Supplementary information Chemicals All

Supplementary information Chemicals All chemicals used in this study were of analytical grade or higher. Reagents used in this study: modified

sequencing grade trypsin (Promega, Madison, WI, USA), 18 O water (96.7% enriched) (Isotec Inc., Miamisburg, OH, USA), Tris buffered phenol (Life Technologies, Carlsbad, CA, USA), and iTRAQ reagents (AB Sciex, Toronto, ON, Canada).

Figure S1. (a) Archaeal and bacterial subfamily richness across individual samples. Archaeal subfamily richness ranged from 7 to 24; bacterial subfamily richness ranged from 346 to 602. All seawater samples were less rich in bacterial taxa compared to the hull biofilms. (b) Proportions of OTUs classified at the family level (nine richest families). Each color block represents the percentage of OTUs detected within a family compared to the total number of OTUs detected within the top nine richest families. Data acquired using the PhyloChip™ Array, version G3.

Quantitative metaproteomics Identified and annotated proteins that were common to both hull biofilms were compared using 18O labeling and iTRAQ® labeling for relative quantitative LC-MS/MS analyses. Five aliquots of protein extracts from both biofilm samples were used for the quantitative metaproteomic analyses. Each sample was processed as previously described for the qualitative metaproteomics analyses and then further subjected to stable isotope labeling. Two labeling techniques were used in this study – proteolytic 18O labeling and chemical labeling by iTRAQ. Three aliquots were used for proteolytic 18O labeling. To ensure that complete labeling was achieved and that there was no bias between heavy and light oxygen labels, two of these aliquots were split after digestion and subjected to forward and reverse labeling. This produced five technical replicates to be analyzed by LC-MS/MS [in three samples peptides from the Ship-2 biofilm were labeled with 18O and peptides from the Ship-1 biofilm were labeled with 16O (forward labeling); in two samples the biofilm peptides from Ship-2 were labeled with 16O and Ship-1 with 18O (reverse labeling)]. Thus, if a protein was quantified in all samples five separate ratios were obtained. Peptides extracted from gel bands were dried in a speed-vac and labeled with 16O or 18O at pH 6 as described previously (Tian et al. 2009; Hajkova et al. 2010, 2011). After trypsin deactivation, the samples were mixed in a 1:1 ratio and desalted using a C18 column (Nest Group, Southborough, MA, USA) according to the manufacturer’s instructions. The mixed peptides were reconstituted in solvent A (0.1% formic acid, 2.0% acetonitrile in water) and analyzed by LC-MS/MS. Acquired spectra were searched by Mascot against an in-house biofilm database (generated from the matched biofilm metagenome) with carbamidomethylation, methionine oxidation and 18O labeling quantification. The precursor and fragment ion tolerance was set to 0.2 Da. Identified peptides were quantified using the Mascot Distiller software (version 2.4.2.0, Matrix Science, London, UK). The same software package also calculated protein ratios from the peptide ratios. The average protein ratios of proteins quantified in three or more technical replicates were statistically analyzed using a one sample t-test to determine if they were different from the theoretical average of 1 (no change). Two aliquots of each biofilm were also labeled with iTRAQ reagents 113 and 121. The aliquots were split into two after digestion to perform forward (Ship-2, reagent 113; Ship-1, reagent 121) and reverse labeling (Ship-2, reagent 121; Ship-1, reagent 113) reactions according to the manufacturer’s instructions. Thus, if

proteins were quantified in all samples four ratios were obtained. The iTRAQ-labeled peptides were mixed in a 1:1 ratio and desalted using a C18 column. The mixed peptides were reconstituted in solvent A and analyzed by LC-MS/MS. All acquired spectra were searched using Mascot against the in-house biofilm database with carbamidomethylation and methionine oxidation set as

Figure S2. (a) Overview of the biofilm metagenome Illumina HiSeq 2000 sequence reads and read filtering from both ships. (b) Biofilm metagenome assembly and annotation pipeline. A total of 49,426,099 and 33,516,399 raw sequence reads were generated, trimmed and assembled into contiguous sequences resulting in the identification of 243,146 and 183,173 ORFs from which 89,504 and 76,123 ORFs could be annotated by SwissProt from Ship-1 and Ship-2. (c) Biofilm metagenome sequencing read classification summary.

