Calcite-accumulating large sulfur bacteria of the genus Achromatium in Sippewissett Salt Marsh

Supplementary information

Verena Salman, Tingting Yang, Tom Berben, Frieder Klein, Esther Angert,

Andreas Teske

Materials and Methods

Sampling details June-July, 2012 Achromatium cells collected from no particular depth were used for light

microscopy, DIC microscopy, DAPI- and FITC-staining/confocal microscopy.

June-August 2013 One push core was used to collect sediment from depth 0-1 cm and 1-2 cm for

phylogenetic community analysis; two additional syringe cores were taken for

sectioning in 2 mm steps for Achromatium cell counts; collected Achromatium

cells from no particular depth were used for 16S rRNA gene sequencing (both, via

multiple displacement amplification and DNA extraction), for Raman analysis, and

fixed for CARD-FISH analysis.

July-August 2014 Three individual push cores from midday, and an additional three individual cores

from midnight were used for microprofiling with sensors; from these six cores cells

were collected at 1 cm intervals for SEM-EDS; one additional syringe core was

collected for sectioning in 2 mm steps for Achromatium cell counts; collected cells

from no particular depth were used for calcium-staining, specific dissolving of

internal inclusions, and additional Raman analysis.

Beam penetration in SEM/EDS

The penetration depth of an electron beam is dependent on the density of the

material and the energy of the electron shell of the element (K edge). The depth (Z)

can then be calculated with the following equation

Z = 0.033 * (E˚2 - Ec2) / ρ

where E˚ is the energy of the electron beam (here 15 keV), Ec is the energy of the

electron shell of the element (4.034 for calcite, and 2.471 for sulfur), and ρ is the

density of the material (ca. 2.7 g/cm3 for calcite, and 1.31 g/cm3 for sulfur according

to (Steudel et al 1990)).

This gives specific penetration depths of:

~2.5 µm for calcite

~5.5 µm for sulfur

The electron beam directed onto an Achromatium cell accordingly penetrates first a

nm-thick outer membrane and wall, then penetrates the sulfur globules of ca 1-2

µm (assumed to be in the periplasm, Pattaragulwanit et al 1998, Strohl et al 1982,

Williams et al 1987, Wirsen and Jannasch 1978) then passes the cytoplasmic

membrane and <2 µm of cytoplasm, before it finally reaches the large calcite

inclusions of ca. 4-5 µm.

In conclusion, even if a cell does not contain (much) sulfur, the beam does not

penetrate any deeper than into the first layer of calcite around the cell periphery. An

increase in S signal/deposits dilutes the Ca signal (CaCO3 and Ca2+) from deeper

regions of the cell, and vice versa, an increased Ca signal indicates that the cell

contains less S in its periphery. Net amounts of Ca and S signal per image thus give

a relative implication on the amount of S versus Ca within the ~5 µm-thick penetration

depth.

Results and Discussion

Figure S1: Microscopic observations of marsh Achromatium cells. (A) FITC staining shows the cytoplasm filling the interstitial space between the intracellular inclusions of a cell that is dividing with a flat division plane. (B) FITC staining and focusing on the cell surface with a confocal fluorescent microscope reveals that the outer rim of the Achromatium cell contains numerous small, spherical and rod-shaped bacteria. (C) Differential interference microscopy of this cell reveals short, translucent filaments attached on the outside.

Figure S2: Two-dimensional structure of the V6 region of Achromatium spp. Representative sequence affiliating to cluster A (A), cluster B (B), and the full-length sequence retrieved in this study of the novel marsh Achromatium (C).

Figure S3: Model illustrating the working hypothesis for the physiological function of calcite in marine, and possibly also freshwater, Achromatium spp. Specific influx/efflux mechanisms for carbonate and/or H+ across the calcite granule membrane remain speculative at this point. Deprotonation of bicarbonate in the cytoplasm may be an enzymatic reaction rather than a spontaneous consequence of proton deficiency during sulfide oxidation, because the pK value for HCO3

-/CO32- is 10.3. Calcite dissolution happens at pH values <6, so the

release of CO32- resulting from dissociation of calcite may lead to a spontaneous

formation of HCO3- in the cytoplasm.

equation S1 HS- + H+ + 1/2 O2 → S0 + H2O

equation S2 5 HS- + 2 NO3- + 7 H+ → 5 S0 + N2 + 6 H2O

equation S3 4 HS- + NO3- + 6 H+ → 4 S0 + NH4

+ + 3 H2O

equation S4 HCO3- → CO3

2- + H+; and CO32- + Ca2+ → CaCO3

equation S5 2 S0 + 3 O2 + 2 H2O → 2 SO42- + 4 H+

equation S6 5 S0 + 4 NO3- + 8 H2O → 5 SO4

2- + 2 N2 + 16 H+

equation S7 4 S0 + 3 NO3- + 7 H2O → 4 SO4

2- + 3 NH4+ + 2 H+

equation S8 CaCO3 → Ca2+ + CO32-; and H+ + CO3

2- → HCO3-

Table S1. Elemental analysis of individual cells from different depths of the sediment, excluding all other elements, such as carbon, oxygen and hydrogen. depth DAY average Ca [%] average S [%] s.d. [%] # of cells sedim./water interface 99.27 0.73 0.42 2 0-1 cm 98.48 1.52 1.92 11 1-2 cm 96.50 3.50 6.3 11 2-3 cm 69.72 30.28 26.4 7 3-4 cm 60.29 39.71 23.9 11

depth NIGHT average Ca [%] average S [%] s.d. [%] # of cells 0-1 cm 88.4 11.6 13.3 3 2-3 cm 1.9 98.1 1.6 3

