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Supporting Information

Materials and Methods

Cloning of cingulin cDNAs. Synthesis of a T. pseudonana cDNA library coupled to

oligo(dT)25 magnetic beads (Invitrogen, USA), and 5‟- and 3‟- rapid amplification of

cDNA ends (RACE) PCR of cingulin cDNAs was performed as described previously (1).

Oligonucleotides for RACE PCR were deduced from the sequence of the cingulin gene

models in the T. pseudonana genome database (see SI table 1 for protein IDs).

Cingulin-specific sense primers for amplifying the 3‟-end of cinY1 were 5‟-GAC

GAT TAC ACT CCT TCC AC-3‟ (first PCR) and 5‟-CGA TTA CTC TCA TTC CAC CAA

C-3‟ (second PCR), of cinY2 were 5‟-ATG ACG GAC ATG GAT ATG G-3‟ (first PCR)

and 5‟-GGA GAA ATA GAA GGT TGG G-3‟ (second PCR), of cinY3 were 5‟-GCA AGG

GTG AGG GAT ACC A-3‟ (first PCR) and 5‟-GGA TAC CAC ATG TTC CAT GAC A-3‟

(second PCR), of cinW1 were 5‟-GAC ATG GAG ACT GGT CTG AC-3‟ (first PCR) and

5‟-GCT GGG GCG GTG ACT AC-3‟ (second PCR), of cinW2 were 5‟-GGG GAG GAC

ACT ACG ATG T-3‟ (first PCR) and 5‟-CGA TGT TGA TGT TGA TGA TGA TGA T-3‟

(second PCR) and of cinW3 were 5‟-AGT GGA GCC GGA GCT GAT-3‟ (first PCR) and

5‟-GTT GGG AGG CCG ATG GAT-3‟ (second PCR).

Cingulin-specific sense primers for amplifying the 5‟-end of cinY1 were 5‟-AAT

GGA GTT GGT GCG AGG G-3‟ (first PCR) and 5‟-GCG AGG GTT TTG TTG GTG C-3‟

(second PCR), of cinY2 were 5‟-TCG TCG TAG GAA CCC C-3‟ (first PCR) and 5‟-CGG

GAG TTT GCT CCG-3‟ (second PCR), of cinY3 were 5‟-CGT AGG AGT ACT GCG G-3‟

(first PCR) and 5‟-GCT GTT CGG CCG TGC C-3‟ (second PCR), of cinW1 were 5‟-TCC

TTC CCA GAC CCA GTG-3‟ (first PCR) and 5‟-GAC CAT CCA TCA TCG CTC C-3‟

(second PCR), of cinW2 were 5‟-ATG TAC ACC CAG TGG CCG T-3‟ (first PCR) and 5‟-

TGG AGT AGT CTC CTC CCC-3‟ (second PCR) and of cinW3 were 5‟-CCC ATC CAC

TGA CCC AGT-3‟ (first PCR) and 5‟-CAT CCA TCG TCA CTC CAG TC-3‟ (second

PCR).

To experimentally validate the predicted intron/exon structures of cingulins,

reverse-transcription PCRs were performed from the T. pseudonana cDNA library using

cingulin-specific oligo pairs. The deduced amino acid sequences derived from the

amplified genes of CinY2, CinY3, and CinW2 perfectly matched the sequences of the

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corresponding gene models in the T. pseudonana genome database. The amino acid

sequences derived from the other amplified cingulin genes contained multiple sequence

differences compared to the genome derived gene models.

All PCR products were ligated to vector pJet1.2 (Fermentas, USA) and

sequenced.

