sulfide F 1 11 34-sulfate + 13-bicarb + 3 mM sulfide + 10 mM molybdate G 3 13 34-sulfate + 13-bicarb + 3 mM sulfide + 20 mM molybdate H 4,5 14,15 32-sulfate + 12-bicarb + 3 mM sulfide Solutions Prepared: Seawater with MOPS buffer: - filtered ~0.5 L of water from the sediment interface of the berry pond - bubbled for ~20 minutes with CO2/N2 - incubated in anoxic chamber for 2-3 hrs - added 25 mL anoxic MOPS (what concentration?) - filter sterilized - added 3 mL to each 5 mL incubation vial 34-S labelled Na2SO4 at 867.8 mM - added 2 mL anoxic milliQ water directly to the vial containing 250 mg material Regular Na2SO4 at 880 mM - to added 250 mg Na2SO4 filled 2 mL volumetric with anoxic milliQ water Na2MO4 dihydrate at 400 mM - 482 mg in 5 mL anoxic milliQ water 10 mM and 100 mM Na2S solutions previously prepared anoxically 13-C NaHCO3 bicarbonate 0.6 M - 250 mg in 5 mL anoxic milliQ water, stoppered immediately to prevent CO2 escape Regular bicarbonate 0.6 M - as above Exact protocol: Condition A: for vials 1 and 11 Volume to remove: 297 uL Add: 97 uL 879.8 mM 34-sulfate 150 uL 10 mM Na2S 50 uL 0.6 M 13-C bicarb 2 mL 15-N2 gas Condition B: for vials 2 and 12 Volume to remove: 372uL Add: 97 uL 879.8 mM 34-sulfate 150 uL 10 mM Na2S 50 uL 0.6 M 13-C bicarb

25

2 mL 15-N2 gas 75 uL 400 mM sodium molybdate Condition C: for vials 3 and 13 Volume to remove: 447uL Add: 97 uL 879.8 mM 34-sulfate 150 uL 10 mM Na2S 50 uL 0.6 M 13-C bicarb 2 mL 15-N2 gas 150 uL 400 mM sodium molybdate Condition D: for vials 4,5 and 14,15 Volume to remove: 296 uL Add: 96 uL 880 mM unlabeled-sulfate 150 uL 10 mM Na2S 50 uL 0.6 M unlabeled bicarb 2 mL unlabeled N2 gas Condition E: for vials 6 and 16 Volume to remove: 237 uL Add: 97 uL 879.8 mM 34-sulfate 90 uL 100 mM Na2S 50 uL 0.6 M 13-C bicarb 2 mL 15-N2 gas Condition F: for vials 7 and 17 Volume to remove: 312 uL Add: 97 uL 879.8 mM 34-sulfate 90 uL 100 mM Na2S 50 uL 0.6 M 13-C bicarb 2 mL 15-N2 gas 75 uL 400 mM sodium molybdate Condition G: for vials 8 and 18 Volume to remove: 362 uL Add: 97 uL 879.8 mM 34-sulfate 90 uL 100 mM Na2S 50 uL 0.6 M 13-C bicarb 2 mL 15-N2 gas 150 uL 400 mM sodium molybdate Condition H: for vials 9.10 and 19,20 Volume to remove: 236 uL Add: 96 uL 880 mM unlabeled-sulfate 90 uL 100 mM Na2S 50 uL 0.6 M unlabeled bicarb 2 mL unlabeled N2 gas All additions were performed as follows:

3 mL of the filter-sterilized MOPS-buffered berry pond water was added to each 5 mL volumetric flask numbered 1-20

The amount of water listed in the "Volume to Remove" above was removed with a micropipettor

5 large sized berries were added to each vial. Vials 1-10 recieved pink berries while vials 11-20 were filled with purple berries

26

The 34-S labeled sodium sulfate or unlabeled sodium sulfate were next added to the appropriate vials (above)

Then, sodium molybdate solutions were added to the appropriate vials

From then on, each vial was processed one by one as follows. First sodium sulfide solution was added by 1 mL syringe, then either 13-C labeled bicarbonate or unlabeled bicarbonate was added by micropipet. Vials were then sealed with a rubber stopper. 2 mL of head space was removed via syringe and 2 mL of either 15-N labeled nitrogen gas or unlabeled nitrogen gas was added via syringe. Vials were then sealed with parafilm and wax. Tape was later applied to prevent the stoppers from coming loose due to any possible build up of pressure.

Vials were removed from the anaerobic chamber and placed into a metal tube rack that was placed in the 30 degree C incubator on a light/dark cycle (the cyano incubator)

27

Figure 1. Green and pink berry collection site within Little Sippewissett Marsh, West Falmouth

MA. Marsh water used for all incubation experiments was collected from this site as well.

28

A B

C D

E

Figure 2. Rarefaction curves for 16S bacterial clone libraries from A) pink berries including sequences from 2010 and 2007 and B) green berries and 18S eukaryal clone libraries from C) pink and D) green berries. E) 16S archaeal rarefaction curve from pink berries.

29

Figure 3. Classification of archaeal 16S sequence from pink berries.

Bacteria8% Crenarchaeota

5%

Euryarchaeota67%

unclassified_Archaea11%

unclassified_Root9%

30

C D

Figure 4. Phylum-level classification of 16S bacterial sequences from A) green and B) pink berries. C) Class-level classification of proteobaceobacteria from C) green and D) pink berries.