Table S2: Pyrosequencing results of phylogenetic community analysis given in percent of total OTUs. The upper two centimeters of the marsh sediment were sequenced separately ('0-1 cm', and '1-2 cm'). To increase the chance of retrieving Achromatium-related data hand-picked Achromatium cells were sequenced ('handpicked'), and data include bacteria that may have been more or less tightly associated with the outside of the Achromatium cells. All taxonomic groups that were dominating in the sediment samples, or were indifferent among all three samples, were highlighted in brown. Groups that were clearly dominating in the handpicked sample over the sediment samples were highlighted in yellow. Note also that the here assigned family name Thiotrichaceae includes the family Achromatiaceae. It was recently suggested to separated the taxon Thiotrichaceae into its validly described families again (Salman et al 2011, Teske and Salman 2014).

Community analysis. Partial 16S rRNA gene sequencing of bulk DNA from the

sediment-associated community in the upper 2 cm of the Achromatium-containing

marsh sediment revealed a dominance of bacteria associated with sulfur cycling

in marine environments (Table S2). We retrieved 5178 reads from the sediment

depth 0-1 cm, and 4682 reads form 1-2 cm. Both sediment layers were dominated

by Proteobacteria (54.7% and 43.3%, respectively), most significantly

Gammaproteobacteria (32.3% and 20.5%) and Deltaproteobacteria (12.4% and

16.4%). Bacteroidetes were also abundant (ca. 17% each, the majority of which

could not be classified at higher taxonomic level than this phylum); and minor

groups were the Cyanobacteria with 5.4% and 4.1%, Rhodobacteraceae of the

Alphaproteobacteria with 5.5% and 2.8%, and Planctomycetes with 2.4% and

4.1%. Among the Gammaproteobacteria, the majority (67.3% and 58.2%)

affiliated with the family Chromatiaceae, which includes anoxygenic phototrophs

that use reduced inorganic substrates such as sulfide (including the partners of

the pink consortia). Another 10.5% and 11.7% affiliated with the family

Alteromonadaceae that contains primarily facultative aerobic heterotrophic

bacteria. Approximately 20% of the Gammproteobacteria remain unclassified at

the family level. Among the Deltaproteobacteria, the family Desulfobacteraceae

that includes primarily strictly anaerobic sulfate reducers dominated the upper 2

cm of sediment with 71.7% and 72.4%; the anaerobic, sulfate-reducing families

Desulfobulbaceae (including the partners of the pink consortia; Wilbanks et al

2014) and Desulfovibrionaceae were represented by 8.3% and 5.4%, and 1.7%

and 0.4%. Members of the Desulfobulbaceae were also recently described as

"cable bacteria" oxidizing sulfide with oxygen via electronic conductance, whereby

they are able to physically separate these two gradients over a mm to cm scale

(Nielsen et al 2010). With this bulk sequencing approach, we only retrieved three reads affiliated with

the Achromatiaceae family in each sediment depth, which reflects the fact that

marsh sediments can be populated by 107-109 bacterial cells per ml or g sediment

(Edgcomb et al 1999, Rublee and Dornseif 1978, Rublee 1982), and

Achromatium make up only a small fraction of the total cell numbers (<103).

Physical enrichment of Achromatium cells, as described above, is thus

recommended to obtain phylogenetic information from this population.

Hypotheses about the formation of calcite. One hypothesis suggested

calcification lowers the pH to convert HCO3- into CO2 to support carbon fixation

(Head et al 1995, Head et al 2000). However, this hypothesis is questionable

because 1) calcite-containing Achromatium populations were detected in an

acidic lake (pH 4.5), where CO2 is the dominating DIC species (Glöckner et al

1999); and 2) not all Achromatium cells are autotrophic or contain RuBiSCo (Gray

et al 1999, Gray et al 2000).

Calcite as a buoyancy mechanism was ruled out before (Head et al 2000),

because cells contained more calcite at the surface and less in deeper layers

(Babenzien 1991, Lauterborn 1915). However, we propose a modified form of this

hypothesis as cells at the sediment surface would need to become heavier in

order to avoid suspension in oxic waters. In Sippewissett, the tide pools fill and

drain gradually, suggesting a benefit for a calcite-anchoring function.

A recent hypothesis proposed that the precipitation of calcite is used to generate

extracellular protons to retrieve bioavailable dissolved sulfide from solid iron

sulfides (Gray 2006). Even though this hypothesis does not hold for a high-sulfide

marine environment, the scenario described for the freshwater realm including

thermodynamic calculations presented for the given nutrient concentrations is

plausible. It was also critically mentioned that calcification (energy sink) is

energetically linked to sulfide oxidation (energy source), and that in high-sulfide

environments like a marine system, calcifying organisms might be energetically

less competitive with non-calcifying bacteria. Hence, internal calcification in

Achromatium was until now only observed in freshwater, low-sulfide environments

(Gray 2006). Our observation coincides with this statement, as we see little calcite

in cells at depths where sulfide is high, but detect an increased accumulation

when cells are in proximity to the sediment surface - in particular when the

surface is highly oxic. Energetically, sediment layers with a higher redox potential

can thus allow competitive calcification in a marine system, and might be initiated

when cells travel upwards and reach zones of higher redox potential, such as the

nitrate-reduction zone.

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