Construction of cingulin-GFP expression vectors. To construct pTpNR-

GFP/fcpNat(-KpnI) for expression of C-terminal GFP fusion proteins under the control of

the nitrate reductase promoter, the egfp gene was amplified by PCR from a plasmid

using sense primer 5′-ATC GCA TGC GGT ACC GGC GGA ATG GTG AGC AAG GGC

GAG G-3′ (SphI site in bold; KpnI site underlined) and antisense primer 5′-GAA TGC

GGC CGC TTA CTT GTA ACA GCT CGT CCA TG-3′ (NotI site underlined). The PCR

product was ligated with the EcoRV and NotI sites of the vector pTpNR (2) in which the

KpnI site had been destroyed. The resulting plasmid, pTpNR-GFP, consisted of the

regulatory sequences of the NR gene flanking the egfp gene, which carries a 5′

extension containing three unique restriction sites (EcoRV, SphI, and KpnI) allowing for

the in-frame insertion of genes. For insertion of the nourseothricin resistance cassette

fcpNat(-KpnI) into plasmid pTpNR-GFP, a nat gene without the internal KpnI site was

generated by PCR using the oligonucleotides (5‟- GAA TGC GGC CGC TCA GGG GCA

GGG CAT GCT CAT G-3‟; 5‟-ACC AAA ATG ACC ACT CTT GAC GAC ACG GCT TAC

CGT TAC CGC ACC AGT-3‟) (NotI site underlined). The PCR product was ligated to

EcoRV and NotI digested vector pTpfcp (2) resulting in plasmid pTpfcp/nat(-KpnI). The

fcpNat(-KpnI) DNA fragment was cut out of plasmid pTpfcp/nat(-Kpn) using

HindIII(blunt) and XbaI and ligated to the SmaI and XbaI digested vector pTpNR-GFP

resulting in the final plasmid pTpNR-GFP/fcpNat(-KpnI).

For the construction of cingulin-GFP expression vectors, the cingulin genes were

PCR amplified from genomic DNA of T. pseudonana and inserted into the EcoRV and

KpnI sites of the vector pTpNR-GFP/fcpNat(-KpnI). The tyrosine rich cingulin genes

inserted into pTpNR-GFP/fcpNat(-KpnI) lacked the RXL domain coding gene segment.

The CinW3 gene inserted into pTpNR-GFP/fcpNat(-KpnI) lacked the gene region that

encodes the C-terminal 19 amino acids.

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The following oligonucleotide pairs were used to amplify cingulin genes or their

derivatives for insertion into EcoRV and KpnI digested pTpNR-GFP/fcpNat(-KpnI):

CinY1: 5‟- GAT ATC ATA ATC ATG AAG TCG ATC ATC GCC CTC TC -3‟ and

5‟- GAT CGA TAT CGT AGT AAG CAT CGT CGT CAT AT-3‟

CinY2: 5‟-GAT TGA TAT CAT AAT CAT GAA GTT AAT CAT CGC CCT CA-„3

and 5‟-GAT CGG TAC CAT TTC TCC TGA CGT ACT CAT CG-3‟

CinY3: 5‟-CTA GAT ATC ATA ATC ATG AAG TTC AGC GCC TCC AT-„3 and 5‟-

TAT GGT ACC CGT CCT CCT TCC GTA TCC-3‟

CinW1: 5‟-CTA GAT ATC ATA ATC ATG AAG ATC GGA TAC TCA TTA GCT

TT-„3 and 5‟-GCC GGT ACC GAA GCC TCC GTA TTC ACG G-3‟

CinW2: 5‟- CTA GAT ATC ATA ATC ATG AAG CTC GCT CTC TTC CTA A-„3

and 5‟-GCC GGT ACC CCA TCC ACT GTA CCA TCC C-3‟

CinW3: 5‟-CTA GAT ATC ATA ATC ATG AAG GCA GCT TTG ATA TTG GC-„3

and 5‟-GCC GGT ACC GTT GGA GCT AGA CGC TTC TGT-3‟

In the oligonucleotide sequences above the KpnI sites are underlined, and the

EcoRV sites are presented in bold.