Figure S3. (a) Fluorescence excitation and (b) emission spectra of biofilm acetone extracts from Ship-1. (c) Fluorescence excitation and (d) emission spectra of biofilm acetone extracts from Ship-2. The second derivatives of the excitation spectra for fluorescence at (e) 630 nm, (f) 642 nm, and (g) 675 nm from Ship-1 (red trace) and Ship-2 (blue trace). (h) Fluorescence spectra from Ship-1 (blue trace, left axis) and Ship-2 (red trace, right axis) under excitation at 466 nm. The spectra were normalized at 668 nm. (i) Biofilm pigment acetone extracts (Ship-1, left; Ship-2, right).

Table S1. Biofilm 16S rDNA profiling comparison: estimation of bacterial phylum, class and family richness based on the number of unique OTUs identified using the PhyloChip™ Array, version G3.a Phylum

Ship-1

Ship-2

Class

Ship-1

Ship-2

Family

Ship-1

Ship-2

Proteobacteria Firmicutes Bacteroidetes Actinobacteria Verrucomicrobia Planctomycetes Cyanobacteria Acidobacteria Chloroflexi Gemmatimonadetes

54.07 11.82 8.84 4.85 4.15 2.27 2.11 1.64 0.94 0.78

54.60 18.30 5.17 5.76 1.78 3.03 3.03 1.37 0.89 0.30

Gammaproteobacteria Alphaproteobacteria Clostridia Deltaproteobacteria Verrucomicrobiae Flavobacteria Actinobacteria Bacteroidia Bacilli Planctomycetacia

35.84 13.62 8.22 3.68 3.52 2.90 2.82 2.43 2.35 1.25

35.06 16.10 14.38 2.61 1.31 1.90 3.62 1.31 2.61 1.90

Pseudomonadaceae Verrucomicrobiaceae Enterobacteriaceae Rhodobacteraceae Oxalobacteraceae Clostridiaceae Bacteroidaceae Aeromonadaceae Peptostreptococcaceae Vibrionaceae

14.55 3.21 3.05 2.97 2.35 2.19 1.96 1.80 1.64 1.49

8.44 1.19 5.76 2.32 3.57 6.54 0.89 1.49 2.32 1.25

a

All numbers indicate the percentage of OTUs based on the total number of OTUs identified in all biofilm samples tested from the same ship’s hull.

variable modifications and precursor and fragment ion tolerance set to 0.2 Da. iTRAQ quantitation was selected. Identified peptides and proteins were quantified using Mascot. The average protein ratios with no normalization and automatic outlier removal were calculated from the peptide ratios. Finally, the average protein ratio from all of the technical replicates were calculated and analyzed by a one sample t-test. Carbon isotope elemental analysis Aliquots of both biofilms were analyzed for total organic carbon (TOC) weight per cent (wt%) and natural stable carbon isotope ratios (δ13CTOC) in

triplicate using a Costech Elemental Combustion System (ECS 4010) interfaced with a Delta Plus XP Isotope Ratio Mass Spectrometer (IRMS) (Thermo Fisher Scientific, Waltham, MA, USA) (Hamdan et al. 2011). Samples were freeze dried in liquid nitrogen, ground, acidified with 10% HCl to drive off inorganic carbon and re-dried at 65°C for 24 h and weighed before analysis. Measured TOC wt% was plotted against an acetanilide standard curve. A normalization equation was generated from USGA and IAEA standards and applied to reference δ13CTOC data to the Vienna Peedee Belemnite scale. Data were expressed in the standard δ-notation as ‰.