31

Figure 5. Maximum likelihood phylogeny of pink berry 16S bacterial sequences belonging to the Chromatiacaea, represented in red on the tree. Bootstrap support based on 50 replicates in RAxML rapid bootstrapping method. The collapsed polytomy of 107 clone sequences includes representatives from both the 2007 and 2010 16S clone libraries constructed from the pink berries. Within this collapsed group there was no bootstrap support for further clustering of these very similar sequences. Sequences labeled pnk37E05 and pur96H12 were obtained from the 2007 clone library.

32

Figure 6. Maximum likelihood phylogeny of pink berry 16S bacterial sequences belonging to the delta-proteobacteria, represented in red on the tree. In green are some, but not all of the green berry delta-proteobacterial isolates. Bootstrap support based on 50 replicates in RAxML rapid bootstrapping method. The collapsed polytomy of 71 clone sequences includes representatives from both the 2007 and 2010 16S clone libraries constructed from the pink berries. Within this collapsed group there was no bootstrap support for further clustering of these very similar sequences. Both the green berry sequences and those red berry sequences labeled _EGW were obtained from our 2010 clone library.

33

Figure 7. Classification of eukaryal 18S sequences from A) pink and B) green berries. Note that 4 out of 86 of green berry 18S clones and 1 out of 91 pink berry 18S clones were bacterial 16S sequences which are not presented here.

34

Figure 8. Maximum likelihood phylogeny of pink (red taxa) and green (green taxa) berries 18S bacterial sequences classified as stramenopiles. Bootstrap support based on 50 replicates in RAxML rapid bootstrapping method.

35

36

Figure 9. CARD FISH hybridization of sectioned pink berries with a nonsense probe (Non338, panel A), a Gammaproteobacteria probe (Gam42a, panel B), a Deltaproteobacteria probe (Delta 495a-c + competitor a-c, panel C) and a Cytophaga probe CF319a, panel D,E,F). The left side of each panel shows the probe signal and autofluorescence, the right side of the panel show the DAPI stain.

37

Figure 10. TEM of 0.01 and 0.150µM-thin pink berry sections. Sulfur globule vacuoles can be seen in (A) and (B) closeup. Rod-shaped bacilli can be seen interspersed with PSB in (C) and

(E). (D) displays what look to be exopolysaccharide matrix. (F) shows PSB binary fission.

38

Figure 11. nanoSIMS shows heavy C,S and N isotope incorporation in pink berries. Multiple

measurements were taken throughout regions of a single 10µM section of each of three berries incubated under the conditions specified on the x-axis. Data are ∂ values derived from

secondary ion mass spectrometry. NO LABEL (n=4 measurements) = light carbonate, sulfate incubation, representing background values in the berries; LABEL (n=9 measurements) = 13carbonate, 34sulfate and 15N2 incubations. +MoO4 (n=7) = sodium molybdate (10mM), a

putative SRB inhibitor. A,B,C indicate statistically significantly different groups. AvB p<0.0001 and AvC p=0.02 for C,S and N.

39

Figure 12. Heavy carbon (A), nitrogen (B) and sulfur (C) accumulation in berry sections

recorded by nanoSIMS. ‘-‘ Un-Labeled treatment berry; ‘+’ Label treatment berry. Note the order of magnitude of the heat map scales and the scale bar on the heat map plane. PSB cells are on

average 2µm wide, corresponding well to the width of enrichment foci in (+)-plots.

40

Figure 13. Green berries reduce acetylene, demonstrating nitrogenase activity. Each data point represents an average of 2 measurements for each replicate (2 per treatment) taken after 50h

of incubation. NC=negative control (media + acetylene). NC values indicate a significant fraction of acetylene is reduced abiotically during incubation.

41

Figure 14. Berry stoichiometry. [A.] Differences in dry biomass of similarly sized green and pink

berries used for elemental analysis suggest red berries are less dense. [B.] Percent C in red and green berries shows no significant difference. [C.] %N is higher in red berries, which

accounts for the finding in [D.], which shows a statistically lower C:N in red berries suggesting less nitrogen limitation.

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Figure 15. Isotope fractionation in the berries. [A.] Natural stable isotope ratios differ between green and red berries. [B.] 13-Carbon enrichment in red berries through carbon fixation trends towards an increase with higher H2S availability. Pariwise Student’s t tests: +0µM vs. +250µM:

t=-2.51, p=0.07; all other, p>0.1.

43

Figure 16. C:N ratio varies between experiments in pink berries. Biomass of pink berries in

experiment 2 was consistently higher and similar to that for green berries, suggesting either low-mass associated error or condition-dependent biotic variation in C:N. A/B indicate statistical

groups.

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Figure 17. Pink berries consume H2S when present at 500µM. Abiotic control represents

medium without pink berries. Berries alone incubated with SO42—rich media do not produce

detectable H2S in batch culture. Molybdate treatment was hypothesized to reduce the H2S production in berries.

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Figure S1. Schematic of experimental design for stable isotope probing of pink berries.

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Figure S2. Schematic of experimental design for sulfide and sulfate consumption time course experiment. Narwahls are awesome.