For expression of the tpSil3-GFP fusion protein, plasmid pTpSil3-GFP was

constructed by replacing the regulatory sequences of the fcp gene (3) with promoter

and terminator regions of the tpSil3 gene. To construct this plasmid the 5‟-end of the

tpSil3 gene together with 820 bp of the tpSil3 promoter region were amplified from

gDNA using the oligonucleotides 5‟-GTG TGG GTG GGA TGA GGA G-3‟ and 5‟-GAA

TAT GAG AAT GAT GCA TGG C-3‟. The 1165 bp PCR product was digested with XhoI

and inserted into the XhoI site of pTpfcp/Sil3-GFP (3), resulting in plasmid pTpSil3-

GFP/fcpt. The tpSil3 terminator was amplified from gDNA using oligonucleotides 5‟-GAA

TGC GGC CGC GTT CAT CAT CTT CAT ATC GTA TG-3‟ and 5‟-TGG GCG TTG GAG

GTG GAG-3‟ that introduced a NotI site (underlined). The 551 bp PCR product was

digested with NotI and inserted into the NotI/SmaI site of pTpSil3-GFP/fcpt resulting in

the target plasmid pTpSil3-GFP.

Transformation of T. pseudonana and selection of transformants was performed

as described previously (2).

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Biosilica isolation. Biosilica from T. pseudonana wild-type cells was isolated as

described previously (1). Biosilica from T. pseudonana strains expressing GFP fusion

proteins was isolated using a slightly modified protocol as is described in the following.

Cells were extracted twice in 2 % SDS, 0.1 M EDTA pH 8 at 55 C for 60 min, and

washed once with 0.1 M EDTA pH 8 after the first SDS extraction step. The material

was then subjected to three washes with 1 mM phenylmethylsulfonyl fluoride (PMSF),

followed by alternating washes with acetone and H2O until the acetone supernatant was

colorless. After the last acetone wash the biosilica was washed three times with H2O,

lyophilized, and stored at -20 °C.

SDS-PAGE and Western blot. Isolated biosilica (50 mg) of T. pseudonana strains

expressing GFP fusion proteins was resuspended in 10 M NH4F, adjusted to pH 4-5 by

drop wise addition of 6 M HCl, and incubated for 30 min. Ammonium fluoride soluble

and insoluble material was separated by centrifugation at 4,000 g for 15 min at 4 oC.

The soluble material was dialyzed (molecular mass cut off 6 - 8 kDa) against 200 mM

ammonium acetate, and then against 100 mM ammonium acetate. The dialysate was

lyophylized twice, and the residue was dissolved in 100 µl H2O yielding the ammonium

fluoride extract. Five microliters of the ammonium fluoride extract was mixed with SDS-

sample buffer and subjected to SDS-PAGE (see below).

The ammonium fluoride insoluble material (i.e., pellet from 4,000 g centrifugation)

was washed twice with 200 mM ammonium acetate, resuspended in 1 ml 100 mM

ammonium acetate, and sonicated (Sonicator 3000, Misonix Inc., USA) for 5-20 s until

the suspension appeared homogenous. The suspension was centrifuged at 14,000 g for

5 min at 4 oC, and the resulting pellet resuspended in 200 µl of a solution containing 1

% SDS and 0.1 M DTT, and incubated for 10 min at 95 °C. Five microliters of sample

was mixed with SDS-sample buffer and subjected to SDS-PAGE.

For analysis of the extracts by “Stains-All” staining (4) the samples were

separated on 15 % Laemmli gels. For Western blot analysis the samples were

separated on a 10 % Laemmli gel, blotted onto nitrocellulose membrane and probed

with anti-GFP antibodies (Living Colours full-length A.v. polyclonal; Clontech, USA).

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Chitinase treatment and monitoring of chitin degradation. Chitinase from

Streptomyces griseus (SIGMA-Aldrich, USA) was added to biosilica or microring

preparations at a final concentration of 1 mgml-1 (0.47-0.65 Umg-1) in a solution

containing 50 mM potassium phosphate pH 6.0, and 0.05 % w/v sodium azide. To

suppress contaminating proteolytic activity 1 mM PMSF or 1x complete protease

inhibitor cocktail (Roche Applied Science, USA) was added. To assess the progress of

chitin degradation, aliquots of the samples were periodically removed and stained with

calcofluor white as described previously (5). Samples were considered “chitin free” if the

blue fluorescence diagnostic for chitin was virtually absent as judged by epifluorescence

microscopy.

Preparation of microrings. Isolated biosilica was resuspended in 10 M NH4F, adjusted

to pH 4-5 using 6 M HCl and incubated for 5-10 min at ambient temperature before

processing for electron microscopy, fluorophotometry, or silica formation experiments.