Table S2. Representation of the Clusters of Orthologous Groups (COGs) from both biofilm metagenomes. COG class

Description

Ship-1

%

Ship-2

%

A B C D E F G H I J K L M N O P Q R S T U V Y Z

RNA processing and modification Chromatin structure and dynamics Energy production and conversion Cell cycle control, cell division, chromosome partitioning Amino acid transport and metabolism Nucleotide transport and metabolism Carbohydrate transport and metabolism Coenzyme transport and metabolism Lipid transport and metabolism Translation, ribosomal structure and biogenesis Transcription Replication, recombination and repair Cell wall/membrane/envelope biogenesis Cell motility Posttranslational modification, protein turnover, chaperones Inorganic ion transport and metabolism Secondary metabolites biosynthesis, transport and catabolism General function prediction only Function unknown Signal transduction mechanisms Intracellular trafficking, secretion, and vesicular transport Defense mechanisms Nuclear structure Cytoskeleton

33 102 7,949 1,046 10,983 2,865 5,369 4,966 5,343 6,038 5,144 6,361 5,754 1,195 4,348 5,864 3,603 11,182 6,116 4,819 2,368 2,072 3 146

0.032 0.098 7.668 1.009 10.594 2.764 5.179 4.790 5.154 5.824 4.962 6.136 5.550 1.153 4.194 5.656 3.475 10.786 5.900 4.648 2.284 1.999 0.003 0.141

108 204 6,861 1,162 9,925 2,493 4,390 4,409 4,738 5,900 4,988 6,168 5,079 1,106 4,253 4,717 2,798 9,979 5,309 4,376 2,321 1,659 7 136

0.116 0.219 7.371 1.248 10.662 2.678 4.716 4.736 5.090 6.338 5.358 6.626 5.456 1.188 4.569 5.067 3.006 10.720 5.703 4.701 2.493 1.782 0.008 0.146

Table S3. Examples of potentially unique biological potential, skewed biological potential and the most abundant read-associated functions based on Pfam, KEGG, COG and GO database annotations of both ship hull biofilm metagenomes.a Ship-1 reads Total number

% of Total

Ship-2 reads Total number

% of Total

Database Identifier

Description

Pfam Pfam Pfam Pfam Pfam Pfam Pfam Pfam Pfam Pfam Pfam Pfam Pfam Pfam

PF00502 PF01385 PF04966 PF00147 PF00001 PF03028 PF00008 PF00028 PF00447 PF00069 PF00072 PF00873 PF00106 PF00005

Phycobilisome protein Probable transposase Carbohydrate-selective porin, OprB family Fibrinogen beta and gamma chains Seven transmembrane receptor (rhodopsin family) Dynein heavy chain EGF-like domain Cadherin domain HSF-type DNA-binding protein Protein kinase domain Response regulator receiver domain AcrB/AcrD/AcrF family Short chain dehydrogenase ABC transporter

27 48 49 66 168 27 90 102 2 261 832 850 878 886

0.026% 0.047% 0.048% 0.065% 0.164% 0.026% 0.088% 0.100% 0.002% 0.255% 0.813% 0.831% 0.858% 0.866%

0 0 0 0 0 1 1 1 93 474 624 662 693 864

0.000% 0.000% 0.000% 0.000% 0.000% 0.001% 0.001% 0.001% 0.103% 0.525% 0.692% 0.734% 0.768% 0.958%

GO GO GO GO

GO:0005272 GO:0030286 GO:0007156 GO:0007186

27 27 79 225

0.019% 0.019% 0.054% 0.155%

1 1 1 9

0.001% 0.001% 0.001% 0.007%

GO GO GO GO GO

GO:0005102 GO:0030089 GO:0016020 GO:0008152 GO:0005524

Sodium channel activity Dynein complex Homophilic cell adhesion G-protein coupled receptor protein signaling pathway Receptor binding Phycobilisome Membrane Metabolic process ATP binding