For SEM the microrings were filtered onto gold coated (prepared by sputter

coating) nucleopore polycarbonate filters (pore size: 0.1 or 0.2 µm, Whatman, UK). The

samples were extensively washed by passing H2O through the filter and then air dried.

For fluorophotometry microrings were pelleted by centrifugation (5 min, 14,000

g), and resuspended in 1 M Tris-HCl pH 7.5.

For silica formation experiments microrings were pelleted by centrifugation (5

min, 14,000 g), the pellet was washed twice with H2O, and resuspended in H2O.

Fluorophotometry. Isolated biosilica from transformant strains expressing GFP fusion

proteins was resuspended in 0.1 M sodium phosphate pH 7.0 containing 1x complete

protease inhibitor cocktail (Roche Applied Science). The suspension was sonicated

(Sonicator 3000, Misonix) until it appeared homogeneous, and then subjected to

fluorescence measurements. The amount of GFP associated with insoluble material

(biosilica or ammonium fluoride insoluble material) was calculated from standard curves

that were generated by adding known amounts of purified GFP-His6 (3) to biosilica or

ammonium fluoride insoluble material obtained from wild type cells.

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Dynamic Light Scattering. The zeta potential of microrings was measured in 50 mM

sodium acetate, pH 5.5, using the HPPS 5001 system (Zetasizer, Malvern Instruments,

UK).

Microring-induced silica formation. To study the formation of silica on microrings

deposited on gold-coated polycarbonate filters, sodium acetate pH 5.5 (50 mM final

concentration) and silicic acid (100 mM final concentration) were mixed immediately

prior to immersion of the filter in this solution. After the desired incubation time at

ambient temperature, the solution was pulled though the filter, the filter was washed

extensively with H2O, air dried, and subjected to scanning electron microscopy.

For silica formation in the presence of polyamines the silicic acid/sodium acetate

pH 5.5 solution was mixed with DAB-Am-16 immediately prior to immersion of the

membrane.

For both silica formation experiments in suspension and on the membrane the

ratio of polyamine to microring was identical (1 mol of polyamine per 2,000 microring

units; 1 microring unit corresponds to 1 mol SiO2 in the biosilica from which the

microrings were isolated).

References

1. Poulsen N, Kröger N. (2004) Silica morphogenesis by alternative processing of

silaffins in the diatom Thalassiosira pseudonana. J Biol Chem 279:42993-42999.

2. Poulsen N, Chesley PM, Kröger N (2006) Molecular genetic manipulation of the

diatom Thalassiosira pseudonana (Bacillariophyceae). J Phycol 42:1059-1065.

3. Poulsen N, Berne C, Spain J, Kröger N (2007) Silica immobilization of an

enzyme via genetic engineering of the diatom Thalassiosira pseudonana. Angew

Chem Int Ed 46:1843-1846.

4. Campbell KP, MacLennan DH, Jorgensen A) (1983) Staining of the Ca2+-binding

proteins Calsequestrin, Calmodulin, Troponin-C, and S-100 with the cationic

carbocyanine dye Stains-All. J Biol Chem 258:11267-11273.

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5. Brunner E, et al. (2009) Chitin-based organic networks: an integral part of cell

wall biosilica in the diatom Thalassiosira pseudonana. Angew Chem Int Ed

48:9724-9727.

6. Bowler C, et al. (2008) The Phaeodactylum genome reveals the evolutionary

history of diatom genomes. Nature 456:239-244.

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Table S1. Silaffin-like proteins from T. pseudonana.

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Table S2. Predicted cingulin-like proteins from P. tricornutum (6) and F. cylindrus (6).

The predicted proteomes of both diatoms were screened for silaffin-like proteins using

the algorithm described in the present work. Cingulin-like proteins were manually

identified from the list of silaffin-like proteins using the following criteria: >5 % Y for

CinY-like proteins, >3 % W and >2 % Y for CinW-like proteins.

Diatom species Protein ID Comments

P. tricornutum 46913 6.0 % Y, 1.7 % W; contains RXL domain

F. cylindrus 246170 5.5 % Y, 0.5 % W; contains RXL domain

F. cylindrus 184436 5.7 % Y, 1.0 % W; contains KXXK and RXL domains

F. cylindrus 260154 5.4 % Y, 0 % W

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Fig. S1. Analysis of the material that was extractable from biosilica isolated from

cingulin-GFP and tpSil3-GFP expressing transformants. (A) “Stains All” stained SDS-

PAGs of ammonium fluoride extracts (E), and SDS/dithiothreitol extracts of the

ammonium fluoride insoluble material (M). (B) Anti-GFP Western blot analysis of

extracts E and M of the tpSil3-GFP expressing transformant. Due to extensive post-

translational modifications of tpSil3 (3) the apparent molecular mass of the tpSil3-GFP

fusion protein is considerably larger than the predicted molecular mass of 48.6 kDa.

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Fig. S2. Epifluorescence microscopy of the ammonium fluoride insoluble material

(AFIM) from cingulin-GFP expressing transformants. GFP fluorescence was imaged

using a Piston GFP bandpass filter set, and Calcofluor White stained chitin was imaged

using a DAPI bandpass filter set. The numbers in the bottom left corners of the image

indicate the camera exposure times for recording the images. (A) Before and (B) after

treatment of AFIM with chitinase.

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Fig. S3. Effect of pronase on microrings prepared from chitin-free biosilica from T.

pseudonana. SEM images of microring preparations after incubation for 24 hours at 37

°C in 100 mM Tris-HCl pH 7.5 (A) and in 100 mM Tris-HCl pH 7.5 containing pronase

(B). The arrowheads indicate remnants of the disintegrated microrings. The dark dots

that are present within and outside the microrings are the pores in the underlying filter

membrane.

Fig. S4. Effect of pronase on biosilica associated microrings (chitin-free) of T.

pseudonana. (A, B) SEM images of microring preparations obtained from biosilica that

has been incubated for 24 hours at 37 °C in (A) 100 mM Tris-HCl pH 7.5, and in (B) 100

mM Tris-HCl pH 7.5 containing pronase. The dark dots that are present within and

outside the microrings are the pores in the underlying filter membrane.

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Fig. S5. SEM analysis (A, C) and electron dispersive X-ray spectroscopy (EDXS) (B, D,

E) of microrings before (A, B) and after incubation with silicic acid for 60 min (C-E). The

rectangular frames indicate the areas selected for EDXS. The dark dots that are present

within and outside the microrings are the pores in the underlying filter membrane.

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Fig. S6. Remineralization of microrings in vitro. SEM images of individual microrings

before and after addition of silicic acid. The times of incubation with silicic acid in the

absence and presence of constant amounts of synthetic polyamine DAB-Am-16 are

indicated. Two images are shown for each microring, one was recorded with the

secondary electron detector (SED) and the other with the in-lens detector (ILD). The

SED is more sensitive to surface morphology than ILD enhancing the contrast of the

contour and the appearance of surface roughness in the recorded images. The dark

dots that are present within and outside the microrings are the pores in the underlying

filter membrane.

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Fig. S7. Structures and elemental analyses of biosilica and biosilica-associated organic

matrices from the diatom Coscinodiscus wailesii. (A) SEM images of valve and girdle

band biosilica and biosilica-derived organic matrix (AFIM) from C. wailesii. The organic

matrices exhibit the same nanopatterns as the parent biosilica structures. The breaks in

the patterns of the biosilica and the AFIM (see asterisks) correspond to the region

where two adjacent girdle bands overlap.

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Fig. S7. (B) SEM images (left column) and EDXS spectra (right column) of C. wailesii

valve biosilica and the derived organic matrix (AFIM) demonstrating the absence of

silica in the organic matrix. The white rectangular frames indicate the areas selected for

EDXS.

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Fig. S8. Remineralization of the insoluble organic matrix of C. wailesii. Top, SEM image

of the organic matrix after incubation for 2 hours with 100 mM silicic acid at pH 5.5. The

white rectangular frames indicate the areas selected for EDXS. Bottom, EDX spectra of

areas within the organic matrix (area 1) and outside the organic matrix (area 2) are

shown.