63 65 7,039 6,123 6,640

0.043% 0.045% 4.844% 4.214% 4.570%

0 0 5,115 5,308 6,509

0.000% 0.000% 4.108% 4.263% 5.228%

COG

COG05076

2

0.002%

25

0.030%

COG COG

COG05169 COG04636

2 40

0.002% 0.043%

92 0

0.110% 0.000%

COG COG COG COG COG

COG00675 COG02217 COG01960 COG01028 COG00841

54 476 577 582 586

0.058% 0.509% 0.616% 0.622% 0.626%

0 338 540 436 461

0.000% 0.405% 0.647% 0.523% 0.553%

KEGG KEGG KEGG KEGG KEGG KEGG KEGG KEGG KEGG KEGG KEGG KEGG KEGG KEGG

K09415 K13348 K09414 K08482 K06813 K04600 K06252 K11525 K06271 K11514 K05575 K02004 K02014 K00540

0 0 0 13 13 44 24 25 26 28 28 630 805 872

0.000% 0.000% 0.000% 0.012% 0.012% 0.042% 0.023% 0.024% 0.025% 0.027% 0.027% 0.597% 0.762% 0.826%

13 14 20 0 0 0 0 0 0 0 0 220 570 495

0.015% 0.016% 0.022% 0.000% 0.000% 0.000% 0.000% 0.000% 0.000% 0.000% 0.000% 0.247% 0.640% 0.556%

KEGG

K00936

977

0.925%

662

0.744%

a

Transcription factor involved in chromatin remodeling Heat shock transcription factor Uncharacterized protein conserved in cyanobacteria Transposase and inactivated derivatives Cation transport ATPase Acyl-CoA dehydrogenases Dehydrogenases with different specificities Cation/multidrug efflux pump Heat shock transcription factor 2 Protein Mpv17 Heat shock transcription factor 1 Circadian clock protein KaiC Cadherin 23 Cadherin EGF LAG seven-pass G-type receptor 1 Tenascin Methyl-accepting chemotaxis protein PixJ Talin Borealin NADH dehydrogenase I subunit 4 Putative ABC transport system permease protein Iron complex outer membrane recepter protein Unclassified metabolism (predicted oxidoreductase) Unclassified metabolism (sensor histidine kinase)

Unique biological potential was defined by the absence of reads in one biofilm. Skewed biological potential was defined as a > 10× difference in the number of reads between both biofilm metagenomes for any annotated functional category. Boldfaced text indicates the most abundant read-associated functions annotated using each database.

Table S4. Identified and gene ontology (GO) annotated proteins from both biofilm metaproteomes. Biological process

GO term number

GO term

Ship-1 proteins

Ship-2 proteins

Photosynthesis

GO:0009535 GO:0015979 GO:0009507 GO:0018298 GO:0009523 GO:0016168 GO:0009522 GO:0009767 GO:0009538 GO:0051539

Chloroplast thylakoid membrane Photosynthesis Chloroplast Protein-chromophore linkage Photosystem II Chlorophyll binding Photosystem I Photosynthetic electron transport chain Photosystem I reaction center 4 Iron, 4 sulfur cluster binding

8 7 7 6 3 1 1 1 1 1

25 22 22 19 17 15 8 6 6 5

RuBisCo

GO:0004497 GO:0016984

Monooxygenase activity Ribulose-bisphosphate carboxylase activity

6 6

12 12

Membrane & membrane transport

GO:0005886 GO:0006810 GO:0030288 GO:0015288 GO:0046930 GO:0015031 GO:0005887 GO:0007156

Plasma membrane Transport Outer membrane-bounded periplasmic space Porin activity Pore complex Protein transport Integral to plasma membrane Homophilic cell adhesion

31 6 1 1 1 7 5 15

33 16 6 5 5 3 1 0

Energy acquisition & metabolism

GO:0005524 GO:0006412 GO:0005525 GO:0003924 GO:0015986 GO:0009853 GO:0045261

45 16 24 21 10 6 9

47 21 20 16 15 13 13

GO:0022900 GO:0006096 GO:0003723 GO:0051258 GO:0008553 GO:0003677 GO:0016491 GO:0005509 GO:0006351 GO:0006355

ATP binding Translation GTP binding GTPase activity ATP synthesis coupled proton transport Photorespiration Proton-transporting ATP synthase complex, F(1) Electron transport chain Glycolysis RNA binding Protein polymerization Hydrogen-exporting ATPase activity DNA binding Oxidoreductase activity Calcium ion binding Transcription, DNA-dependent Regulation of transcription, DNA-dependent

2 4 9 9 7 15 5 10 6 5

9 8 8 7 7 6 5 3 2 2

GO:0005634 GO:0005759 GO:0005764 GO:0042470 GO:0048471 GO:0005788 GO:0005737 GO:0005856 GO:0005840 GO:0005829 GO:0005874 GO:0005911 GO:0006457 GO:0007018 GO:0003774 GO:0016477

Nucleus Mitochondrial matrix Lysosome Melanosome Perinuclear region of cytoplasm Endoplasmic reticulum lumen Cytoplasm Cytoskeleton Ribosome Cytosol Microtubule Cell-cell junction Protein folding Microtubule-based movement Motor activity Cell migration

15 5 8 9 7 11 52 5 9 12 10 6 10 10 6 5

8 2 0 2 0 5 25 4 9 8 7 0 2 7 0 0

Cytoplasmic components & structures

Table S5. Quantitative biofilm metaproteome results from Mascot Distiller.

Table S6. Relative pigment composition estimated from Gaussian fitting and deconvolution of the fluorescence and excitation spectra of biofilm pigment acetone extracts.

Ship-1 Ship-2 Ship-2/Ship-1 ratio

Chl a

Chl b

Chl c

Pheo a

1.000 1.000 1.000

0.038 0.008 0.211

0.105 0.189 1.800

0.125 0.062 0.496

Table S7. Whole biofilm carbon and nitrogen elemental analyses.

Ship-1.1 Ship-1.2 Ship-1.3 Ship-2.1 Ship-2.2 Ship-2.3

% Nitrogen

% TOC

Component

Norm δ13C

Stable carbon isotope ratio (‰)

2.021 2.138 2.014 1.160 1.053 1.041

11.420 11.453 11.005 8.796 8.143 8.403

Carbon Carbon Carbon Carbon Carbon Carbon

−18.370 −18.198 −18.548 −19.642 −20.007 −20.010

−18.372 −19.887

Spectroscopic analysis of photosynthetic pigments Aliquots of both biofilm samples (0.1 g) were mixed with 1.0 ml of acetone and vortexed for 1 min to extract photosynthetic pigments. The samples were cleared by centrifugation and the extracted pigments were analyzed by fluorescence spectroscopy. The spectra were recorded with a SPEX Fluorog-3 fluorometer (Horiba Instruments, Inc., Edison, NJ, USA) using a 0.5 cm rectangular quartz cuvette at ambient temperature by collecting the emission at a right angle to the excitation light with the excitation and emission spectral slit width equal to 4 nm. To eliminate the contribution of higher orders of excitation light in the emission light path, a combination of cut-off filters (SZS22 and OS18, LOMO, St Petersburg, Russia) was used. The best excitation and emission wavelengths for each spectrum and their combinations were estimated by 2D scanning of the entire luminescence. To ensure quality of the spectral identifications, each fluorescence spectrum was recorded at two to three different excitation wavelengths. To increase the spectral resolution and verify the purity of the identified spectral signatures for each pigment, the second derivatives of the excitation and emission spectra were calculated. The pigment assignment was based on the identity of the estimated combination of both excitation and emission spectra and their second derivatives to the spectra of chromatographically purified pigments in the same solvent (Porra 1991; Lebedev et al. 2001). The pheophytin to chlorophyll a ratio in the same samples was estimated by the subtraction of chlorophyll a bands from the total excitation spectrum of the two pigments and dividing the amount of pheophytin from the amount of chlorophyll a after normalization of total and chlorophyll a excitation spectra at a maximum of 429 nm (Lebedev et al. 1989). For the comparison of pigment amounts in different samples, the excitation spectra were normalized to the contribution of chlorophyll a and were corrected for the molar extinction coefficient and fluorescence quantum yield of each pigment (Porra 1991). In these calculations each molecule was considered as an independent oscillator (dipole approximation) and used molar extinction coefficients (in mM−1 cm−1) at Soret maxima and fluorescence quantum yields as 118, 115, 132, 71, 212 and 0.27, 0.27, 0.10, 0.14, 0.14 for chl a, pheo a, chl b, chl c1, and chl c3, respectively. To calculate the contributions of individual bands, the complex spectra were fitted with

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